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Henden C, Fjerdingstad HB, Bjørnsen EG, Thiruchelvam-Kyle L, Daws MR, Inngjerdingen M, Glover JC, Dissen E. NK-cell cytotoxicity toward pluripotent stem cells and their neural progeny: impacts of activating and inhibitory receptors and KIR/HLA mismatch. Stem Cells 2025; 43:sxae083. [PMID: 39708357 PMCID: PMC11929945 DOI: 10.1093/stmcls/sxae083] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/03/2020] [Accepted: 11/14/2024] [Indexed: 12/23/2024]
Abstract
Pluripotent stem cells provide opportunities for treating injuries and previously incurable diseases. A major concern is the immunogenicity of stem cells and their progeny. Here, we have dissected the molecular mechanisms that allow natural killer (NK) cells to respond to human pluripotent stem cells, investigating a wide selection of activating and inhibitory NK-cell receptors and their ligands. Reporter cells expressing the activating receptor NKG2D responded strongly to embryonic stem (ES) cell lines and induced pluripotent stem (iPS) cell lines, whereas reporter cells expressing the activating receptors NKp30, NKp46, KIR2DS1, KIR2DS2, and KIR2DS4 did not respond. Human ES and iPS cells invariably expressed several ligands for NKG2D. Expression of HLA-C and HLA-E was lacking or low, insufficient to trigger reporter cells expressing the inhibitory receptors KIR2DL1, -2DL2, or -2DL3. Similar results were obtained for the pluripotent embryonic carcinoma cell lines NTERA-2 and 2102Ep, and also iPS-cell-derived neural progenitor cells. Importantly, neural progenitor cells and iPS-cell-derived motoneurons also expressed B7H6, the ligand for the activating receptor NKp30. In line with these observations, IL-2-stimulated NK cells showed robust cytotoxic responses to ES and iPS cells as well as to iPS-cell-derived motoneurons. No significant differences in cytotoxicity levels were observed between KIR/HLA matched and mismatched combinations of NK cells and pluripotent targets. Together, these data indicate that pluripotent stem cells and their neural progeny are targets for NK-cell killing both by failing to sufficiently express ligands for inhibitory receptors and by expression of ligands for activating receptors.
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Affiliation(s)
- Camilla Henden
- Department of Molecular Medicine, Institute of Basic Medical Sciences, University of Oslo, N-0317 Oslo, Norway
| | - Hege B Fjerdingstad
- Department of Molecular Medicine, Institute of Basic Medical Sciences, University of Oslo, N-0317 Oslo, Norway
- Norwegian Center for Stem Cell Research, Department of Immunology and Transfusion Medicine, Oslo University Hospital, N-0317 Oslo, Norway
| | - Elisabeth G Bjørnsen
- Department of Molecular Medicine, Institute of Basic Medical Sciences, University of Oslo, N-0317 Oslo, Norway
| | - Lavanya Thiruchelvam-Kyle
- Department of Molecular Medicine, Institute of Basic Medical Sciences, University of Oslo, N-0317 Oslo, Norway
| | - Michael R Daws
- Department of Molecular Medicine, Institute of Basic Medical Sciences, University of Oslo, N-0317 Oslo, Norway
| | - Marit Inngjerdingen
- Department of Pharmacology, Institute of Clinical Medicine, University of Oslo, N-0317 Oslo, Norway
| | - Joel C Glover
- Department of Molecular Medicine, Institute of Basic Medical Sciences, University of Oslo, N-0317 Oslo, Norway
- Norwegian Center for Stem Cell Research, Department of Immunology and Transfusion Medicine, Oslo University Hospital, N-0317 Oslo, Norway
| | - Erik Dissen
- Department of Molecular Medicine, Institute of Basic Medical Sciences, University of Oslo, N-0317 Oslo, Norway
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Bogomiakova ME, Bogomazova AN, Lagarkova MA. Dysregulation of Immune Tolerance to Autologous iPSCs and Their Differentiated Derivatives. BIOCHEMISTRY. BIOKHIMIIA 2024; 89:799-816. [PMID: 38880643 DOI: 10.1134/s0006297924050031] [Citation(s) in RCA: 1] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/12/2023] [Revised: 12/21/2023] [Accepted: 02/13/2024] [Indexed: 06/18/2024]
Abstract
Induced pluripotent stem cells (iPSCs), capable of differentiating into any cell type, are a promising tool for solving the problem of donor organ shortage. In addition, reprogramming technology makes it possible to obtain a personalized, i.e., patient-specific, cell product transplantation of which should not cause problems related to histocompatibility of the transplanted tissues and organs. At the same time, inconsistent information about the main advantage of autologous iPSC-derivatives - lack of immunogenicity - still casts doubt on the possibility of using such cells beyond immunosuppressive therapy protocols. This review is devoted to immunogenic properties of the syngeneic and autologous iPSCs and their derivatives, as well as to the reasons for dysregulation of their immune tolerance.
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Affiliation(s)
- Margarita E Bogomiakova
- Federal Research and Clinical Center of Physical-Chemical Medicine, Federal Medical Biological Agency, Moscow, 119435, Russia.
- Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Federal Research and Clinical Center of Physical-Chemical Medicine, Federal Medical Biological Agency, Moscow, 119435, Russia
| | - Alexandra N Bogomazova
- Federal Research and Clinical Center of Physical-Chemical Medicine, Federal Medical Biological Agency, Moscow, 119435, Russia
- Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Federal Research and Clinical Center of Physical-Chemical Medicine, Federal Medical Biological Agency, Moscow, 119435, Russia
| | - Maria A Lagarkova
- Federal Research and Clinical Center of Physical-Chemical Medicine, Federal Medical Biological Agency, Moscow, 119435, Russia
- Lomonosov Moscow State University, Moscow, 119991, Russia
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Li W, Zhou M, Wang L, Huang L, Chen X, Sun X, Liu T. Evaluation of the safety and efficiency of cytotoxic T cell therapy sensitized by tumor antigens original from T-ALL-iPSC in vivo. CANCER INNOVATION 2024; 3:e95. [PMID: 38948536 PMCID: PMC11212296 DOI: 10.1002/cai2.95] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 04/29/2023] [Revised: 06/17/2023] [Accepted: 07/04/2023] [Indexed: 07/02/2024]
Abstract
Background Since RNA sequencing has shown that induced pluripotent stem cells (iPSCs) share a common antigen profile with tumor cells, cancer vaccines that focus on iPSCs have made promising progress in recent years. Previously, we showed that iPSCs derived from leukemic cells of patients with primary T cell acute lymphoblastic leukemia (T-ALL) have a gene expression profile similar to that of T-ALL cell lines. Methods Mice with T-ALL were treated with dendritic and T (DC-T) cells loaded with intact and complete antigens from T-ALL-derived iPSCs (T-ALL-iPSCs). We evaluated the safety and antitumor efficiency of autologous tumor-derived iPSC antigens by flow cytometry, cytokine release assay, acute toxicity experiments, long-term toxicity experiments, and other methods. Results Our results indicate that complete tumor antigens from T-ALL-iPSCs could inhibit the growth of inoculated tumors in immunocompromised mice without causing acute and long-term toxicity. Conclusion T-ALL-iPSC-based treatment is safe and can be used as a potential strategy for leukemia immunotherapy.
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Affiliation(s)
- Weiran Li
- Department of Tumor Immunotherapy, Shenzhen Luohu People's HospitalThe Third Affiliated Hospital of Shenzhen UniversityShenzhenGuangdongChina
- Cell Quality Testing Laboratory of Shenzhen Luohu Hospital GroupShenzhenGuangdongChina
| | - Meiling Zhou
- Department of Tumor Immunotherapy, Shenzhen Luohu People's HospitalThe Third Affiliated Hospital of Shenzhen UniversityShenzhenGuangdongChina
- Cell Quality Testing Laboratory of Shenzhen Luohu Hospital GroupShenzhenGuangdongChina
| | - Lu Wang
- Department of Tumor Immunotherapy, Shenzhen Luohu People's HospitalThe Third Affiliated Hospital of Shenzhen UniversityShenzhenGuangdongChina
- Cell Quality Testing Laboratory of Shenzhen Luohu Hospital GroupShenzhenGuangdongChina
| | - Liying Huang
- Department of Tumor Immunotherapy, Shenzhen Luohu People's HospitalThe Third Affiliated Hospital of Shenzhen UniversityShenzhenGuangdongChina
| | - Xuemei Chen
- Department of Tumor Immunotherapy, Shenzhen Luohu People's HospitalThe Third Affiliated Hospital of Shenzhen UniversityShenzhenGuangdongChina
- Cell Quality Testing Laboratory of Shenzhen Luohu Hospital GroupShenzhenGuangdongChina
| | - Xizhuo Sun
- Department of Tumor Immunotherapy, Shenzhen Luohu People's HospitalThe Third Affiliated Hospital of Shenzhen UniversityShenzhenGuangdongChina
| | - Tao Liu
- Department of Tumor Immunotherapy, Shenzhen Luohu People's HospitalThe Third Affiliated Hospital of Shenzhen UniversityShenzhenGuangdongChina
- Cell Quality Testing Laboratory of Shenzhen Luohu Hospital GroupShenzhenGuangdongChina
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Bogomiakova ME, Sekretova EK, Anufrieva KS, Khabarova PO, Kazakova AN, Bobrovsky PA, Grigoryeva TV, Eremeev AV, Lebedeva OS, Bogomazova AN, Lagarkova MA. iPSC-derived cells lack immune tolerance to autologous NK-cells due to imbalance in ligands for activating and inhibitory NK-cell receptors. Stem Cell Res Ther 2023; 14:77. [PMID: 37038186 PMCID: PMC10088155 DOI: 10.1186/s13287-023-03308-5] [Citation(s) in RCA: 9] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/12/2022] [Accepted: 03/28/2023] [Indexed: 04/12/2023] Open
Abstract
BACKGROUND Dozens of transplants generated from pluripotent stem cells are currently in clinical trials. The creation of patient-specific iPSCs makes personalized therapy possible due to their main advantage of immunotolerance. However, some reports have claimed recently that aberrant gene expression followed by proteome alterations and neoantigen formation can result in iPSCs recognition by autologous T-cells. Meanwhile, the possibility of NK-cell activation has not been previously considered. This study focused on the comparison of autologous and allogeneic immune response to iPSC-derived cells and isogeneic parental somatic cells used for reprogramming. METHODS We established an isogeneic cell model consisting of parental dermal fibroblasts, fibroblast-like iPSC-derivatives (iPS-fibro) and iPS-fibro lacking beta-2-microglobulin (B2M). Using the cells obtained from two patients, we analyzed the activation of autologous and allogeneic T-lymphocytes and NK-cells co-cultured with target cells. RESULTS Here we report that cells differentiated from iPSCs can be recognized by NK-cells rather than by autologous T-cells. We observed that iPS-fibro elicited a high level of NK-cell degranulation and cytotoxicity, while isogeneic parental skin fibroblasts used to obtain iPSCs barely triggered an NK-cell response. iPSC-derivatives with B2M knockout did not cause an additional increase in NK-cell activation, although they were devoid of HLA-I, the major inhibitory molecules for NK-cells. Transcriptome analysis revealed a significant imbalance of ligands for activating and inhibitory NK-cell receptors in iPS-fibro. Compared to parental fibroblasts, iPSC-derivatives had a reduced expression of HLA-I simultaneously with an increased gene expression of major activating ligands, such as MICA, NECTIN2, and PVR. The lack of inhibitory signals might be due to insufficient maturity of cells differentiated from iPSCs. In addition, we showed that pretreatment of iPS-fibro with proinflammatory cytokine IFNγ restored the ligand imbalance, thereby reducing the degranulation and cytotoxicity of NK-cells. CONCLUSION In summary, we showed that iPSC-derived cells can be sensitive to the cytotoxic potential of autologous NK-cells regardless of HLA-I status. Thus, the balance of ligands for NK-cell receptors should be considered prior to iPSC-based cell therapies. Trial registration Not applicable.
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Affiliation(s)
- Margarita E Bogomiakova
- Lopukhin Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency, 1a Malaya Pirogovskaya, Moscow, Russia, 119435.
- Faculty of Biology, Lomonosov Moscow State University, 1-12 Leninskie Gory, Moscow, Russia, 119991.
- Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Lopukhin Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency, 1a Malaya Pirogovskaya, Moscow, Russia, 119435.
| | - Elizaveta K Sekretova
- Lopukhin Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency, 1a Malaya Pirogovskaya, Moscow, Russia, 119435
- Faculty of Biology, Lomonosov Moscow State University, 1-12 Leninskie Gory, Moscow, Russia, 119991
| | - Ksenia S Anufrieva
- Lopukhin Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency, 1a Malaya Pirogovskaya, Moscow, Russia, 119435
- Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Lopukhin Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency, 1a Malaya Pirogovskaya, Moscow, Russia, 119435
| | - Polina O Khabarova
- Faculty of Biology, Lomonosov Moscow State University, 1-12 Leninskie Gory, Moscow, Russia, 119991
| | - Anastasia N Kazakova
- Lopukhin Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency, 1a Malaya Pirogovskaya, Moscow, Russia, 119435
| | - Pavel A Bobrovsky
- Lopukhin Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency, 1a Malaya Pirogovskaya, Moscow, Russia, 119435
- Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Lopukhin Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency, 1a Malaya Pirogovskaya, Moscow, Russia, 119435
| | | | - Artem V Eremeev
- Lopukhin Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency, 1a Malaya Pirogovskaya, Moscow, Russia, 119435
- Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Lopukhin Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency, 1a Malaya Pirogovskaya, Moscow, Russia, 119435
| | - Olga S Lebedeva
- Lopukhin Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency, 1a Malaya Pirogovskaya, Moscow, Russia, 119435
- Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Lopukhin Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency, 1a Malaya Pirogovskaya, Moscow, Russia, 119435
| | - Alexandra N Bogomazova
- Lopukhin Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency, 1a Malaya Pirogovskaya, Moscow, Russia, 119435
- Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Lopukhin Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency, 1a Malaya Pirogovskaya, Moscow, Russia, 119435
| | - Maria A Lagarkova
- Lopukhin Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency, 1a Malaya Pirogovskaya, Moscow, Russia, 119435
- Faculty of Biology, Lomonosov Moscow State University, 1-12 Leninskie Gory, Moscow, Russia, 119991
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Oh JY, Kim H, Lee HJ, Lee K, Barreda H, Kim HJ, Shin E, Bae EH, Kaur G, Zhang Y, Kim E, Lee JY, Lee RH. MHC Class I Enables MSCs to Evade NK-Cell-Mediated Cytotoxicity and Exert Immunosuppressive Activity. Stem Cells 2022; 40:870-882. [PMID: 35852488 PMCID: PMC9512104 DOI: 10.1093/stmcls/sxac043] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/31/2021] [Accepted: 05/20/2022] [Indexed: 01/07/2023]
Abstract
Allogeneic mesenchymal stem/stromal cells (MSCs) are frequently used in clinical trials due to their low expression of major histocompatibility complex (MHC) class I and lack of MHC class II. However, the levels of MHC classes I and II in MSCs are increased by inflammatory stimuli, raising concerns over potential adverse effects associated with allogeneic cell therapy. Also, it is unclear how the host immune response to MHC-mismatched MSCs affects the therapeutic efficacy of the cells. Herein, using strategies to manipulate MHC genes in human bone marrow-derived MSCs via the CRISPR-Cas9 system, plasmids, or siRNAs, we found that inhibition of MHC class I-not MHC class II-in MSCs lowered the survival rate of MSCs and their immunosuppressive potency in mice with experimental autoimmune uveoretinitis, specifically by increasing MSC vulnerability to natural killer (NK)-cell-mediated cytotoxicity. A subsequent survey of MSC batches derived from 6 human donors confirmed a significant correlation between MSC survival rate and susceptibility to NK cells with the potency of MSCs to increase MHC class I level upon stimulation. Our overall results demonstrate that MHC class I enables MSCs to evade NK-cell-mediated cytotoxicity and exert immunosuppressive activity.
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Affiliation(s)
- Joo Youn Oh
- Department of Ophthalmology, Seoul National University College of Medicine, Seoul, Korea,Laboratory of Ocular Regenerative Medicine and Immunology, Biomedical Research Institute, Seoul National University Hospital, Seoul, Korea
| | - Hyemee Kim
- Department of Molecular and Cellular Medicine, Institute for Regenerative Medicine, College of Medicine, Texas A&M University, College Station, TX, USA
| | - Hyun Ju Lee
- Laboratory of Ocular Regenerative Medicine and Immunology, Biomedical Research Institute, Seoul National University Hospital, Seoul, Korea
| | - Kangin Lee
- ToolGen, Inc., Geumcheon-gu, Seoul, Korea
| | - Heather Barreda
- Department of Molecular and Cellular Medicine, Institute for Regenerative Medicine, College of Medicine, Texas A&M University, College Station, TX, USA
| | - Hyeon Ji Kim
- Laboratory of Ocular Regenerative Medicine and Immunology, Biomedical Research Institute, Seoul National University Hospital, Seoul, Korea
| | - Eunji Shin
- ToolGen, Inc., Geumcheon-gu, Seoul, Korea
| | - Eun-Hye Bae
- Department of Molecular and Cellular Medicine, Institute for Regenerative Medicine, College of Medicine, Texas A&M University, College Station, TX, USA
| | - Gagandeep Kaur
- Department of Molecular and Cellular Medicine, Institute for Regenerative Medicine, College of Medicine, Texas A&M University, College Station, TX, USA
| | - Yu Zhang
- Department of Molecular and Cellular Medicine, Institute for Regenerative Medicine, College of Medicine, Texas A&M University, College Station, TX, USA
| | - Eunjae Kim
- Department of Molecular and Cellular Medicine, Institute for Regenerative Medicine, College of Medicine, Texas A&M University, College Station, TX, USA
| | | | - Ryang Hwa Lee
- Corresponding author: Ryang Hwa Lee, Molecular and Cellular Medicine Department, Institute for Regenerative Medicine, College of Medicine, Texas A&M University, 1114 TAMU, 206 Olsen Boulevard, College Station, TX 77845, USA.
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Melendez E, Chondronasiou D, Mosteiro L, Martínez de Villarreal J, Fernández-Alfara M, Lynch CJ, Grimm D, Real FX, Alcamí J, Climent N, Pietrocola F, Serrano M. Natural killer cells act as an extrinsic barrier for in vivo reprogramming. Development 2022; 149:275063. [PMID: 35420133 PMCID: PMC9124575 DOI: 10.1242/dev.200361] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/15/2021] [Accepted: 03/21/2022] [Indexed: 01/20/2023]
Abstract
ABSTRACT
The ectopic expression of the transcription factors OCT4, SOX2, KLF4 and MYC (OSKM) enables reprogramming of differentiated cells into pluripotent embryonic stem cells. Methods based on partial and reversible in vivo reprogramming are a promising strategy for tissue regeneration and rejuvenation. However, little is known about the barriers that impair reprogramming in an in vivo context. We report that natural killer (NK) cells significantly limit reprogramming, both in vitro and in vivo. Cells and tissues in the intermediate states of reprogramming upregulate the expression of NK-activating ligands, such as MULT1 and ICAM1. NK cells recognize and kill partially reprogrammed cells in a degranulation-dependent manner. Importantly, in vivo partial reprogramming is strongly reduced by adoptive transfer of NK cells, whereas it is significantly increased by their depletion. Notably, in the absence of NK cells, the pancreatic organoids derived from OSKM-expressing mice are remarkably large, suggesting that ablating NK surveillance favours the acquisition of progenitor-like properties. We conclude that NK cells pose an important barrier for in vivo reprogramming, and speculate that this concept may apply to other contexts of transient cellular plasticity.
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Affiliation(s)
- Elena Melendez
- Institute for Research in Biomedicine (IRB Barcelona), Barcelona Institute of Science and Technology (BIST), Barcelona 08028, Spain
| | - Dafni Chondronasiou
- Institute for Research in Biomedicine (IRB Barcelona), Barcelona Institute of Science and Technology (BIST), Barcelona 08028, Spain
| | - Lluc Mosteiro
- Department of Discovery Oncology, Genentech, South San Francisco, CA 94080, USA
| | - Jaime Martínez de Villarreal
- Epithelial Carcinogenesis Group, Spanish National Cancer Research Centre (CNIO), Madrid 28029, Spain
- CIBERONC, Madrid 28029, Spain
| | - Marcos Fernández-Alfara
- Institute for Research in Biomedicine (IRB Barcelona), Barcelona Institute of Science and Technology (BIST), Barcelona 08028, Spain
| | - Cian J. Lynch
- Institute for Research in Biomedicine (IRB Barcelona), Barcelona Institute of Science and Technology (BIST), Barcelona 08028, Spain
| | - Dirk Grimm
- Department of Infectious Diseases/Virology, Medical Faculty, University of Heidelberg, Heidelberg 69120, Germany
- BioQuant, Cluster of Excellence CellNetworks, University of Heidelberg, Heidelberg 69120, Germany
- German Center for Infection Research (DZIF) and German Center for Cardiovascular Research (DZHK), partner site Heidelberg, Heidelberg 69120, Germany
| | - Francisco X. Real
- Epithelial Carcinogenesis Group, Spanish National Cancer Research Centre (CNIO), Madrid 28029, Spain
- CIBERONC, Madrid 28029, Spain
- Department of Medicine and Life Sciences, Universitat Pompeu Fabra, Barcelona 08003, Spain
| | - José Alcamí
- HIV Unit, Hospital Clínic - Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona 08036, Spain
- AIDS Immunopathology Unit, National Center for Microbiology, Institute of Health Carlos III, Majadahonda (Madrid) 28220, Spain
- CIBER de Enfermedades Infecciosas, Instituto de Salud Carlos III, Madrid 28029, Spain
| | - Núria Climent
- HIV Unit, Hospital Clínic - Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona 08036, Spain
- AIDS Immunopathology Unit, National Center for Microbiology, Institute of Health Carlos III, Majadahonda (Madrid) 28220, Spain
- Fundació Clínic per a la Recerca Biomèdica (FCRB), Barcelona 08036, Spain
| | - Federico Pietrocola
- Institute for Research in Biomedicine (IRB Barcelona), Barcelona Institute of Science and Technology (BIST), Barcelona 08028, Spain
- Department of Biosciences and Nutrition, Karolinska Institutet, Huddinge 14152, Sweden
| | - Manuel Serrano
- Institute for Research in Biomedicine (IRB Barcelona), Barcelona Institute of Science and Technology (BIST), Barcelona 08028, Spain
- Catalan Institution for Research and Advanced Studies (ICREA), Barcelona 08010, Spain
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Rossbach B, Hariharan K, Mah N, Oh SJ, Volk HD, Reinke P, Kurtz A. Human iPSC-Derived Renal Cells Change Their Immunogenic Properties during Maturation: Implications for Regenerative Therapies. Cells 2022; 11:cells11081328. [PMID: 35456007 PMCID: PMC9032821 DOI: 10.3390/cells11081328] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/15/2022] [Revised: 04/11/2022] [Accepted: 04/12/2022] [Indexed: 12/24/2022] Open
Abstract
The success of human induced pluripotent stem cell (hiPSC)-based therapy critically depends on understanding and controlling the immunological effects of the hiPSC-derived transplant. While hiPSC-derived cells used for cell therapy are often immature with post-grafting maturation, immunological properties may change, with adverse effects on graft tolerance and control. In the present study, the allogeneic and autologous cellular immunity of hiPSC-derived progenitor and terminally differentiated cells were investigated in vitro. In contrast to allogeneic primary cells, hiPSC-derived early renal progenitors and mature renal epithelial cells are both tolerated not only by autologous but also by allogeneic T cells. These immune-privileged properties result from active immunomodulation and low immune visibility, which decrease during the process of cell maturation. However, autologous and allogeneic natural killer (NK) cell responses are not suppressed by hiPSC-derived renal cells and effectively change NK cell activation status. These findings clearly show a dynamic stage-specific dependency of autologous and allogeneic T and NK cell responses, with consequences for effective cell therapies. The study suggests that hiPSC-derived early progenitors may provide advantageous immune-suppressive properties when applied in cell therapy. The data furthermore indicate a need to suppress NK cell activation in allogeneic as well as autologous settings.
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Affiliation(s)
- Bella Rossbach
- Berlin Institute of Health Center for Regenerative Therapies (BCRT), Charité Universitätsmedizin Berlin, 13353 Berlin, Germany; (K.H.); (S.-J.O.); (H.-D.V.); (P.R.)
- Fraunhofer Institute for Biomedical Engineering (IBMT), Fraunhofer-Forum Berlin, 10178 Berlin, Germany;
- Correspondence: (B.R.); (A.K.)
| | - Krithika Hariharan
- Berlin Institute of Health Center for Regenerative Therapies (BCRT), Charité Universitätsmedizin Berlin, 13353 Berlin, Germany; (K.H.); (S.-J.O.); (H.-D.V.); (P.R.)
- Fraunhofer Institute for Biomedical Engineering (IBMT), Fraunhofer Project Center for Stem Cell Processing, 97082 Würzburg, Germany
| | - Nancy Mah
- Fraunhofer Institute for Biomedical Engineering (IBMT), Fraunhofer-Forum Berlin, 10178 Berlin, Germany;
| | - Su-Jun Oh
- Berlin Institute of Health Center for Regenerative Therapies (BCRT), Charité Universitätsmedizin Berlin, 13353 Berlin, Germany; (K.H.); (S.-J.O.); (H.-D.V.); (P.R.)
| | - Hans-Dieter Volk
- Berlin Institute of Health Center for Regenerative Therapies (BCRT), Charité Universitätsmedizin Berlin, 13353 Berlin, Germany; (K.H.); (S.-J.O.); (H.-D.V.); (P.R.)
- Institute for Medical Immunology (IMI), Charité Universitätsmedizin Berlin, 13353 Berlin, Germany
| | - Petra Reinke
- Berlin Institute of Health Center for Regenerative Therapies (BCRT), Charité Universitätsmedizin Berlin, 13353 Berlin, Germany; (K.H.); (S.-J.O.); (H.-D.V.); (P.R.)
- Berlin Center for Advanced Therapies (BeCat), Charité Universitätsmedizin Berlin, 13353 Berlin, Germany
| | - Andreas Kurtz
- Berlin Institute of Health Center for Regenerative Therapies (BCRT), Charité Universitätsmedizin Berlin, 13353 Berlin, Germany; (K.H.); (S.-J.O.); (H.-D.V.); (P.R.)
- Fraunhofer Institute for Biomedical Engineering (IBMT), Fraunhofer-Forum Berlin, 10178 Berlin, Germany;
- Correspondence: (B.R.); (A.K.)
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8
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Wang G, Heimendinger P, Ramelmeier RA, Wang W. Pluripotent stem cell-based cell therapies: current applications and future prospects. CURRENT OPINION IN BIOMEDICAL ENGINEERING 2022. [DOI: 10.1016/j.cobme.2022.100390] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/18/2022]
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9
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Çekiç D, Yılmaz ŞN, Bölgen N, Ünal S, Duce MN, Bayrak G, Demir D, Türkegün M, Sarı A, Demir Y, Ünal Ş. Impact of injectable chitosan cryogel microspherescaffolds on differentiation and proliferation of adiposederived mesenchymal stem cells into fat cells. J Biomater Appl 2021; 36:1335-1345. [PMID: 34965760 DOI: 10.1177/08853282211048284] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/22/2023]
Abstract
Difficulty in the clinical practice of stem cell therapy is often experienced in achieving desired target tissue cell differentiation and migration of stem cells to other tissue compartments where they are destroyed or die. This study was performed to evaluate if mesenchymal stem cells (MSCs) may differentiate into desired cell types when injected after combined with an injectable cryogel scaffold and to investigate if this scaffold may help in preventing cells from passing into different tissue compartments. MSCs were obtained from fat tissue of the rabbits as autografts and nuclei and cytoplasms of these cells were labeled with BrdU and PKH26. In Group 1, only-scaffold; in Group 2, only-MSCs; and in Group 3, combined stem cell/scaffold were injected to the right malar area of the rabbits. At postoperative 3 weeks, volumes of the injected areas were calculated by computer-tomography scans and histopathological evaluation was performed. The increase in the volume of the right malar areas was more in Group 3. In histopathological evaluation, chitosan cryogel microspheres were observed microscopically within the tissue and the scaffold was only partially degraded. Normal tissue form was seen in Group 2. Cells differentiated morphologically into fat cells were detected in Groups 2 and 3. Injectable chitosan cryogel microspheres were used in vivo for the first time in this study. As it was demonstrated to be useful in carrying MSCs to the reconstructed area, help cell differentiation to desired cells and prevent migration to other tissue compartments, it may be used for reconstructive purposes in the future.
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Affiliation(s)
- Duran Çekiç
- Faculty of Medicine, Department of Plastic, Reconstructive and Aesthetic Surgery, Mersin University, Turkey
| | | | - Nimet Bölgen
- Faculty of Engineering, Department of Chemical Engineering, Mersin University, Turkey
| | - Selma Ünal
- Faculty of Engineering, Department of Chemical Engineering, Mersin University, Turkey
| | - Meltem Nass Duce
- Faculty of Medicine, Department of Radiology, Mersin University, Turkey
| | - Gülsen Bayrak
- Faculty of Medicine, Department of Histology, Mersin University, Turkey
| | - Didem Demir
- Faculty of Engineering, Department of Chemical Engineering, Mersin University, Turkey
| | - Merve Türkegün
- Faculty of Medicine, Department of Biostatistics and Medical Informatics, Mersin University, Turkey
| | - Alper Sarı
- Faculty of Medicine, Department of Plastic, Reconstructive and Aesthetic Surgery, Mersin University, Turkey
| | - Yavuz Demir
- Faculty of Medicine, Department of Plastic, Reconstructive and Aesthetic Surgery, Mersin University, Turkey
| | - Şakir Ünal
- Faculty of Medicine, Department of Plastic, Reconstructive and Aesthetic Surgery, Mersin University, Turkey
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10
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Lu S, Feng Q. CAR-NK cells from engineered pluripotent stem cells: Off-the-shelf therapeutics for all patients. Stem Cells Transl Med 2021; 10 Suppl 2:S10-S17. [PMID: 34724715 PMCID: PMC8560199 DOI: 10.1002/sctm.21-0135] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/09/2021] [Revised: 05/04/2021] [Accepted: 05/16/2021] [Indexed: 12/12/2022] Open
Abstract
Clinical success of adoptive cell therapy with chimeric antigen receptor (CAR) T cells for treating hematological malignancies has revolutionized the field of cellular immunotherapy. However, due to the nature of utilizing autologous T cells, affordability and availability are major hurdles, in addition to scientific challenges relating to CAR-T therapy optimization. Natural killer (NK) cell is a specialized immune effector cell type that recognizes and kills targets without human leukocyte antigen (HLA) restriction and prior sensitization. CAR-NK cells do not cause graft vs host disease and can be obtained from unrelated donors as well as pluripotent stem cells (PSC), representing an ideal off-the-shelf therapeutics readily available for patients. Furthermore, unlike cytotoxic T cells, NK cells specifically target and eliminate cancer stem cells, which are the cells causing relapse and metastasis. PSCs can be genetically manipulated and engineered with CARs at the pluripotent stage, which allows the establishment of permanent, stable, and clonal PSC-CAR lines for the manufacture of unlimited homogenous CAR-NK cells. Multiple master PSC-CAR cell banks targeting a variety of antigens for cancer, viral infection, and autoimmune diseases provide inexhaustible cell sources for all patients. Development of a next-generation 3D bioreactor platform for PSC expansion and NK cell production overcomes major barriers related to cost and scalability for CAR-NK product.
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Affiliation(s)
| | - Qiang Feng
- HebeCell CorporationNatickMassachusettsUSA
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11
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Petrus-Reurer S, Romano M, Howlett S, Jones JL, Lombardi G, Saeb-Parsy K. Immunological considerations and challenges for regenerative cellular therapies. Commun Biol 2021; 4:798. [PMID: 34172826 PMCID: PMC8233383 DOI: 10.1038/s42003-021-02237-4] [Citation(s) in RCA: 49] [Impact Index Per Article: 12.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/09/2020] [Accepted: 05/17/2021] [Indexed: 02/06/2023] Open
Abstract
The central goal of regenerative medicine is to replace damaged or diseased tissue with cells that integrate and function optimally. The capacity of pluripotent stem cells to produce unlimited numbers of differentiated cells is of considerable therapeutic interest, with several clinical trials underway. However, the host immune response represents an important barrier to clinical translation. Here we describe the role of the host innate and adaptive immune responses as triggers of allogeneic graft rejection. We discuss how the immune response is determined by the cellular therapy. Additionally, we describe the range of available in vitro and in vivo experimental approaches to examine the immunogenicity of cellular therapies, and finally we review potential strategies to ameliorate immune rejection. In conclusion, we advocate establishment of platforms that bring together the multidisciplinary expertise and infrastructure necessary to comprehensively investigate the immunogenicity of cellular therapies to ensure their clinical safety and efficacy.
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Affiliation(s)
- Sandra Petrus-Reurer
- Department of Surgery, University of Cambridge, and NIHR Cambridge Biomedical Research Centre, Cambridge, United Kingdom.
| | - Marco Romano
- Peter Gorer Department of Immunobiology, School of Immunology and Microbial Sciences, King's College London, Guy's Hospital, London, United Kingdom
| | - Sarah Howlett
- Department of Clinical Neuroscience, University of Cambridge, Cambridge, United Kingdom
| | - Joanne Louise Jones
- Department of Clinical Neuroscience, University of Cambridge, Cambridge, United Kingdom
| | - Giovanna Lombardi
- Peter Gorer Department of Immunobiology, School of Immunology and Microbial Sciences, King's College London, Guy's Hospital, London, United Kingdom
| | - Kourosh Saeb-Parsy
- Department of Surgery, University of Cambridge, and NIHR Cambridge Biomedical Research Centre, Cambridge, United Kingdom.
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12
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Evolution of Stem Cells in Cardio-Regenerative Therapy. Stem Cells 2021. [DOI: 10.1007/978-3-030-77052-5_7] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/26/2022]
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13
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Ishigaki H, Pham VL, Terai J, Sasamura T, Nguyen CT, Ishida H, Okahara J, Kaneko S, Shiina T, Nakayama M, Itoh Y, Ogasawara K. No Tumorigenicity of Allogeneic Induced Pluripotent Stem Cells in Major Histocompatibility Complex-matched Cynomolgus Macaques. Cell Transplant 2021; 30:963689721992066. [PMID: 33588604 PMCID: PMC7894586 DOI: 10.1177/0963689721992066] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/18/2019] [Revised: 12/11/2020] [Accepted: 01/12/2021] [Indexed: 12/14/2022] Open
Abstract
Tumorigenicity of induced pluripotent stem cells (iPSCs) is anticipated when cells derived from iPSCs are transplanted. It has been reported that iPSCs formed a teratoma in vivo in autologous transplantation in a nonhuman primate model without immunosuppression. However, there has been no study on tumorigenicity in major histocompatibility complex (MHC)-matched allogeneic iPSC transplantation with immune-competent hosts. To examine the tumorigenicity of allogeneic iPSCs, we generated four iPSC clones carrying a homozygous haplotype of the MHC. Two clones were derived from female fibroblasts by using a retrovirus and the other two clones were derived from male peripheral blood mononuclear cells by using Sendai virus (episomal approach). The iPSC clones were transplanted into allogenic MHC-matched immune-competent cynomolgus macaques. After transplantation of the iPSCs into subcutaneous tissue of an MHC-matched female macaque and into four testes of two MHC-matched male macaques, histological analysis showed no tumor, inflammation, or regenerative change in the excised tissues 3 months after transplantation, despite the results that iPSCs formed teratomas in immune-deficient mice and in autologous transplantation as previously reported. The results in the present study suggest that there is no tumorigenicity of iPSCs in MHC-matched allogeneic transplantation in clinical application.
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Affiliation(s)
- Hirohito Ishigaki
- Division of Pathology and Disease Regulation, Department of Pathology, Shiga University of Medical Science, Otsu, Shiga, Japan
| | - Van Loi Pham
- Division of Pathology and Disease Regulation, Department of Pathology, Shiga University of Medical Science, Otsu, Shiga, Japan
- Biomolecular and Genetic Unit, Department of Hematology, Choray Hospital, Ho Chi Minh City, Vietnam
| | - Jun Terai
- Division of Pathology and Disease Regulation, Department of Pathology, Shiga University of Medical Science, Otsu, Shiga, Japan
| | - Takako Sasamura
- Division of Pathology and Disease Regulation, Department of Pathology, Shiga University of Medical Science, Otsu, Shiga, Japan
| | - Cong Thanh Nguyen
- Division of Pathology and Disease Regulation, Department of Pathology, Shiga University of Medical Science, Otsu, Shiga, Japan
| | - Hideaki Ishida
- Division of Pathology and Disease Regulation, Department of Pathology, Shiga University of Medical Science, Otsu, Shiga, Japan
| | - Junko Okahara
- Central Institute for Experimental Animals, Kawasaki, Kanagawa, Japan
| | - Shin Kaneko
- Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan
| | - Takashi Shiina
- Division of Basic Medical Science and Molecular Medicine, Department of Molecular Life Science, Tokai University School of Medicine, Isehara, Kanagawa, Japan
| | - Misako Nakayama
- Division of Pathology and Disease Regulation, Department of Pathology, Shiga University of Medical Science, Otsu, Shiga, Japan
| | - Yasushi Itoh
- Division of Pathology and Disease Regulation, Department of Pathology, Shiga University of Medical Science, Otsu, Shiga, Japan
| | - Kazumasa Ogasawara
- Division of Pathology and Disease Regulation, Department of Pathology, Shiga University of Medical Science, Otsu, Shiga, Japan
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14
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Strategies for Cancer Immunotherapy Using Induced Pluripotency Stem Cells-Based Vaccines. Cancers (Basel) 2020; 12:cancers12123581. [PMID: 33266109 PMCID: PMC7760556 DOI: 10.3390/cancers12123581] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/31/2020] [Revised: 11/24/2020] [Accepted: 11/27/2020] [Indexed: 12/14/2022] Open
Abstract
Despite improvements in cancer therapy, metastatic solid tumors remain largely incurable. Immunotherapy has emerged as a pioneering and promising approach for cancer therapy and management, and in particular intended for advanced tumors unresponsive to current therapeutics. In cancer immunotherapy, components of the immune system are exploited to eliminate cancer cells and treat patients. The recent clinical successes of immune checkpoint blockade and chimeric antigen receptor T cell therapies represent a turning point in cancer treatment. Despite their potential success, current approaches depend on efficient tumor antigen presentation which are often inaccessible, and most tumors turn refractory to current immunotherapy. Patient-derived induced pluripotent stem cells (iPSCs) have been shown to share several characteristics with cancer (stem) cells (CSCs), eliciting a specific anti-tumoral response when injected in rodent cancer models. Indeed, artificial cellular reprogramming has been widely compared to the biogenesis of CSCs. Here, we will discuss the state-of-the-art on the potential implication of cellular reprogramming and iPSCs for the design of patient-specific immunotherapeutic strategies, debating the similarities between iPSCs and cancer cells and introducing potential strategies that could enhance the efficiency and therapeutic potential of iPSCs-based cancer vaccines.
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15
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Strategies for Genetically Engineering Hypoimmunogenic Universal Pluripotent Stem Cells. iScience 2020; 23:101162. [PMID: 32502965 PMCID: PMC7270609 DOI: 10.1016/j.isci.2020.101162] [Citation(s) in RCA: 29] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/06/2020] [Revised: 04/07/2020] [Accepted: 05/11/2020] [Indexed: 01/18/2023] Open
Abstract
Despite progress in developing cell therapies, such as T cell or stem cell therapies to treat diseases, immunoincompatibility remains a major barrier to clinical application. Given the fact that a host's immune system may reject allogeneic transplanted cells, methods have been developed to genetically modify patients' primary cells. To advance beyond this time-consuming and costly approach, recent research efforts focus on generating universal pluripotent stem cells to benefit a broader spectrum of patients. In this review, we first summarize current achievements to harness immunosuppressive mechanisms in cells to reduce immunogenicity. Then, we discuss several recent studies demonstrating the feasibility of genetically modifying pluripotent stem cells to escape immune attack and summarize the methods to evaluate hypoimmunogenicity. Although challenges remain, progress to develop genetically engineered universal pluripotent stem cells holds the promise of expediting their use in future gene and cell therapeutics and regenerative medicine.
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16
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Benabdallah B, Désaulniers-Langevin C, Colas C, Li Y, Rousseau G, Guimond JV, Haddad E, Beauséjour C. Natural Killer Cells Prevent the Formation of Teratomas Derived From Human Induced Pluripotent Stem Cells. Front Immunol 2019; 10:2580. [PMID: 31787975 PMCID: PMC6854018 DOI: 10.3389/fimmu.2019.02580] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/26/2019] [Accepted: 10/17/2019] [Indexed: 01/14/2023] Open
Abstract
The safe utilization of induced pluripotent stem cell (iPSC) derivatives in clinical use is attributed to the complete elimination of the risk of forming teratomas after transplantation. The extent by which such a risk exists in immune-competent hosts is mostly unknown. Here, using humanized mice reconstituted with fetal hematopoietic stem cells and autologous thymus tissue (bone-liver-thymus humanized mice [Hu-BLT]) or following the adoptive transfer of peripheral blood mononuclear cells(PBMCs) (Hu-AT), we evaluated the capacity of immune cells to prevent or eliminate teratomas derived from human iPSCs (hiPSCs). Our results showed that the injection of hiPSCs failed to form teratomas in Hu-AT mice reconstituted with allogeneic or autologous PBMCs or purified natural killer (NK) cells alone. However, teratomas were observed in Hu-AT mice reconstituted with autologous PBMCs depleted from NK cells. In line with these results, Hu-BLT, which do not have functional NK cells, could not prevent the growth of teratomas. Finally, we found that established teratomas were not targeted by NK cells and instead were efficiently rejected by allogeneic but not autologous T cells in Hu-AT mice. Overall, our findings suggest that autologous hiPSC-derived therapies are unlikely to form teratomas in the presence of NK cells.
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Affiliation(s)
| | | | - Chloé Colas
- Centre de recherche du CHU Sainte-Justine, Montreal, QC, Canada
| | - Yuanyi Li
- Centre de recherche du CHU Sainte-Justine, Montreal, QC, Canada
| | - Guy Rousseau
- Centre de recherche du CIUSSS du Nord-de-Île-de Montréal, Montreal, QC, Canada
- Département de Pharmacologie et Physiologie, Faculté de Médecine, Université de Montréal, Montreal, QC, Canada
| | - Jean V. Guimond
- CIUSSS du Centre-Sud-de-l'Ile-de-Montréal, Montreal, QC, Canada
| | - Elie Haddad
- Centre de recherche du CHU Sainte-Justine, Montreal, QC, Canada
- Département de Pédiatrie, Faculté de Médecine, Université de Montréal, Montreal, QC, Canada
| | - Christian Beauséjour
- Centre de recherche du CHU Sainte-Justine, Montreal, QC, Canada
- Département de Pharmacologie et Physiologie, Faculté de Médecine, Université de Montréal, Montreal, QC, Canada
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17
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Nakamura Y, Miyagawa S, Yoshida S, Sasawatari S, Toyofuku T, Toda K, Sawa Y. Natural killer cells impede the engraftment of cardiomyocytes derived from induced pluripotent stem cells in syngeneic mouse model. Sci Rep 2019; 9:10840. [PMID: 31346220 PMCID: PMC6658523 DOI: 10.1038/s41598-019-47134-3] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/17/2018] [Accepted: 07/11/2019] [Indexed: 02/06/2023] Open
Abstract
Transplantation of cardiomyocytes derived from induced pluripotent stem cell (iPSC-CMs) is a promising approach for increasing functional CMs during end-stage heart failure. Although major histocompatibility complex (MHC) class I matching between donor cells and recipient could reduce acquired immune rejection, innate immune responses may have negative effects on transplanted iPSC-CMs. Here, we demonstrated that natural killer cells (NKCs) infiltrated in iPSC-CM transplants even in a syngeneic mouse model. The depletion of NKCs using an anti-NKC antibody rescued transplanted iPSC-CMs, suggesting that iPSC-CMs activated NKC-mediated innate immunity. Surprisingly, iPSC-CMs lost inhibitory MHCs but not activating ligands for NKCs. Re-expression of MHC class I induced by IFN-γ as well as suppression of activating ligands by an antibody rescued the transplanted iPSC-CMs. Thus, NKCs impede the engraftment of transplanted iPSC-CMs because of lost MHC class I, and our results provide a basis for an approach to improve iPSC-CM engraftment.
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Affiliation(s)
- Yuki Nakamura
- Department of Cardiovascular Surgery, Osaka University Graduate School of Medicine, Suita, Osaka, 565-0871, Japan
| | - Shigeru Miyagawa
- Department of Cardiovascular Surgery, Osaka University Graduate School of Medicine, Suita, Osaka, 565-0871, Japan
| | - Shohei Yoshida
- Department of Cardiovascular Surgery, Osaka University Graduate School of Medicine, Suita, Osaka, 565-0871, Japan
| | - Shigemi Sasawatari
- Department of Immunology and Regenerative Medicine, Osaka University Graduate School of Medicine, Suita, Osaka, 565-0871, Japan
| | - Toshihiko Toyofuku
- Department of Immunology and Regenerative Medicine, Osaka University Graduate School of Medicine, Suita, Osaka, 565-0871, Japan
| | - Koichi Toda
- Department of Cardiovascular Surgery, Osaka University Graduate School of Medicine, Suita, Osaka, 565-0871, Japan
| | - Yoshiki Sawa
- Department of Cardiovascular Surgery, Osaka University Graduate School of Medicine, Suita, Osaka, 565-0871, Japan.
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18
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Cisneros T, Dillard DW, Qu X, Arredondo-Guerrero J, Castro M, Schaffert S, Martin R, Esquivel CO, Krams SM, Martinez OM. Differential role of natural killer group 2D in recognition and cytotoxicity of hepatocyte-like cells derived from embryonic stem cells and induced pluripotent stem cells. Am J Transplant 2019; 19:1652-1662. [PMID: 30549427 PMCID: PMC6543818 DOI: 10.1111/ajt.15217] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/07/2018] [Revised: 11/28/2018] [Accepted: 11/30/2018] [Indexed: 01/25/2023]
Abstract
Stem cell-based approaches have the potential to address the organ shortage in transplantation. Whereas both embryonic stem cells and induced pluripotent stem cells have been utilized as cellular sources for differentiation and lineage specification, their relative ability to be recognized by immune effector cells is unclear. We determined the expression of immune recognition molecules on hepatocyte-like cells (HLC) generated from murine embryonic stem cells and induced pluripotent stem cells, compared to adult hepatocytes, and we evaluated the impact on recognition by natural killer (NK) cells. We report that HLC lack MHC class I expression, and that embryonic stem cell-derived HLC have higher expression of the NK cell activating ligands Rae1, H60, and Mult1 than induced pluripotent stem cell-derived HLC and adult hepatocytes. Moreover, the lack of MHC class I renders embryonic stem cell-derived HLC, and induced pluripotent stem cell-derived HLC, susceptible to killing by syngeneic and allogeneic NK cells. Both embryonic stem cell-derived HLC, and induced pluripotent stem cell-derived HLC, are killed by NK cells at higher levels than adult hepatocytes. Finally, we demonstrate that the NK cell activation receptor, NKG2D, plays a key role in NK cell cytotoxicity of embryonic stem cell-derived HLC, but not induced pluripotent stem cell-derived HLC.
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Affiliation(s)
- Trinidad Cisneros
- Stanford Immunology, Stanford University School of
Medicine, Stanford, CA, USA,Department of Surgery/Division of Abdominal
Transplantation, Stanford University School of Medicine, Stanford, CA, USA
| | - Danielle W. Dillard
- Department of Surgery/Division of Abdominal
Transplantation, Stanford University School of Medicine, Stanford, CA, USA
| | - Xiumei Qu
- Department of Surgery/Division of Abdominal
Transplantation, Stanford University School of Medicine, Stanford, CA, USA
| | | | - Martha Castro
- Stanford Immunology, Stanford University School of
Medicine, Stanford, CA, USA
| | - Steven Schaffert
- Stanford Center for Biomedical Informatics Research,
Stanford University School of Medicine, Stanford, CA, USA
| | - Renata Martin
- Department of Biology, Stanford University School of
Medicine, Stanford, CA, USA
| | - Carlos O. Esquivel
- Department of Surgery/Division of Abdominal
Transplantation, Stanford University School of Medicine, Stanford, CA, USA
| | - Sheri M. Krams
- Stanford Immunology, Stanford University School of
Medicine, Stanford, CA, USA,Department of Surgery/Division of Abdominal
Transplantation, Stanford University School of Medicine, Stanford, CA, USA
| | - Olivia M. Martinez
- Stanford Immunology, Stanford University School of
Medicine, Stanford, CA, USA,Department of Surgery/Division of Abdominal
Transplantation, Stanford University School of Medicine, Stanford, CA, USA
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19
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NanoCsA improves the survival of human iPSC transplant in hemiparkinsonian rats. Brain Res 2019; 1719:124-132. [PMID: 31153914 DOI: 10.1016/j.brainres.2019.05.040] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/05/2019] [Revised: 05/28/2019] [Accepted: 05/29/2019] [Indexed: 12/14/2022]
Abstract
Increasing evidence has supported that transplantation of human stem cells induces neuroprotective and reparative effects in animal models of Parkinson's disease (PD). However, without systemic immunosuppressive therapy, most of these grafted cells are rejected by the hosts. Long term and systemic injection of cyclosporine-A (CsA) is required to maintain the survival of grafted cells. The purpose this study is to examine a new treatment strategy to suppress the immunorejection by locally co-grafting of polylactic/glycolic acid nanoparticles containing CsA (NanoCsA) with differentiated human induced pluripotent stem cells (iPSCs). In the in vitro media, NanoCsA provided sustained release of CsA for >6 weeks. The differentiated human iPSCs were co-grafted with NanoCsA or NanoVeh (nanoparticle without CsA) to the striatum of unilaterally 6-hydroxydopamine -lesioned rats. NanoCsA/iPSCs co-graft significantly improved locomotor activity compared to NanoVeh/iPSCs co-grafts or iPSC grafts + sytemic CsA at 1 month after transplantation. Brain tissues were collected for measurements of tyrosine hydroxylase (TH) and human marker Stem121 immunoreactivity. Cografting with NanoCsA/iPSCs, compared to NanoVeh/iPSCs, significantly increased TH and Stem121 immunoreactivity as well as tumor formation in the lesioned striatum. Taken together, our study supports that NanoCsA provides long-lasting CsA release and reduces immunorejection of human iPSCs xenograft in a 6-hydroxydopamine rat model of PD.
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20
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Hypoimmunogenic derivatives of induced pluripotent stem cells evade immune rejection in fully immunocompetent allogeneic recipients. Nat Biotechnol 2019; 37:252-258. [PMID: 30778232 DOI: 10.1038/s41587-019-0016-3] [Citation(s) in RCA: 482] [Impact Index Per Article: 80.3] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/23/2018] [Accepted: 12/28/2018] [Indexed: 12/21/2022]
Abstract
Autologous induced pluripotent stem cells (iPSCs) constitute an unlimited cell source for patient-specific cell-based organ repair strategies. However, their generation and subsequent differentiation into specific cells or tissues entail cell line-specific manufacturing challenges and form a lengthy process that precludes acute treatment modalities. These shortcomings could be overcome by using prefabricated allogeneic cell or tissue products, but the vigorous immune response against histo-incompatible cells has prevented the successful implementation of this approach. Here we show that both mouse and human iPSCs lose their immunogenicity when major histocompatibility complex (MHC) class I and II genes are inactivated and CD47 is over-expressed. These hypoimmunogenic iPSCs retain their pluripotent stem cell potential and differentiation capacity. Endothelial cells, smooth muscle cells, and cardiomyocytes derived from hypoimmunogenic mouse or human iPSCs reliably evade immune rejection in fully MHC-mismatched allogeneic recipients and survive long-term without the use of immunosuppression. These findings suggest that hypoimmunogenic cell grafts can be engineered for universal transplantation.
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21
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Abstract
The retina is a very fine and layered neural tissue, which vitally depends on the preservation of cells, structure, connectivity and vasculature to maintain vision. There is an urgent need to find technical and biological solutions to major challenges associated with functional replacement of retinal cells. The major unmet challenges include generating sufficient numbers of specific cell types, achieving functional integration of transplanted cells, especially photoreceptors, and surgical delivery of retinal cells or tissue without triggering immune responses, inflammation and/or remodeling. The advances of regenerative medicine enabled generation of three-dimensional tissues (organoids), partially recreating the anatomical structure, biological complexity and physiology of several tissues, which are important targets for stem cell replacement therapies. Derivation of retinal tissue in a dish creates new opportunities for cell replacement therapies of blindness and addresses the need to preserve retinal architecture to restore vision. Retinal cell therapies aimed at preserving and improving vision have achieved many improvements in the past ten years. Retinal organoid technologies provide a number of solutions to technical and biological challenges associated with functional replacement of retinal cells to achieve long-term vision restoration. Our review summarizes the progress in cell therapies of retina, with focus on human pluripotent stem cell-derived retinal tissue, and critically evaluates the potential of retinal organoid approaches to solve a major unmet clinical need—retinal repair and vision restoration in conditions caused by retinal degeneration and traumatic ocular injuries. We also analyze obstacles in commercialization of retinal organoid technology for clinical application.
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22
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Gröschel C, Sasse A, Monecke S, Röhrborn C, Elsner L, Didié M, Reupke V, Bunt G, Lichtman AH, Toischer K, Zimmermann WH, Hasenfuß G, Dressel R. CD8 +-T Cells With Specificity for a Model Antigen in Cardiomyocytes Can Become Activated After Transverse Aortic Constriction but Do Not Accelerate Progression to Heart Failure. Front Immunol 2018; 9:2665. [PMID: 30498501 PMCID: PMC6249381 DOI: 10.3389/fimmu.2018.02665] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/09/2018] [Accepted: 10/29/2018] [Indexed: 12/17/2022] Open
Abstract
Heart failure due to pressure overload is frequently associated with inflammation. In addition to inflammatory responses of the innate immune system, autoimmune reactions of the adaptive immune system appear to be triggered in subgroups of patients with heart failure as demonstrated by the presence of autoantibodies against myocardial antigens. Moreover, T cell-deficient and T cell-depleted mice have been reported to be protected from heart failure induced by transverse aortic constriction (TAC) and we have shown recently that CD4+-helper T cells with specificity for an antigen in cardiomyocytes accelerate TAC-induced heart failure. In this study, we set out to investigate the potential contribution of CD8+-cytotoxic T cells with specificity to a model antigen (ovalbumin, OVA) in cardiomyocytes to pressure overload-induced heart failure. In 78% of cMy-mOVA mice with cardiomyocyte-specific OVA expression, a low-grade OVA-specific cellular cytotoxicity was detected after TAC. Adoptive transfer of OVA-specific CD8+-T cells from T cell receptor transgenic OT-I mice before TAC did not increase the risk of OVA-specific autoimmunity in cMy-mOVA mice. After TAC, again 78% of the mice displayed an OVA-specific cytotoxicity with on average only a three-fold higher killing of OVA-expressing target cells. More CD8+ cells were present after TAC in the myocardium of cMy-mOVA mice with OT-I T cells (on average 17.5/mm2) than in mice that did not receive OVA-specific CD8+-T cells (3.6/mm2). However, the extent of fibrosis was similar in both groups. Functionally, as determined by echocardiography, the adoptive transfer of OVA-specific CD8+-T cells did not significantly accelerate the progression from hypertrophy to heart failure in cMy-mOVA mice. These findings argue therefore against a major impact of cytotoxic T cells with specificity for autoantigens of cardiomyocytes in pressure overload-induced heart failure.
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Affiliation(s)
- Carina Gröschel
- Institute of Cellular and Molecular Immunology, University Medical Center Göttingen, Göttingen, Germany.,DZHK (German Center for Cardiovascular Research), Partner Site Göttingen, Göttingen, Germany
| | - André Sasse
- Institute of Cellular and Molecular Immunology, University Medical Center Göttingen, Göttingen, Germany
| | - Sebastian Monecke
- Institute of Cellular and Molecular Immunology, University Medical Center Göttingen, Göttingen, Germany.,DZHK (German Center for Cardiovascular Research), Partner Site Göttingen, Göttingen, Germany
| | - Charlotte Röhrborn
- Institute of Cellular and Molecular Immunology, University Medical Center Göttingen, Göttingen, Germany
| | - Leslie Elsner
- Institute of Cellular and Molecular Immunology, University Medical Center Göttingen, Göttingen, Germany
| | - Michael Didié
- DZHK (German Center for Cardiovascular Research), Partner Site Göttingen, Göttingen, Germany.,Institute of Pharmacology and Toxicology, University Medical Center Göttingen, Göttingen, Germany.,Department of Cardiology and Pneumology, University Medical Center Göttingen, Göttingen, Germany
| | - Verena Reupke
- Central Animal Facility, University Medical Center Göttingen, Göttingen, Germany
| | - Gertrude Bunt
- Clinical Optical Microscopy, Department of Neuropathology, University Medical Center Göttingen, Göttingen, Germany
| | - Andrew H Lichtman
- Department of Pathology, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, United States
| | - Karl Toischer
- DZHK (German Center for Cardiovascular Research), Partner Site Göttingen, Göttingen, Germany.,Department of Cardiology and Pneumology, University Medical Center Göttingen, Göttingen, Germany
| | - Wolfram-Hubertus Zimmermann
- DZHK (German Center for Cardiovascular Research), Partner Site Göttingen, Göttingen, Germany.,Institute of Pharmacology and Toxicology, University Medical Center Göttingen, Göttingen, Germany
| | - Gerd Hasenfuß
- DZHK (German Center for Cardiovascular Research), Partner Site Göttingen, Göttingen, Germany.,Department of Cardiology and Pneumology, University Medical Center Göttingen, Göttingen, Germany
| | - Ralf Dressel
- Institute of Cellular and Molecular Immunology, University Medical Center Göttingen, Göttingen, Germany.,DZHK (German Center for Cardiovascular Research), Partner Site Göttingen, Göttingen, Germany
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23
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Duffy C, Prugue C, Glew R, Smith T, Howell C, Choi G, Cook AD. Feasibility of Induced Pluripotent Stem Cell Therapies for Treatment of Type 1 Diabetes. TISSUE ENGINEERING PART B-REVIEWS 2018; 24:482-492. [PMID: 29947303 DOI: 10.1089/ten.teb.2018.0124] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/02/2023]
Abstract
IMPACT STATEMENT This review of iPSCs to treat T1D provides a current assessment of the challenges and potential for this proposed new therapy.
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Affiliation(s)
- Caden Duffy
- Department of Chemical Engineering, Brigham Young University , Provo, Utah
| | - Cesar Prugue
- Department of Chemical Engineering, Brigham Young University , Provo, Utah
| | - Rachel Glew
- Department of Chemical Engineering, Brigham Young University , Provo, Utah
| | - Taryn Smith
- Department of Chemical Engineering, Brigham Young University , Provo, Utah
| | - Calvin Howell
- Department of Chemical Engineering, Brigham Young University , Provo, Utah
| | - Gina Choi
- Department of Chemical Engineering, Brigham Young University , Provo, Utah
| | - Alonzo D Cook
- Department of Chemical Engineering, Brigham Young University , Provo, Utah
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24
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Voutsadakis IA. Expression and function of immune ligand-receptor pairs in NK cells and cancer stem cells: therapeutic implications. Cell Oncol (Dordr) 2018; 41:107-121. [PMID: 29470831 DOI: 10.1007/s13402-018-0373-9] [Citation(s) in RCA: 22] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 02/09/2018] [Indexed: 12/15/2022] Open
Abstract
BACKGROUND The interplay between the immune system and cancer cells has come to the forefront of cancer therapeutics, with novel immune blockade inhibitors being approved for the treatment of an increasing list of cancers. However, the majority of cancer patients still display or develop resistance to these promising drugs. It is possible that cancer stem cells (CSCs) are contributing to this therapeutic resistance. Although CSCs usually represent a small percentage of the total number of cancer cells, they are endowed with the ability of self-renewal and to produce differentiated progeny. Additionally, they have shown the capacity to establish tumors after transplantation to animals, even in small numbers. CSCs have also been found to be resistant to various anti-cancer therapies, including chemotherapy, radiation therapy and, more recently, immunotherapy. This is true despite the sensitivity of CSCs to lysis in vitro by natural killer (NK) cells, the main effector cells of the innate immune system. In this paper the expression of ligands specific for NK cells on CSCs, the intracellular network responsible for the expression of the NK cytotoxicity receptors, and the status of activation of NK cells in the tumor micro-environment are reviewed. The aim of this review is to highlight potential strategies for overcoming CSC immune resistance, thereby enhancing the efficacy of current and future anti-cancer therapies. THERAPEUTIC IMPLICATIONS NK cell activation in the tumor micro-environment through drugs neutralizing inhibitory immune receptors, and combined with other drugs harnessing the potential of the adaptive immune system, could be the most effective approach for attacking both stem cell and non-stem cell cancer populations.
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Affiliation(s)
- Ioannis A Voutsadakis
- Algoma District Cancer Program, Sault Area Hospital, Sault Ste. Marie, ON, Canada. .,Division of Clinical Sciences, Northern Ontario School of Medicine, Sudbury, ON, Canada. .,Division of Medical Oncology, Sault Area Hospital, 750 Great Northern Road, Sault Ste Marie, ON, P6B 0A8, Canada.
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25
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Svirshchevskaya EV, Poltavtsev AM, Os’mak GZ, Poltavtseva RA. Activation of NK Cells in Mixed Cultures of Wharton’s Jelly Mesenchymal Stromal Cells and Peripheral Blood Lymphocytes. Bull Exp Biol Med 2018; 164:339-343. [DOI: 10.1007/s10517-018-3985-1] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/05/2016] [Indexed: 12/23/2022]
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26
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Precise immune tolerance for hPSC derivatives in clinical application. Cell Immunol 2017; 326:15-23. [PMID: 28866278 DOI: 10.1016/j.cellimm.2017.08.005] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/03/2017] [Revised: 08/03/2017] [Accepted: 08/04/2017] [Indexed: 11/22/2022]
Abstract
Human pluripotent stem cells (hPSCs) promise a foreseeing future for regeneration medicine and cell replacement therapy with their abilities to produce almost any types of somatic cells of the body. The complicated immunogenicity of hPSC derivatives and context dependent responses in variable transplantations greatly hurdle the practical application of hPSCs in clinic. Especially for applications of hPSCs, induction of immune tolerance at the same time increases the risks of tumorigenesis. Over the past few years, thanks to the progress in immunology and practices in organ transplantation, endeavors on exploring strategies to induce long term protection of allogeneic transplants have shed light on overcoming this barrier. Novel genetic engineering techniques also allow to precisely cradle the immune response of transplantation. Here we reviewed the current understanding on immunogenicity, and efforts have been attempted on inducing immune tolerance for hPSC derivatives, with extra focus on modifying the graft cells. We also glimpse on employing cutting-edge genome editing technologies for this purpose, which will potentially endow hPSC derivatives with the nature of wide spectrum drugs for therapy.
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27
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Chhabra A. Inherent Immunogenicity or Lack Thereof of Pluripotent Stem Cells: Implications for Cell Replacement Therapy. Front Immunol 2017; 8:993. [PMID: 28868053 PMCID: PMC5563324 DOI: 10.3389/fimmu.2017.00993] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/16/2017] [Accepted: 08/03/2017] [Indexed: 01/07/2023] Open
Abstract
Donor-specific induced pluripotent stem cells (iPSCs) offer opportunities for personalized cell replacement therapeutic approaches due to their unlimited self-renewal potential and ability to differentiate into different somatic cells. A significant progress has been made toward generating iPSC lines that are free of integrating viral vectors, development of xeno-free culture conditions, and differentiation of pluripotent stem cells (PSCs) into functional somatic cell lineages. Since donor-specific iPSC lines are genetically identical to the individual, they are expected to be immunologically matched and these iPSC lines and their cellular derivatives are not expected to be immunologically rejected. However, studies in mouse models, utilizing rejection of teratomas as a model, have claimed that syngenic iPSC lines, especially the iPSC lines derived with integrating viral vectors, could be inherently immunogenic. This manuscript reviews current understanding of inherent immunogenicity of PSC lines, especially that of the human iPSC lines and their cellular derivatives, and strategies to overcome it.
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Affiliation(s)
- Arvind Chhabra
- Department of Medicine, University of Connecticut Health Center (UCONN Health), Farmington, CT, United States
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28
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Johannsen H, Muppala V, Gröschel C, Monecke S, Elsner L, Didié M, Zimmermann WH, Dressel R. Immunological Properties of Murine Parthenogenetic Stem Cells and Their Differentiation Products. Front Immunol 2017; 8:924. [PMID: 28824647 PMCID: PMC5543037 DOI: 10.3389/fimmu.2017.00924] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/30/2017] [Accepted: 07/20/2017] [Indexed: 12/27/2022] Open
Abstract
The perspective to transplant grafts derived from pluripotent stem cells has gained much attention in recent years. Parthenogenetic stem cells (PSCs) are an alternative pluripotent stem cell type that is attractive as source of grafts for allogeneic transplantations because most PSCs are haploidentical for the major histocompatibility complex (MHC). This reduced immunogenetic complexity of PSCs could tremendously simplify the search for MHC-matched allogeneic stem cells. In this study, we have characterized immunological properties of the MHC haploidentical PSC line A3 (H2d/d) and the heterologous PSC line A6 (H2b/d). Both PSC lines largely lack MHC class I molecules, which present peptides to cytotoxic T lymphocytes (CTLs) and serve as ligands for inhibitory natural killer (NK) receptors. They express ligands for activating NK receptors, including the NKG2D ligand RAE-1, and the DNAM-1 ligands CD112 and CD155. Consequently, both PSC lines are highly susceptible to killing by IL-2-activated NK cells. In vitro-differentiated cells acquire resistance and downregulate ligands for activating NK receptors but fail to upregulate MHC class I molecules. The PSC line A6 and differentiated A6 cells are largely resistant to CTLs derived from T cell receptor transgenic OT-I mice after pulsing of the targets with the appropriate peptide. The high susceptibility to killing by activated NK cells may constitute a general feature of pluripotent stem cells as it has been also found with other pluripotent stem cell types. This activity potentially increases the safety of transplantations, if grafts contain traces of undifferentiated cells that could be tumorigenic in the recipient.
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Affiliation(s)
- Hannah Johannsen
- Institute of Cellular and Molecular Immunology, University Medical Center Göttingen, Göttingen, Germany
| | - Vijayakumar Muppala
- Institute of Pharmacology and Toxicology, University Medical Center Göttingen, Göttingen, Germany
| | - Carina Gröschel
- Institute of Cellular and Molecular Immunology, University Medical Center Göttingen, Göttingen, Germany.,DZHK (German Center for Cardiovascular Research), Partner Site Göttingen, Göttingen, Germany
| | - Sebastian Monecke
- Institute of Cellular and Molecular Immunology, University Medical Center Göttingen, Göttingen, Germany.,DZHK (German Center for Cardiovascular Research), Partner Site Göttingen, Göttingen, Germany
| | - Leslie Elsner
- Institute of Cellular and Molecular Immunology, University Medical Center Göttingen, Göttingen, Germany
| | - Michael Didié
- Institute of Pharmacology and Toxicology, University Medical Center Göttingen, Göttingen, Germany.,DZHK (German Center for Cardiovascular Research), Partner Site Göttingen, Göttingen, Germany
| | - Wolfram-Hubertus Zimmermann
- Institute of Pharmacology and Toxicology, University Medical Center Göttingen, Göttingen, Germany.,DZHK (German Center for Cardiovascular Research), Partner Site Göttingen, Göttingen, Germany
| | - Ralf Dressel
- Institute of Cellular and Molecular Immunology, University Medical Center Göttingen, Göttingen, Germany.,DZHK (German Center for Cardiovascular Research), Partner Site Göttingen, Göttingen, Germany
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29
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Gröschel C, Hübscher D, Nolte J, Monecke S, Sasse A, Elsner L, Paulus W, Trenkwalder C, Polić B, Mansouri A, Guan K, Dressel R. Efficient Killing of Murine Pluripotent Stem Cells by Natural Killer (NK) Cells Requires Activation by Cytokines and Partly Depends on the Activating NK Receptor NKG2D. Front Immunol 2017; 8:870. [PMID: 28890717 PMCID: PMC5582315 DOI: 10.3389/fimmu.2017.00870] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/14/2017] [Accepted: 07/10/2017] [Indexed: 12/14/2022] Open
Abstract
Natural killer (NK) cells play an important role as cytotoxic effector cells, which scan the organism for infected or tumorigenic cells. Conflicting data have been published whether NK cells can also kill allogeneic or even autologous pluripotent stem cells (PSCs) and which receptors are involved. A clarification of this question is relevant since an activity of NK cells against PSCs could reduce the risk of teratoma growth after transplantation of PSC-derived grafts. Therefore, the hypothesis has been tested that the activity of NK cells against PSCs depends on cytokine activation and specifically on the activating NK receptor NKG2D. It is shown that a subcutaneous injection of autologous iPSCs failed to activate NK cells against these iPSCs and can give rise to teratomas. In agreement with this result, several PSC lines, including two iPSC, two embryonic stem cell (ESC), and two so-called multipotent adult germline stem cell (maGSC) lines, were largely resistant against resting NK cells although differences in killing were found at low level. All PSC lines were killed by interleukin (IL)-2-activated NK cells, and maGSCs were better killed than the other PSC types. The PSCs expressed ligands of the activating NK receptor NKG2D and NKG2D-deficient NK cells from Klrk1-/- mice were impaired in their cytotoxic activity against PSCs. The low-cytotoxic activity of resting NK cells was almost completely dependent on NKG2D. The cytotoxic activity of IL-2-activated NKG2D-deficient NK cells against PSCs was reduced, indicating that also other activating receptors on cytokine-activated NK cells must be engaged by ligands on PSCs. Thus, NKG2D is an important activating receptor involved in killing of murine PSCs. However, NK cells need to be activated by cytokines before they efficiently target PSCs and then also other NK receptors become relevant. These features of NK cells might be relevant for transplantation of PSC-derived grafts since NK cells have the capability to kill undifferentiated cells, which might be present in grafts in trace amounts.
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Affiliation(s)
- Carina Gröschel
- Institute of Cellular and Molecular Immunology, University Medical Center Göttingen, Göttingen, Germany.,DZHK (German Center for Cardiovascular Research), Göttingen, Germany
| | - Daniela Hübscher
- DZHK (German Center for Cardiovascular Research), Göttingen, Germany.,Department of Cardiology and Pneumology, University Medical Center Göttingen, Göttingen, Germany
| | - Jessica Nolte
- Institute of Human Genetics, University Medical Center Göttingen, Göttingen, Germany
| | - Sebastian Monecke
- Institute of Cellular and Molecular Immunology, University Medical Center Göttingen, Göttingen, Germany.,DZHK (German Center for Cardiovascular Research), Göttingen, Germany
| | - André Sasse
- Institute of Cellular and Molecular Immunology, University Medical Center Göttingen, Göttingen, Germany
| | - Leslie Elsner
- Institute of Cellular and Molecular Immunology, University Medical Center Göttingen, Göttingen, Germany
| | - Walter Paulus
- Department of Clinical Neurophysiology, University Medical Center Göttingen, Göttingen, Germany
| | | | - Bojan Polić
- Department of Histology and Embryology, Faculty of Medicine, University of Rijeka, Rijeka, Croatia
| | - Ahmed Mansouri
- Department of Clinical Neurophysiology, University Medical Center Göttingen, Göttingen, Germany.,Department of Molecular Cell Biology, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany
| | - Kaomei Guan
- Department of Cardiology and Pneumology, University Medical Center Göttingen, Göttingen, Germany.,Institute of Pharmacology and Toxicology, Technische Universität Dresden, Dresden, Germany
| | - Ralf Dressel
- Institute of Cellular and Molecular Immunology, University Medical Center Göttingen, Göttingen, Germany.,DZHK (German Center for Cardiovascular Research), Göttingen, Germany
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30
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Abstract
Human embryonic stem cells (hESCs) can undergo unlimited self-renewal and differentiate into all cell types in human body, and therefore hold great potential for cell therapy of currently incurable diseases including neural degenerative diseases, heart failure, and macular degeneration. This potential is further underscored by the promising safety and efficacy data from the ongoing clinical trials of hESC-based therapy of macular degeneration. However, one main challenge for the clinical application of hESC-based therapy is the allogeneic immune rejection of hESC-derived cells by the recipient. The breakthrough of the technology to generate autologous-induced pluripotent stem cells (iPSCs) by nuclear reprogramming of patient’s somatic cells raised the possibility that autologous iPSC-derived cells can be transplanted into the patients without the concern of immune rejection. However, accumulating data indicate that certain iPSC-derived cells can be immunogenic. In addition, the genomic instability associated with iPSCs raises additional safety concern to use iPSC-derived cells in human cell therapy. In this review, we will discuss the mechanism underlying the immunogenicity of the pluripotent stem cells and recent progress in developing immune tolerance strategies of human pluripotent stem cell (hPSC)-derived allografts. The successful development of safe and effective immune tolerance strategy will greatly facilitate the clinical development of hPSC-based cell therapy.
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Affiliation(s)
- Xin Liu
- Center for Regenerative and Translational Medicine, Guangdong Provincial Academy of Chinese Medical Sciences, The Second Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangzhou, China.,Division of Biological Sciences, University of California, San Diego, La Jolla, CA, United States
| | - Wenjuan Li
- Center for Regenerative and Translational Medicine, Guangdong Provincial Academy of Chinese Medical Sciences, The Second Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangzhou, China
| | - Xuemei Fu
- The Eighth Affiliated Hospital of Sun Yat-sen University, Shenzhen, China
| | - Yang Xu
- Center for Regenerative and Translational Medicine, Guangdong Provincial Academy of Chinese Medical Sciences, The Second Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangzhou, China.,Division of Biological Sciences, University of California, San Diego, La Jolla, CA, United States
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31
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Hübscher D, Kaiser D, Elsner L, Monecke S, Dressel R, Guan K. The Tumorigenicity of Multipotent Adult Germline Stem Cells Transplanted into the Heart Is Affected by Natural Killer Cells and by Cyclosporine A Independent of Its Immunosuppressive Effects. Front Immunol 2017; 8:67. [PMID: 28220117 PMCID: PMC5292627 DOI: 10.3389/fimmu.2017.00067] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/23/2016] [Accepted: 01/16/2017] [Indexed: 12/18/2022] Open
Abstract
Transplantation of stem cells represents an upcoming therapy for many degenerative diseases. For clinical use, transplantation of pluripotent stem cell-derived cells should lead to integration of functional grafts without immune rejection or teratoma formation. Our previous studies showed that the risk of teratoma formation is highly influenced by the immune system of the recipients. In this study, we have observed a higher teratoma formation rate when undifferentiated so-called multipotent adult germline stem cells (maGSCs) were transplanted into the heart of T, B, and natural killer (NK) cell-deficient RAG2−/−γc−/− mice than in RAG2−/− mice, which still have NK cells. Notably, in both strains, the teratoma formation rate was significantly reduced by the immunosuppressive drug cyclosporine A (CsA). Thus, CsA had a profound effect on teratoma formation independent of its immunosuppressive effects. The transplantation into RAG2−/− mice led to an activation of NK cells, which reached the maximum 14 days after transplantation and was not affected by CsA. The in vivo-activated NK cells efficiently killed YAC-1 and also maGSC target cells. This NK cell activation was confirmed in C57BL/6 wild-type mice whether treated with CsA or not. Sham operations in wild-type mice indicated that the inflammatory response to open heart surgery rather than the transplantation of maGSCs activated the NK cell system. An activation of NK cells during the transplantation of stem cell-derived in vitro differentiated grafts might be clinically beneficial by reducing the risk of teratoma formation by residual pluripotent cells.
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Affiliation(s)
- Daniela Hübscher
- Department of Cardiology and Pneumology, University Medical Center Göttingen, Göttingen, Germany; DZHK (German Center for Cardiovascular Research), Partner Site Göttingen, Göttingen, Germany
| | - Diana Kaiser
- Department of Cardiology and Pneumology, University Medical Center Göttingen , Göttingen , Germany
| | - Leslie Elsner
- Institute of Cellular and Molecular Immunology, University Medical Center Göttingen , Göttingen , Germany
| | - Sebastian Monecke
- DZHK (German Center for Cardiovascular Research), Partner Site Göttingen, Göttingen, Germany; Institute of Cellular and Molecular Immunology, University Medical Center Göttingen, Göttingen, Germany
| | - Ralf Dressel
- DZHK (German Center for Cardiovascular Research), Partner Site Göttingen, Göttingen, Germany; Institute of Cellular and Molecular Immunology, University Medical Center Göttingen, Göttingen, Germany
| | - Kaomei Guan
- Department of Cardiology and Pneumology, University Medical Center Göttingen, Göttingen, Germany; Institute of Pharmacology and Toxicology, Technische Universität Dresden, Dresden, Germany
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32
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Miyagawa S, Fukushima S, Imanishi Y, Kawamura T, Mochizuki-Oda N, Masuda S, Sawa Y. Building A New Treatment For Heart Failure-Transplantation of Induced Pluripotent Stem Cell-derived Cells into the Heart. Curr Gene Ther 2016; 16:5-13. [PMID: 26785736 PMCID: PMC4997929 DOI: 10.2174/1566523216666160119094143] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/22/2015] [Revised: 01/07/2016] [Accepted: 01/08/2016] [Indexed: 02/08/2023]
Abstract
Advanced cardiac failure is a progressive intractable disease and is the main cause of mortality and morbidity worldwide. Since this pathology is represented by a definite decrease in cardiomyocyte number, supplementation of functional cardiomyocytes into the heart would hypothetically be an ideal therapeutic option. Recently, unlimited in vitro production of human functional cardiomyocytes was established by using induced pluripotent stem cell (iPSC) technology, which avoids the use of human embryos. A number of basic studies including ours have shown that transplantation of iPSC-derived cardiomyocytes (iPSC-CMs) into the damaged heart leads to recovery of cardiac function, thereby establishing “proof-of-concept” of this iPSC-transplantation therapy. However, considering clinical application of this therapy, its feasibility, safety, and therapeutic efficacy need to be further investigated in the pre-clinical stage. This review summarizes up-to-date important topics related to safety and efficacy of iPSC-CMs transplantation therapy for cardiac disease and discusses the prospects for this treatment in clinical studies.
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Affiliation(s)
| | | | | | | | | | | | - Yoshiki Sawa
- Department of Cardiovascular Surgery, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka, 565-0871, Japan.
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33
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Rohani L, Fabian C, Holland H, Naaldijk Y, Dressel R, Löffler-Wirth H, Binder H, Arnold A, Stolzing A. Generation of human induced pluripotent stem cells using non-synthetic mRNA. Stem Cell Res 2016; 16:662-72. [DOI: 10.1016/j.scr.2016.03.008] [Citation(s) in RCA: 28] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/28/2015] [Revised: 02/28/2016] [Accepted: 03/17/2016] [Indexed: 11/24/2022] Open
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34
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Li CW, Pan WT, Ju JC, Wang GJ. An endothelial cultured condition medium embedded porous PLGA scaffold for the enhancement of mouse embryonic stem cell differentiation. ACTA ACUST UNITED AC 2016; 11:025015. [PMID: 27068738 DOI: 10.1088/1748-6041/11/2/025015] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
In this study, we have developed a microporous poly(lactic-co-glycolic acid) (PLGA) scaffold that combines a continuous release property and a three-dimensional (3D) scaffolding technique for the precise and efficient formation of endothelial cell lineage from embryonic stem cells (ESCs). Eight PLGA scaffolds (14.29%, 16.67%, 20% and 25% concentrations of PLGA solutions) mixed with two crystal sizes of sodium chloride (NaCl) were fabricated by leaching. Then, vascular endothelial cell conditioned medium (ECCM) mixed with gelatin was embedded into the scaffold for culturing of mouse embryonic stem cells (mESCs). The 14.29% PLGA scaffolds fabricated using non-ground NaCl particles (NG-PLGA) and the 25% PLGA containing scaffolds fabricated using ground NaCl particles (G-PLGA) possessed minimum and maximum moisture content and bovine serum albumin (BSA) content properties, respectively. These two groups of scaffolds were used for future experiments in this study. Cell culture results demonstrated that the proposed porous scaffolds without growth factors were sufficient to induce mouse ESCs to differentiate into endothelial-like cells in the early culture stages, and combined with embedded ECCM could provide a long-term inducing system for ESC differentiation.
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Affiliation(s)
- Ching-Wen Li
- PhD Program in Tissue Engineering and Regenerative Medicine, National Chung-Hsing University, Taichung 40227, Taiwan
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35
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Immunological Properties of Corneal Epithelial-Like Cells Derived from Human Embryonic Stem Cells. PLoS One 2016; 11:e0150731. [PMID: 26977925 PMCID: PMC4792422 DOI: 10.1371/journal.pone.0150731] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/20/2015] [Accepted: 02/17/2016] [Indexed: 12/21/2022] Open
Abstract
Transplantation of ex vivo expanded corneal limbal stem cells (LSCs) has been the main treatment for limbal stem cell deficiency, although the shortage of donor corneal tissues remains a major concern for its wide application. Due to the development of tissue engineering, embryonic stem cells (ESCs)-derived corneal epithelial-like cells (ESC-CECs) become a new direction for this issue. However, the immunogenicity of ESC-CECs is a critical matter to be solved. In the present study, we explored the immunological properties of ESC-CECs, which were differentiated from ESCs. The results showed that ESC-CECs had a similar character and function with LSCs both in vitro and in vivo. In ESC-CECs, a large number of genes related with immune response were down-regulated. The expressions of MHC-I, MHC-II, and co-stimulatory molecules were low, but the expression of HLA-G was high. The ESC-CECs were less responsible for T cell proliferation and NK cell lysis in vitro, and there was less immune cell infiltration after transplantation in vivo compared with LSCs. Moreover, the immunological properties were not affected by interferon-γ. All these results indicated a low immunogenicity of ESC-CECs, and they can be promising in clinical use.
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36
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Kruse V, Hamann C, Monecke S, Cyganek L, Elsner L, Hübscher D, Walter L, Streckfuss-Bömeke K, Guan K, Dressel R. Human Induced Pluripotent Stem Cells Are Targets for Allogeneic and Autologous Natural Killer (NK) Cells and Killing Is Partly Mediated by the Activating NK Receptor DNAM-1. PLoS One 2015; 10:e0125544. [PMID: 25950680 PMCID: PMC4423859 DOI: 10.1371/journal.pone.0125544] [Citation(s) in RCA: 43] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/21/2014] [Accepted: 03/25/2015] [Indexed: 02/07/2023] Open
Abstract
Human induced pluripotent stem cells (hiPSCs) could be used to generate autologous cells for therapeutic purposes, which are expected to be tolerated by the recipient. However, iPSC-derived grafts are at risk of giving rise to teratomas in the host, if residuals of tumorigenic cells are not rejected by the recipient. We have analyzed the susceptibility of hiPSC lines to allogeneic and autologous natural killer (NK) cells. IL-2-activated, in contrast to resting NK cells killed hiPSC lines efficiently (P = 1.69 x 10(-39)). Notably, the specific lysis of the individual hiPSC lines by IL-2-activated NK cells was significantly different (P = 1.72 x 10(-6)) and ranged between 46 % and 64 % in 51Cr-release assays when compared to K562 cells. The hiPSC lines were killed by both allogeneic and autologous NK cells although autologous NK cells were less efficient (P=8.63 x 10(-6)). Killing was partly dependent on the activating NK receptor DNAM-1 (P = 8.22 x 10(-7)). The DNAM-1 ligands CD112 and CD155 as well as the NKG2D ligands MICA and MICB were expressed on the hiPSC lines. Low amounts of human leukocyte antigen (HLA) class I proteins, which serve as ligands for inhibitory and activating NK receptors were also detected. Thus, the susceptibility to NK cell killing appears to constitute a common feature of hiPSCs. Therefore, NK cells might reduce the risk of teratoma formation even after autologous transplantations of pluripotent stem cell-derived grafts that contain traces of pluripotent cells.
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Affiliation(s)
- Vanessa Kruse
- Department of Cardiology and Pneumology, University Medical Center Göttingen, Göttingen, Germany
| | - Carina Hamann
- Department of Cellular and Molecular Immunology, University Medical Center Göttingen, Göttingen, Germany
- DZHK (German Center for Cardiovascular Research), Partner site Göttingen, Germany
| | - Sebastian Monecke
- Department of Cellular and Molecular Immunology, University Medical Center Göttingen, Göttingen, Germany
- DZHK (German Center for Cardiovascular Research), Partner site Göttingen, Germany
| | - Lukas Cyganek
- Department of Cardiology and Pneumology, University Medical Center Göttingen, Göttingen, Germany
- DZHK (German Center for Cardiovascular Research), Partner site Göttingen, Germany
| | - Leslie Elsner
- Department of Cellular and Molecular Immunology, University Medical Center Göttingen, Göttingen, Germany
| | - Daniela Hübscher
- Department of Cardiology and Pneumology, University Medical Center Göttingen, Göttingen, Germany
- DZHK (German Center for Cardiovascular Research), Partner site Göttingen, Germany
| | - Lutz Walter
- Primate Genetics Laboratory, German Primate Center, Göttingen, Germany
| | - Katrin Streckfuss-Bömeke
- Department of Cardiology and Pneumology, University Medical Center Göttingen, Göttingen, Germany
- DZHK (German Center for Cardiovascular Research), Partner site Göttingen, Germany
| | - Kaomei Guan
- Department of Cardiology and Pneumology, University Medical Center Göttingen, Göttingen, Germany
- DZHK (German Center for Cardiovascular Research), Partner site Göttingen, Germany
- * E-mail: (RD); (KG)
| | - Ralf Dressel
- Department of Cellular and Molecular Immunology, University Medical Center Göttingen, Göttingen, Germany
- DZHK (German Center for Cardiovascular Research), Partner site Göttingen, Germany
- * E-mail: (RD); (KG)
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Bolton EM, Bradley JA. Avoiding immunological rejection in regenerative medicine. Regen Med 2015; 10:287-304. [DOI: 10.2217/rme.15.11] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022] Open
Abstract
One of the major goals of regenerative medicine is repair or replacement of diseased and damaged tissues by transfer of differentiated stem cells or stem cell-derived tissues. The possibility that these tissues will be destroyed by immunological rejection remains a challenge that can only be overcome through a better understanding of the nature and expression of potentially immunogenic molecules associated with cell replacement therapy and the mechanisms and pathways resulting in their immunologic rejection. This review draws on clinical experience of organ and tissue transplantation, and on transplantation immunology research to consider practical approaches for avoiding and overcoming the possibility of rejection of stem cell-derived tissues.
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Affiliation(s)
- Eleanor M Bolton
- Department of Surgery, University of Cambridge, Box 202, Addenbrooke's Hospital, Cambridge, CB2 0QQ, UK
| | - John Andrew Bradley
- Department of Surgery, University of Cambridge, Box 202, Addenbrooke's Hospital, Cambridge, CB2 0QQ, UK
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Controlling immune rejection is a fail-safe system against potential tumorigenicity after human iPSC-derived neural stem cell transplantation. PLoS One 2015; 10:e0116413. [PMID: 25706286 PMCID: PMC4338009 DOI: 10.1371/journal.pone.0116413] [Citation(s) in RCA: 33] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/03/2014] [Accepted: 12/09/2014] [Indexed: 02/07/2023] Open
Abstract
Our previous work reported functional recovery after transplantation of mouse and human induced pluripotent stem cell-derived neural stem/progenitor cells (hiPSC-NS/PCs) into rodent models of spinal cord injury (SCI). Although hiPSC-NS/PCs proved useful for the treatment of SCI, the tumorigenicity of the transplanted cells must be resolved before they can be used in clinical applications. The current study sought to determine the feasibility of ablation of the tumors formed after hiPSC-NS/PC transplantation through immunoregulation. Tumorigenic hiPSC-NS/PCs were transplanted into the intact spinal cords of immunocompetent BALB/cA mice with or without immunosuppressant treatment. In vivo bioluminescence imaging was used to evaluate the chronological survival and growth of the transplanted cells. The graft survival rate was 0% in the group without immunosuppressants versus 100% in the group with immunosuppressants. Most of the mice that received immunosuppressants exhibited hind-limb paralysis owing to tumor growth at 3 months after iPSC-NS/PC transplantation. Histological analysis showed that the tumors shared certain characteristics with low-grade gliomas rather than with teratomas. After confirming the progression of the tumors in immunosuppressed mice, the immunosuppressant agents were discontinued, resulting in the complete rejection of iPSC-NS/PC-derived masses within 42 days after drug cessation. In accordance with the tumor rejection, hind-limb motor function was recovered in all of the mice. Moreover, infiltration of microglia and lymphocytes was observed during the course of tumor rejection, along with apoptosis of iPSC-NS/PC-generated cells. Thus, immune rejection can be used as a fail-safe system against potential tumorigenicity after transplantation of iPSC-NS/PCs to treat SCI.
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Tolerance Induction and Reversal of Diabetes in Mice Transplanted with Human Embryonic Stem Cell-Derived Pancreatic Endoderm. Cell Stem Cell 2015; 16:148-57. [DOI: 10.1016/j.stem.2014.12.001] [Citation(s) in RCA: 71] [Impact Index Per Article: 7.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/13/2014] [Revised: 10/30/2014] [Accepted: 12/02/2014] [Indexed: 02/06/2023]
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Dppa3 expression is critical for generation of fully reprogrammed iPS cells and maintenance of Dlk1-Dio3 imprinting. Nat Commun 2015; 6:6008. [PMID: 25613421 PMCID: PMC4354275 DOI: 10.1038/ncomms7008] [Citation(s) in RCA: 43] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/30/2014] [Accepted: 12/01/2014] [Indexed: 11/22/2022] Open
Abstract
Reprogramming of mouse somatic cells into induced pluripotent stem cells (iPSCs) often generates partially reprogrammed iPSCs (pre-iPSCs), low-grade chimera forming iPSCs (lg-iPSCs) and fully reprogrammed, high-grade chimera production competent iPSCs (hg-iPSCs). Lg-iPSC transcriptome analysis revealed misregulated Dlk1-Dio3 cluster gene expression and subsequently the imprinting defect at the Dlk1-Dio3 locus. Here, we show that germ-cell marker Dppa3 is present only in lg-iPSCs and hg-iPSCs, and that induction with exogenous Dppa3 enhances reprogramming kinetics, generating all hg-iPSCs, similar to vitamin C (Vc). Conversely, Dppa3-null fibroblasts show reprogramming block at pre-iPSCs state and Dlk1-Dio3 imprinting defect. At the molecular level, we show that Dppa3 is associated with Dlk1-Dio3 locus and identify that Dppa3 maintains imprinting by antagonizing Dnmt3a binding. Our results further show molecular parallels between Dppa3 and Vc in Dlk1-Dio3 imprinting maintenance and suggest that early activation of Dppa3 is one of the cascades through which Vc facilitates the generation of fully reprogrammed iPSCs. Reprogramming of mouse somatic cells into iPSCs often generates pre-iPSCs, low-grade iPSCs that show abnormal Dlk1-Dio3 imprinting, and fully reprogrammed, high-grade iPSCs. Here, the authors show that germ-cell marker Dppa3 enhances reprogramming kinetics, critical for the maintenance of Dlk1-Dio3 imprinting and generation of fully reprogrammed iPSCs.
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Abstract
Natural killer (NK) cells are effector cells of the innate immune system that can lyse target cells without prior sensitization and have an important role in host defense to pathogens and transformed cells. A balance between negative and positive signals transmitted via germ line-encoded inhibitory and activating receptors controls the function of NK cells. Although the concept of "missing-self" would suggest that NK cells could target foreign allografts, the prevailing dogma has been that NK cells are not active participants in the mechanisms that culminate in the rejection of solid organ allografts. Recent studies, however, challenge this conclusion and instead implicate NK cells in contributing to both graft rejection and tolerance to an allograft. In this review, we highlight recent studies with the goal of understanding the complex NK cell interactions that impact alloimmunity.
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Affiliation(s)
- Uzi Hadad
- Division of Abdominal Transplantation, Department of Surgery and Stanford Immunology, Stanford University School of Medicine, Stanford, CA, USA
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43
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Tan Y, Ooi S, Wang L. Immunogenicity and tumorigenicity of pluripotent stem cells and their derivatives: genetic and epigenetic perspectives. Curr Stem Cell Res Ther 2014; 9:63-72. [PMID: 24160683 PMCID: PMC3873036 DOI: 10.2174/1574888x113086660068] [Citation(s) in RCA: 44] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/31/2013] [Revised: 10/19/2013] [Accepted: 10/22/2013] [Indexed: 12/18/2022]
Abstract
One aim of stem cell-based therapy is to utilize pluripotent stem cells (PSCs) as a supplementary source of cells
to repair or replace tissues or organs that have ceased to function due to severe tissue damage. However, PSC-based therapy
requires extensive research to ascertain if PSC derivatives are functional without the risk of tumorigenicity, and also
do not engender severe immune rejection that threatens graft survival and function. Recently, the suitability of induced
pluripotent stem cells applied for patient-tailored cell therapy has been questioned since the discovery of several genetic
and epigenetic aberrations during the reprogramming process. Hence, it is crucial to understand the effect of these abnormalities
on the immunogenicity and survival of PSC grafts. As induced PSC-based therapy represents a hallmark for the
potential solution to prevent and arrest immune rejection, this review also summarizes several up-to-date key findings in
the field.
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Affiliation(s)
| | | | - Lisheng Wang
- Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, 451 Smyth Road, Ottawa K1H8M5, Canada.
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Natural killer cell subsets differentially reject embryonic stem cells based on licensing. Transplantation 2014; 97:992-8. [PMID: 24704665 DOI: 10.1097/tp.0000000000000063] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022]
Abstract
BACKGROUND Embryonic stem cells (ESC) and induced pluripotent stem cells provide great promise to the future of medicine. Because immune rejection represents a major obstacle to the success of all stem cell-based therapies, many recent studies have sought to determine the key immune mediators involved in ESC rejection. The role of natural killer (NK) cells and specifically the role of NK cell licensing is not well understood in ESC rejection. METHODS Mouse or human ESCs were subjected to cytotoxicity assays involving their respective species-matched activated NK cells. Mouse ESCs were then transplanted to allogeneic recipients after depletion of NK cell subsets in the host. ESC engraftment was analyzed by bioluminescent imaging. RESULTS Depletion of all NK cells in vivo resulted in the greatest amount of ESC engraftment, confirming a role for NK cells in ESC rejection. Importantly, depletion of the Ly49C/I or Ly49G2 NK cell subsets resulted in differential ESC engraftment and rejection. This indicates that NK cell rejection of allogeneic ESC is highly differential based on the presence of licensed NK cells. Blocking NKG2D in vitro resulted in less killing of mESC by allogeneic NK cells, indicating NKG2D is a likely mechanism for NK-mediated killing of mESC. CONCLUSIONS In this study, we show that expression of inhibitory Ly49s correlates with the ability of NK cells to kill murine ESC in an NKG2D-dependent manner. This further suggests that the rejection of similar stem cell transplants in humans will be dependent upon the presence of licensed NK cells.
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MHC-matched induced pluripotent stem cells can attenuate cellular and humoral immune responses but are still susceptible to innate immunity in pigs. PLoS One 2014; 9:e98319. [PMID: 24927426 PMCID: PMC4057111 DOI: 10.1371/journal.pone.0098319] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/20/2013] [Accepted: 04/30/2014] [Indexed: 12/16/2022] Open
Abstract
Recent studies have revealed negligible immunogenicity of induced pluripotent stem (iPS) cells in syngeneic mice and in autologous monkeys. Therefore, human iPS cells would not elicit immune responses in the autologous setting. However, given that human leukocyte antigen (HLA)-matched allogeneic iPS cells would likely be used for medical applications, a more faithful model system is needed to reflect HLA-matched allogeneic settings. Here we examined whether iPS cells induce immune responses in the swine leukocyte antigen (SLA)-matched setting. iPS cells were generated from the SLA-defined C1 strain of Clawn miniature swine, which were confirmed to develop teratomas in mice, and transplanted into the testes (n = 4) and ovary (n = 1) of C1 pigs. No teratomas were found in pigs on 47 to 125 days after transplantation. A Mixed lymphocyte reaction revealed that T-cell responses to the transplanted MHC-matched (C1) iPS cells were significantly lower compared to allogeneic cells. The humoral immune responses were also attenuated in the C1-to-C1 setting. More importantly, even MHC-matched iPS cells were susceptible to innate immunity, NK cells and serum complement. iPS cells lacked the expression of SLA class I and sialic acids. The in vitro cytotoxic assay showed that C1 iPS cells were targeted by NK cells and serum complement of C1. In vivo, the C1 iPS cells developed larger teratomas in NK-deficient NOG (T-B-NK-) mice (n = 10) than in NK-competent NOD/SCID (T-B-NK+) mice (n = 8) (p<0.01). In addition, C1 iPS cell failed to form teratomas after incubation with the porcine complement-active serum. Taken together, MHC-matched iPS cells can attenuate cellular and humoral immune responses, but still susceptible to innate immunity in pigs.
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Phillips LK, Gould EA, Babu H, Krams SM, Palmer TD, Martinez OM. Natural killer cell-activating receptor NKG2D mediates innate immune targeting of allogeneic neural progenitor cell grafts. Stem Cells 2014; 31:1829-39. [PMID: 23733329 DOI: 10.1002/stem.1422] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/21/2012] [Accepted: 01/10/2013] [Indexed: 12/25/2022]
Abstract
Cell replacement therapy holds promise for a number of untreatable neurological or psychiatric diseases but the immunogenicity of cellular grafts remains controversial. Emerging stem cell and reprogramming technologies can be used to generate autologous grafts that minimize immunological concerns but autologous grafts may carry an underlying genetic vulnerability that reduces graft efficacy or survival. Healthy allogeneic grafts are an attractive and commercially scalable alternative if immunological variables can be controlled. Stem cells and immature neural progenitor cells (NPC) do not express major histocompatibility complex (MHC) antigens and can evade adaptive immune surveillance. Nevertheless, in an experimental murine model, allogeneic NPCs do not survive and differentiate as well as syngeneic grafts, even when traditional immunosuppressive treatments are used. In this study, we show that natural killer (NK) cells recognize the lack of self-MHC antigens on NPCs and pose a barrier to NPC transplantation. NK cells readily target both syngeneic and allogeneic NPC, and killing is modulated primarily by NK-inhibiting "self" class I MHC and NK-activating NKG2D-ligand expression. The absence of NKG2D signaling in NK cells significantly improves NPC-derived neuron survival and differentiation. These data illustrate the importance of innate immune mechanisms in graft outcome and the potential value of identifying and targeting NK cell-activating ligands that may be expressed by stem cell derived grafts.
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Affiliation(s)
- Lori K Phillips
- Program in Immunology Stanford University School of Medicine University of Colorado, Boulder, Colorado, USA
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Liao MC, Diaconu M, Monecke S, Collombat P, Timaeus C, Kuhlmann T, Paulus W, Trenkwalder C, Dressel R, Mansouri A. Embryonic stem cell-derived neural progenitors as non-tumorigenic source for dopaminergic neurons. World J Stem Cells 2014; 6:248-255. [PMID: 24772251 PMCID: PMC3999782 DOI: 10.4252/wjsc.v6.i2.248] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/17/2013] [Revised: 11/24/2013] [Accepted: 01/14/2014] [Indexed: 02/06/2023] Open
Abstract
AIM: To find a safe source for dopaminergic neurons, we generated neural progenitor cell lines from human embryonic stem cells.
METHODS: The human embryonic stem (hES) cell line H9 was used to generate human neural progenitor (HNP) cell lines. The resulting HNP cell lines were differentiated into dopaminergic neurons and analyzed by quantitative real-time polymerase chain reaction and immunofluorescence for the expression of neuronal differentiation markers, including beta-III tubulin (TUJ1) and tyrosine hydroxylase (TH). To assess the risk of teratoma or other tumor formation, HNP cell lines and mouse neuronal progenitor (MNP) cell lines were injected subcutaneously into immunodeficient SCID/beige mice.
RESULTS: We developed a fairly simple and fast protocol to obtain HNP cell lines from hES cells. These cell lines, which can be stored in liquid nitrogen for several years, have the potential to differentiate in vitro into dopaminergic neurons. Following day 30 of differentiation culture, the majority of the cells analyzed expressed the neuronal marker TUJ1 and a high proportion of these cells were positive for TH, indicating differentiation into dopaminergic neurons. In contrast to H9 ES cells, the HNP cell lines did not form tumors in immunodeficient SCID/beige mice within 6 mo after subcutaneous injection. Similarly, no tumors developed after injection of MNP cells. Notably, mouse ES cells or neuronal cells directly differentiated from mouse ES cells formed teratomas in more than 90% of the recipients.
CONCLUSION: Our findings indicate that neural progenitor cell lines can differentiate into dopaminergic neurons and bear no risk of generating teratomas or other tumors in immunodeficient mice.
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Jiang Z, Han Y, Cao X. Induced pluripotent stem cell (iPSCs) and their application in immunotherapy. Cell Mol Immunol 2013; 11:17-24. [PMID: 24336163 DOI: 10.1038/cmi.2013.62] [Citation(s) in RCA: 49] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/20/2013] [Revised: 12/02/2013] [Accepted: 12/03/2013] [Indexed: 02/06/2023] Open
Abstract
The ever-improving technology to generate induced pluripotent stem cells (iPSCs) has increased their potential use as novel candidates for disease modeling, drug screening, regenerative medicine and cell therapy. Indeed, iPSCs offer extensive capacity for self-renewal without the ethical concerns faced by embryonic stem cells (ESCs). With respect to potential applications in the immune system, many studies provide evidence to support that there are exclusive advantages to using iPSCs over other systems. Both hematopoietic stem cells and several types of mature immune cells have successfully been reprogrammed to iPSCs and vice versa, paving a path toward our ability to effectively model patient-specific diseases and provide potentially alternative cell sources for transfusion medicine. Despite these potential advances, some limitations regarding the use of iPSCs in the clinic still remain, including the immunogenicity of iPSCs and their derivatives, which is currently under debate in the field. In this review, we mainly focus on discussing the recent progress being made in the latest differentiation methods and clinical implications of iPSCs with respect to the immune system. Additionally, current issues regarding the clinical application of iPSCs are addressed, especially the controversy surrounding immunogenicity, along with various other perspectives.
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49
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Belyaev NN, Abramova VA. Transmission of "split anergy" from tumor infiltrating to peripheral NK cells in a manner similar to "infectious tolerance". Med Hypotheses 2013; 82:129-33. [PMID: 24332531 DOI: 10.1016/j.mehy.2013.11.019] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/10/2013] [Accepted: 05/19/2013] [Indexed: 01/02/2023]
Abstract
According to a new paradigm of carcinogenesis, a tumor arises not from transformed cell, but only from tumor initiating cells called cancer stem cells (CSCs), which can originate from tissue stem cells. CSC are resistant to conventional therapy and after treatment form new tumors and give rise to metastases. Only natural killer (NK) cells are capable of lysing CSCs, but within different tumor types these cells experience a condition known as "split anergy", whereby the NK cells lose the ability to kill CSCs and being to produce cytokines. As a result, uncontrolled tumor growth arises and tumor stroma accumulates anergic NK cells. We hypothesize that anergic tumor infiltrating NK (TINK) cells transmit their property to naïve NK cells by infecting" them with a state of "split anergy" in a similar manner as T conventional cells are transformed into T regulatory cells during the process of "infectious tolerance". Anergic TINK cells egress from the tumor stroma via the lymphatic system, where they reach regional lymph nodes and transmit their properties to naïve NK cells, which in turn become anergic toward CSCs and lose immunosurveillance functions. The mechanisms proposed for this hypothesis and the methodological approaches for confirming the idea are presented in this issue.
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Affiliation(s)
- Nikolai N Belyaev
- Laboratory of Molecular Immunology and Immunobiotechnology, M.A. Aitkhozhin Institute of Molecular Biology and Biochemistry, Almaty, Kazakhstan.
| | - Vera A Abramova
- Laboratory of Molecular Immunology and Immunobiotechnology, M.A. Aitkhozhin Institute of Molecular Biology and Biochemistry, Almaty, Kazakhstan
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50
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Spallanzani RG, Dalotto-Moreno T, Raffo Iraolagoitia XL, Ziblat A, Domaica CI, Avila DE, Rossi LE, Fuertes MB, Battistone MA, Rabinovich GA, Salatino M, Zwirner NW. Expansion of CD11b(+)Ly6G (+)Ly6C (int) cells driven by medroxyprogesterone acetate in mice bearing breast tumors restrains NK cell effector functions. Cancer Immunol Immunother 2013; 62:1781-95. [PMID: 24114144 PMCID: PMC11028897 DOI: 10.1007/s00262-013-1483-x] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/03/2013] [Accepted: 10/01/2013] [Indexed: 12/13/2022]
Abstract
The progesterone analog medroxyprogesterone acetate (MPA) is widely used as a hormone replacement therapy in postmenopausal women and as contraceptive. However, prolonged administration of MPA is associated with increased incidence of breast cancer through ill-defined mechanisms. Here, we explored whether exposure to MPA during mammary tumor growth affects myeloid-derived suppressor cells (MDSCs; CD11b(+)Gr-1(+), mostly CD11b(+)Ly6G(+)Ly6C(int) and CD11b(+)Ly6G(-)Ly6C(high) cells) and natural killer (NK) cells, potentially restraining tumor immunosurveillance. We used the highly metastatic 4T1 breast tumor (which does not express the classical progesterone receptor and expands MDSCs) to challenge BALB/c mice in the absence or in the presence of MPA. We observed that MPA promoted the accumulation of NK cells in spleens of tumor-bearing mice, but with reduced degranulation ability and in vivo cytotoxic activity. Simultaneously, MPA induced a preferential expansion of CD11b(+)Ly6G(+)Ly6C(int) cells in spleen and bone marrow of 4T1 tumor-bearing mice. In vitro, MPA promoted nuclear mobilization of the glucocorticoid receptor (GR) in 4T1 cells and endowed these cells with the ability to promote a preferential differentiation of bone marrow cells into CD11b(+)Ly6G(+)Ly6C(int) cells that displayed suppressive activity on NK cell degranulation. Sorted CD11b(+)Gr-1(+) cells from MPA-treated tumor-bearing mice exhibited higher suppressive activity on NK cell degranulation than CD11b(+)Gr-1(+) cells from vehicle-treated tumor-bearing mice. Thus, MPA, acting through the GR, endows tumor cells with an enhanced capacity to expand CD11b(+)Ly6G(+)Ly6C(int) cells that subsequently display a stronger suppression of NK cell-mediated anti-tumor immunity. Our results describe an alternative mechanism by which MPA may affect immunosurveillance and have potential implication in breast cancer incidence.
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MESH Headings
- Animals
- Antigens, Ly/immunology
- Antigens, Ly/metabolism
- Antineoplastic Agents, Hormonal/pharmacology
- Blotting, Western
- Breast Neoplasms/drug therapy
- Breast Neoplasms/immunology
- Breast Neoplasms/pathology
- CD11b Antigen/immunology
- CD11b Antigen/metabolism
- Cell Differentiation
- Cell Proliferation
- Cytotoxicity, Immunologic
- Female
- Flow Cytometry
- Fluorescent Antibody Technique
- Humans
- Killer Cells, Natural/drug effects
- Killer Cells, Natural/immunology
- Killer Cells, Natural/metabolism
- Medroxyprogesterone Acetate/pharmacology
- Mice
- Mice, Inbred BALB C
- Mice, Inbred C57BL
- Myeloid Cells/drug effects
- Myeloid Cells/immunology
- Myeloid Cells/metabolism
- Receptors, Glucocorticoid/metabolism
- STAT3 Transcription Factor/metabolism
- Tumor Cells, Cultured
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Affiliation(s)
- Raúl Germán Spallanzani
- Laboratorio de Fisiopatología de la Inmunidad Innata, Instituto de Biología y Medicina Experimental (IBYME), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Vuelta de Obligado 2490, C1428ADN Ciudad de Buenos Aires, Argentina
| | | | - Ximena Lucía Raffo Iraolagoitia
- Laboratorio de Fisiopatología de la Inmunidad Innata, Instituto de Biología y Medicina Experimental (IBYME), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Vuelta de Obligado 2490, C1428ADN Ciudad de Buenos Aires, Argentina
| | - Andrea Ziblat
- Laboratorio de Fisiopatología de la Inmunidad Innata, Instituto de Biología y Medicina Experimental (IBYME), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Vuelta de Obligado 2490, C1428ADN Ciudad de Buenos Aires, Argentina
| | - Carolina Inés Domaica
- Laboratorio de Fisiopatología de la Inmunidad Innata, Instituto de Biología y Medicina Experimental (IBYME), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Vuelta de Obligado 2490, C1428ADN Ciudad de Buenos Aires, Argentina
| | - Damián Ezequiel Avila
- Laboratorio de Fisiopatología de la Inmunidad Innata, Instituto de Biología y Medicina Experimental (IBYME), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Vuelta de Obligado 2490, C1428ADN Ciudad de Buenos Aires, Argentina
- Cátedra de Inmunología, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, La Plata, Argentina
| | - Lucas Ezequiel Rossi
- Laboratorio de Fisiopatología de la Inmunidad Innata, Instituto de Biología y Medicina Experimental (IBYME), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Vuelta de Obligado 2490, C1428ADN Ciudad de Buenos Aires, Argentina
| | - Mercedes Beatriz Fuertes
- Laboratorio de Fisiopatología de la Inmunidad Innata, Instituto de Biología y Medicina Experimental (IBYME), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Vuelta de Obligado 2490, C1428ADN Ciudad de Buenos Aires, Argentina
- Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires (UBA), Buenos Aires, Argentina
| | | | - Gabriel Adrián Rabinovich
- Laboratorio de Inmunopatología, IBYME, CONICET, Buenos Aires, Argentina
- Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires (UBA), Buenos Aires, Argentina
| | - Mariana Salatino
- Laboratorio de Inmunopatología, IBYME, CONICET, Buenos Aires, Argentina
| | - Norberto Walter Zwirner
- Laboratorio de Fisiopatología de la Inmunidad Innata, Instituto de Biología y Medicina Experimental (IBYME), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Vuelta de Obligado 2490, C1428ADN Ciudad de Buenos Aires, Argentina
- Departamento de Microbiología, Parasitología e Inmunología, Facultad de Medicina, UBA, Buenos Aires, Argentina
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