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Selvi S, Real CM, Gentiluomo M, Balounova K, Vokacova K, Cumova A, Mohlenikova-Duchonova B, Rizzato C, Halasova E, Vodickova L, Smolkova B, Hemminki K, Campa D, Vodicka P. Genomic instability, DNA damage response and telomere homeostasis in pancreatic cancer. Semin Cancer Biol 2025; 113:59-73. [PMID: 40378535 DOI: 10.1016/j.semcancer.2025.05.005] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/05/2024] [Revised: 04/16/2025] [Accepted: 05/04/2025] [Indexed: 05/19/2025]
Abstract
Pancreatic cancer (PC) is becoming one of the most serious health problems at present, but its causes and risk factors are still unclear. One of the drivers in pancreatic carcinogenesis is altered genomic (DNA) integrity with subsequent genomic instability in cancer cells. The latter comprises a) DNA damage response and DNA repair mechanisms, b) DNA replication and mitosis, c) epigenetic regulation, and d) telomere maintenance. In our review we addressed the above aspects in relation to the most abundant and severe form of PC, pancreatic ductal adenocarcinoma (PDAC). In summary, the interactions between the DNA damage response, telomere homeostasis and mitotic regulation are not comprehensively understood at present, including the epigenetic factors entering the trait of genomic stability maintenance. In addition, the complexity of telomere homeostasis in relation to PDAC risk, prognosis and prediction also warrants further investigations.
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Affiliation(s)
- Saba Selvi
- Institute of Experimental Medicine, Czech Academy of Sciences, Videnska 1083, Prague 4 14200, Czech Republic; Department of Genetics and Microbiology, Faculty of Science, Charles University, Prague 12800, Czech Republic
| | - Carmen Macías Real
- Cancer Predisposition and Biomarkers Group, Instituto de Investigacion Sanitaria de Santiago, Santiago de Compostela, Spain
| | | | - Katerina Balounova
- Institute of Experimental Medicine, Czech Academy of Sciences, Videnska 1083, Prague 4 14200, Czech Republic; Department of Physiology, Faculty of Science, Charles University, Prague, Czech Republic
| | - Klara Vokacova
- Institute of Experimental Medicine, Czech Academy of Sciences, Videnska 1083, Prague 4 14200, Czech Republic
| | - Andrea Cumova
- Cancer Research Institute, Biomedical Research Center, Slovak Academy of Sciences, Dubravska cesta 9, Bratislava 84505, Slovakia
| | | | - Cosmeri Rizzato
- Department of Biology, University of Pisa, Pisa 56123, Italy
| | - Erika Halasova
- Biomedical Centre Martin, Jessenius Faculty of Medicine in Martin, Comenius University in Bratislava, Mala Hora 4, Martin 03601, Slovakia
| | - Ludmila Vodickova
- Biomedical Centre Martin, Jessenius Faculty of Medicine in Martin, Comenius University in Bratislava, Mala Hora 4, Martin 03601, Slovakia; Biomedical Center, Faculty of Medicine in Pilsen, Charles University, Alej Svobody 1655, Pilsen 32300, Czech Republic; Institute of Biology and Medical Genetics, First Faculty of Medicine, Charles University, Albertov 4, Prague 12800, Czech Republic
| | - Bozena Smolkova
- Cancer Research Institute, Biomedical Research Center, Slovak Academy of Sciences, Dubravska cesta 9, Bratislava 84505, Slovakia
| | - Kari Hemminki
- Biomedical Center, Faculty of Medicine in Pilsen, Charles University, Alej Svobody 1655, Pilsen 32300, Czech Republic; Division of Cancer Epidemiology, German Cancer Research Center (DKFZ), Heidelberg, FRG 69120, Germany
| | - Daniele Campa
- Department of Biology, University of Pisa, Pisa 56123, Italy
| | - Pavel Vodicka
- Biomedical Centre Martin, Jessenius Faculty of Medicine in Martin, Comenius University in Bratislava, Mala Hora 4, Martin 03601, Slovakia; Biomedical Center, Faculty of Medicine in Pilsen, Charles University, Alej Svobody 1655, Pilsen 32300, Czech Republic; Institute of Biology and Medical Genetics, First Faculty of Medicine, Charles University, Albertov 4, Prague 12800, Czech Republic.
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Nagao K, Watanuki M, Hayashi H, Kawamata N, Kuroiwa K, Narita H, Okamura R, Shimada S, Arai N, Kawaguchi Y, Yanagisawa K, Hattori N. Clinical impact of donor telomere length after umbilical cord blood transplantation. Cytotherapy 2025; 27:626-632. [PMID: 39918489 DOI: 10.1016/j.jcyt.2025.01.011] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/01/2024] [Revised: 01/10/2025] [Accepted: 01/21/2025] [Indexed: 05/24/2025]
Abstract
BACKGROUND AND AIMS Several studies have shown that the telomere length of engrafted donor cells affects the clinical outcomes in patients with hematologic diseases after allogeneic stem-cell transplantation (allo-SCT). However, the relationship between donor telomere length and clinical outcomes after umbilical-cord blood transplantation (UCBT) remains unknown. The study aim was to assess the relationship between donor telomere length and transplantation outcomes. METHODS We measured donor-derived relative telomere length (RTL) in 75 patients after single-unit UCBT and evaluated the association between telomere length and transplantation outcomes. RESULTS Compared with patients with shorter RTL, patients with longer RTL had a higher risk of bacterial and bloodstream infections [hazard ratio (HR), 4.79; 95% confidence interval (CI), 1.70-13.46; P = 0.003 and HR, 3.43; 95% CI, 1.19-9.82; P = 0.022, respectively] and was possibly associated with reduced relapse (HR 0.44, 95% CI 0.15-1.27, P = 0.13) by multivariate analysis. CONCLUSIONS Patients after UCBT who received engrafted donor cells with longer RTL had a higher risk of bacterial and bloodstream infections. The measured donor-derived RTL at engraftment after UCBT may predict clinical outcomes.
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Affiliation(s)
- Kazuki Nagao
- Division of Hematology, Department of Medicine, Showa University School of Medicine, Tokyo, Japan
| | - Megumi Watanuki
- Division of Hematology, Department of Medicine, Showa University School of Medicine, Tokyo, Japan
| | - Hidenori Hayashi
- Division of Hematology, Department of Medicine, Showa University School of Medicine, Tokyo, Japan
| | - Natsuki Kawamata
- Division of Hematology, Department of Medicine, Showa University School of Medicine, Tokyo, Japan
| | - Kai Kuroiwa
- Division of Hematology, Department of Medicine, Showa University School of Medicine, Tokyo, Japan
| | - Hinako Narita
- Division of Hematology, Department of Medicine, Showa University School of Medicine, Tokyo, Japan
| | - Reiko Okamura
- Division of Hematology, Department of Medicine, Showa University School of Medicine, Tokyo, Japan
| | - Shotaro Shimada
- Division of Hematology, Department of Medicine, Showa University School of Medicine, Tokyo, Japan
| | - Nana Arai
- Division of Hematology, Department of Medicine, Showa University School of Medicine, Tokyo, Japan
| | - Yukiko Kawaguchi
- Division of Hematology, Department of Medicine, Showa University School of Medicine, Tokyo, Japan
| | - Kouji Yanagisawa
- Division of Hematology, Department of Medicine, Showa University School of Medicine, Tokyo, Japan
| | - Norimichi Hattori
- Division of Hematology, Department of Medicine, Showa University School of Medicine, Tokyo, Japan.
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Wolf SE, Hastings WJ, Ye Q, Etzel L, Apsley AT, Chiaro C, Heim CC, Heller T, Noll JG, Schreier HMC, Shenk CE, Shalev I. Cross-tissue comparison of telomere length and quality metrics of DNA among individuals aged 8 to 70 years. PLoS One 2024; 19:e0290918. [PMID: 38386656 PMCID: PMC10883573 DOI: 10.1371/journal.pone.0290918] [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: 08/24/2023] [Accepted: 01/03/2024] [Indexed: 02/24/2024] Open
Abstract
Telomere length (TL) is an important biomarker of cellular aging, yet its links with health outcomes may be complicated by use of different tissues. We evaluated within- and between-individual variability in TL and quality metrics of DNA across five tissues using a cross-sectional dataset ranging from 8 to 70 years (N = 197). DNA was extracted from all tissue cells using the Gentra Puregene DNA Extraction Kit. Absolute TL (aTL) in kilobase pairs was measured in buccal epithelial cells, saliva, dried blood spots (DBS), buffy coat, and peripheral blood mononuclear cells (PBMCs) using qPCR. aTL significantly shortened with age for all tissues except saliva and buffy coat, although buffy coat was available for a restricted age range (8 to 15 years). aTL did not significantly differ across blood-based tissues (DBS, buffy coat, PBMC), which had significantly longer aTL than buccal cells and saliva. Additionally, aTL was significantly correlated for the majority of tissue pairs, with partial Spearman's correlations controlling for age and sex ranging from ⍴ = 0.18 to 0.51. We also measured quality metrics of DNA including integrity, purity, and quantity of extracted DNA from all tissues and explored whether controlling for DNA metrics improved predictions of aTL. We found significant tissue variation: DNA from blood-based tissues had high DNA integrity, more acceptable A260/280 and A260/230 values, and greater extracted DNA concentrations compared to buccal cells and saliva. Longer aTL was associated with lower DNA integrity, higher extracted DNA concentrations, and higher A260/230, particularly for saliva. Model comparisons suggested that incorporation of quality DNA metrics improves models of TL, although relevant metrics vary by tissue. These findings highlight the merits of using blood-based tissues and suggest that incorporation of quality DNA metrics as control variables in population-based studies can improve TL predictions, especially for more variable tissues like buccal and saliva.
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Affiliation(s)
- Sarah E. Wolf
- Department of Biobehavioral Health, Penn State University, University Park, Pennsylvania, United States of America
| | - Waylon J. Hastings
- Department of Biobehavioral Health, Penn State University, University Park, Pennsylvania, United States of America
- Department of Psychiatry and Behavioral Science, Tulane University School of Medicine, New Orleans, Louisiana, United States of America
| | - Qiaofeng Ye
- Department of Biobehavioral Health, Penn State University, University Park, Pennsylvania, United States of America
| | - Laura Etzel
- Department of Biobehavioral Health, Penn State University, University Park, Pennsylvania, United States of America
| | - Abner T. Apsley
- Department of Biobehavioral Health, Penn State University, University Park, Pennsylvania, United States of America
| | - Christopher Chiaro
- Department of Biobehavioral Health, Penn State University, University Park, Pennsylvania, United States of America
| | - Christine C. Heim
- Charité–Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, and Humboldt-Universität zu Berlin, Berlin Institute of Health, Institute of Medical Psychology, Berlin, Germany
| | - Thomas Heller
- Department of Biobehavioral Health, Penn State University, University Park, Pennsylvania, United States of America
| | - Jennie G. Noll
- Department of Psychology, University of Rochester, Rochester, NY, United States of America
- Department of Human Development and Family Studies, The Pennsylvania State University, University Park, PA, United States of America
| | - Hannah M. C. Schreier
- Department of Biobehavioral Health, Penn State University, University Park, Pennsylvania, United States of America
| | - Chad E. Shenk
- Department of Human Development and Family Studies, The Pennsylvania State University, University Park, PA, United States of America
- Department of Pediatrics, The Pennsylvania State University College of Medicine, Hershey, PA, United States of America
| | - Idan Shalev
- Department of Biobehavioral Health, Penn State University, University Park, Pennsylvania, United States of America
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Saretzki G. Measuring telomerase activity using TRAP assays. Methods Cell Biol 2023; 181:127-149. [PMID: 38302235 DOI: 10.1016/bs.mcb.2022.12.009] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/03/2024]
Abstract
Telomerase is a reverse transcriptase that consists of the telomerase reverse transcriptase (TERT) protein and the telomerase RNA component TERC which also harbors the template region for telomere synthesis. In its canonical function the enzyme adds single-stranded telomeric hexanucleotides de novo to the ends of linear chromosomes, telomeres, in telomerase-positive cells such as germline, stem- and cancer cells. This potential biochemical activity of telomerase can be measured with the help of a telomerase repeat amplification protocol (TRAP) which often includes a PCR amplification due to the low abundance of telomerase in most cells and tissues. The current chapter describes various TRAP methods to detect telomerase activity (TA) using gel-based methods, its advantages and deficits, how to perform an ELISA-based TRAP assay and how best to interpret its results. Since development of the TRAP assay in 1994, there have been numerous modifications and adaptations of the method from real-time PCR analysis, isothermal amplification and nanotechnology to CRISPR/Cas-based methods which will be briefly mentioned. However, it is not possible to cover all different TRAP methods and thus there is no comprehensiveness claimed by this chapter. Instead, the author describes various aspects of using TRAP assays including required controls, sample preparation, etc. in order to avoid pitfalls and set-backs in applying this rather complex and demanding technique. The TRAP assay is particularly important to support clinical diagnosis of cancer, analyze tumor therapy as well as to evaluate various approaches to inhibit TA as a form of anti-cancer therapy.
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Affiliation(s)
- Gabriele Saretzki
- Biosciences Institute, Newcastle University, Campus for Ageing and Vitality, Newcastle upon Tyne, United Kingdom.
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Yoshida Y, Takeda Y, Yamahara K, Yamamoto H, Takagi T, Kuramoto Y, Nakano-Doi A, Nakagomi T, Soma T, Matsuyama T, Doe N, Yoshimura S. Enhanced angiogenic properties of umbilical cord blood primed by OP9 stromal cells ameliorates neurological deficits in cerebral infarction mouse model. Sci Rep 2023; 13:262. [PMID: 36609640 PMCID: PMC9822952 DOI: 10.1038/s41598-023-27424-7] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/15/2022] [Accepted: 12/29/2022] [Indexed: 01/09/2023] Open
Abstract
Umbilical cord blood (UCB) transplantation shows proangiogenic effects and contributes to symptom amelioration in animal models of cerebral infarction. However, the effect of specific cell types within a heterogeneous UCB population are still controversial. OP9 is a stromal cell line used as feeder cells to promote the hematoendothelial differentiation of embryonic stem cells. Hence, we investigated the changes in angiogenic properties, underlying mechanisms, and impact on behavioral deficiencies caused by cerebral infarction in UCB co-cultured with OP9 for up to 24 h. In the network formation assay, only OP9 pre-conditioned UCB formed network structures. Single-cell RNA sequencing and flow cytometry analysis showed a prominent phenotypic shift toward M2 in the monocytic fraction of OP9 pre-conditioned UCB. Further, OP9 pre-conditioned UCB transplantation in mice models of cerebral infarction facilitated angiogenesis in the peri-infarct lesions and ameliorated the associated symptoms. In this study, we developed a strong, fast, and feasible method to augment the M2, tissue-protecting, pro-angiogenic features of UCB using OP9. The ameliorative effect of OP9-pre-conditioned UCB in vivo could be partly due to promotion of innate angiogenesis in peri-infarct lesions.
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Affiliation(s)
- Yasunori Yoshida
- grid.272264.70000 0000 9142 153XDepartment of Neurosurgery, Hyogo Medical University, 1-1 Mukogawa, Nishinomiya, Hyogo 663-8501 Japan
| | - Yuki Takeda
- grid.272264.70000 0000 9142 153XDepartment of Neurosurgery, Hyogo Medical University, 1-1 Mukogawa, Nishinomiya, Hyogo 663-8501 Japan
| | - Kenichi Yamahara
- Laboratory of Molecular and Cellular Therapy, Institute for Advanced Medical Sciences, Hyogo Medical University, 1-1 Mukogawa, Nishinomiya, Hyogo, 663-8501, Japan.
| | - Hanae Yamamoto
- grid.272264.70000 0000 9142 153XLaboratory of Molecular and Cellular Therapy, Institute for Advanced Medical Sciences, Hyogo Medical University, 1-1 Mukogawa, Nishinomiya, Hyogo 663-8501 Japan
| | - Toshinori Takagi
- grid.272264.70000 0000 9142 153XDepartment of Neurosurgery, Hyogo Medical University, 1-1 Mukogawa, Nishinomiya, Hyogo 663-8501 Japan
| | - Yoji Kuramoto
- grid.272264.70000 0000 9142 153XDepartment of Neurosurgery, Hyogo Medical University, 1-1 Mukogawa, Nishinomiya, Hyogo 663-8501 Japan
| | - Akiko Nakano-Doi
- Laboratory of Neurogenesis and CNS Repair, Institute for Advanced Medical Sciences, Hyogo Medial University, 1-1 Mukogawa, Nishinomiya, Hyogo 663-8501 Japan
| | - Takayuki Nakagomi
- Laboratory of Neurogenesis and CNS Repair, Institute for Advanced Medical Sciences, Hyogo Medial University, 1-1 Mukogawa, Nishinomiya, Hyogo 663-8501 Japan
| | - Toshihiro Soma
- grid.272264.70000 0000 9142 153XDepartment of Hematology, Hyogo Medical University, 1-1 Mukogawa, Nishinomiya, Hyogo 663-8501 Japan
| | - Tomohiro Matsuyama
- grid.272264.70000 0000 9142 153XDepartment of Therapeutic Progress in Brain Diseases, Hyogo Medical University, 1-1 Mukogawa, Nishinomiya, Hyogo 663-8501 Japan
| | - Nobutaka Doe
- Laboratory of Neurogenesis and CNS Repair, Institute for Advanced Medical Sciences, Hyogo Medial University, 1-1 Mukogawa, Nishinomiya, Hyogo 663-8501 Japan ,grid.272264.70000 0000 9142 153XDepartment of Occupational Therapy, School of Rehabilitation, Hyogo Medical University, 1-3-6 Minatojima, Chuo-Ku, Kobe, Hyogo 650-8530 Japan
| | - Shinichi Yoshimura
- grid.272264.70000 0000 9142 153XDepartment of Neurosurgery, Hyogo Medical University, 1-1 Mukogawa, Nishinomiya, Hyogo 663-8501 Japan
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Zhu X, Sun Q, Tan WS, Cai H. Removal of CD34− cells to increase self-renewal symmetric division and expansion ex vivo of cord blood CD34+ cells through reducing the TGF-β1. Process Biochem 2022. [DOI: 10.1016/j.procbio.2021.12.002] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/25/2022]
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Kuhikar R, Khan N, Khare SP, Fulzele A, Melinkeri S, Kale V, Limaye L. Neutrophils generated in vitro from hematopoietic stem cells isolated from apheresis samples and umbilical cord blood form neutrophil extracellular traps. Stem Cell Res 2020; 50:102150. [PMID: 33450673 DOI: 10.1016/j.scr.2020.102150] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/08/2019] [Revised: 11/05/2020] [Accepted: 12/27/2020] [Indexed: 11/22/2022] Open
Abstract
Neutrophils release neutrophil extracellular traps (NET) comprising of decondensed chromatin that immobilizes and kills pathogens. In vitro generation of neutrophils on a large scale from hematopoietic stem cells (HSCs) may be a useful strategy for treating neutropenic patients in future, though it is not in clinical practice yet. Microbial infections lead to major cause of morbidity and mortality in these patients. Despite the importance of NET in preventing infection, efficacy of in vitro-generated neutrophils from HSCs to form NET is not tested. We show that functional neutrophils could be generated in vitro from HSCs/MNCs isolated from umbilical cord blood (UCB) and apheresis-derived peripheral blood (APBL). Neutrophils generated from UCB showed properties comparable to those isolated from peripheral blood. We also show that isolation of HSCs is not absolutely essential for in vitro neutrophil generation. Further, we show that neutrophils generated from HSCs express PADI4 enzyme and their NET-forming ability is comparable to peripheral blood neutrophils. Taken together, our data show that fully functional neutrophils can be generated in vitro from HSCs. NET-forming ability of in vitro-generated neutrophils is an important parameter to determine their functionality and thus, should be studied along with other standard functional assays.
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Affiliation(s)
- Rutuja Kuhikar
- National Centre for Cell Science, NCCS Complex, Savitribai Phule Pune University Campus, Pune 411007, India
| | - Nikhat Khan
- National Centre for Cell Science, NCCS Complex, Savitribai Phule Pune University Campus, Pune 411007, India
| | - Satyajeet P Khare
- Symbiosis School of Biological Sciences, Symbiosis International (Deemed University), Lavale, Pune 412115, India
| | - Amit Fulzele
- Division of Biological Sciences, University of California at San Diego, La Jolla, CA 92093, USA
| | - Sameer Melinkeri
- Blood and Marrow Transplant Unit, Deenanath Mangeshkar Hospital, Erandawne, Pune 411004, India
| | - Vaijayanti Kale
- National Centre for Cell Science, NCCS Complex, Savitribai Phule Pune University Campus, Pune 411007, India; Symbiosis Centre for Stem Cell Research, Symbiosis School of Biological Sciences, Symbiosis Knowledge Village, Lavale, Pune 412115, India
| | - Lalita Limaye
- National Centre for Cell Science, NCCS Complex, Savitribai Phule Pune University Campus, Pune 411007, India.
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Um S, Ha J, Choi SJ, Oh W, Jin HJ. Prospects for the therapeutic development of umbilical cord blood-derived mesenchymal stem cells. World J Stem Cells 2020; 12:1511-1528. [PMID: 33505598 PMCID: PMC7789129 DOI: 10.4252/wjsc.v12.i12.1511] [Citation(s) in RCA: 22] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/30/2020] [Revised: 10/23/2020] [Accepted: 11/12/2020] [Indexed: 02/06/2023] Open
Abstract
Umbilical cord blood (UCB) is a primitive and abundant source of mesenchymal stem cells (MSCs). UCB-derived MSCs have a broad and efficient therapeutic capacity to treat various diseases and disorders. Despite the high latent self-renewal and differentiation capacity of these cells, the safety, efficacy, and yield of MSCs expanded for ex vivo clinical applications remains a concern. However, immunomodulatory effects have emerged in various disease models, exhibiting specific mechanisms of action, such as cell migration and homing, angiogenesis, anti-apoptosis, proliferation, anti-cancer, anti-fibrosis, anti-inflammation and tissue regeneration. Herein, we review the current literature pertaining to the UCB-derived MSC application as potential treatment strategies, and discuss the concerns regarding the safety and mass production issues in future applications.
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Affiliation(s)
- Soyoun Um
- Research Team for Immune Cell Therapy, Biomedical Research Institute, MEDIPOST Co., Ltd., Seongnam 13494, South Korea
| | - Jueun Ha
- Research Team for Osteoarthritis, Biomedical Research Institute, MEDIPOST Co., Ltd., Seongnam 13494, South Korea
| | - Soo Jin Choi
- Biomedical Research Institute, MEDIPOST Co., Ltd., Seongnam 13494, South Korea
| | - Wonil Oh
- Biomedical Research Institute, MEDIPOST Co., Ltd., Seongnam 13494, South Korea
| | - Hye Jin Jin
- Biomedical Research Institute, MEDIPOST Co., Ltd., Seongnam 13494, South Korea
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Emiloju OE, Potdar R, Jorge V, Gupta S, Varadi G. Clinical Advancement and Challenges of ex vivo Expansion of Human Cord Blood Cells. Clin Hematol Int 2019; 2:18-26. [PMID: 34595439 PMCID: PMC8432338 DOI: 10.2991/chi.d.191121.001] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/05/2019] [Accepted: 11/16/2019] [Indexed: 02/02/2023] Open
Abstract
Apart from peripheral blood stem cell (PBSC), umbilical cord blood (UCB) is now a recognized source of stem cells for transplantation. UCB is an especially important source of stem cells for minority populations, which would otherwise be unable to find appropriately matched adult donors. UCB has fewer mature T lymphocytes compared with peripheral blood, thus making a UCB transplantation (UCBT) with a greater degree of HLA mismatch possible. The limited cell dose per UCB sample is however associated with delayed engraftment and a higher risk of graft failure, especially in adult recipients. This lower cell dose can be optimized by performing double unit UCBT, ex vivo UCB expansion prior to transplant and enhancement of the capabilities of the stem cells to home to the bone marrow. UCB contains naïve and immature T cells, thus posing significant challenges with increased risk of infections, graft versus host diseases (GVHD) and relapse following UCBT. Cell engineering techniques have been developed to circumnavigate the immaturity of the T cells, and include virus-specific cytotoxic T cells (VSTs), T cells transduced with disease-specific chimeric antigen receptor (CAR T cells) and regulatory T cell (Tregs) engineering. In this article, we review the advances in UCB ex vivo expansion and engineering to improve engraftment and reduce complications. As further research continues to find ways to overcome the current challenges, outcomes from UCBT will likely improve.
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Affiliation(s)
| | - Rashmika Potdar
- Hematology and Oncology Department, Albert Einstein Medical Center, Philadelphia, PA, USA
| | - Vinicius Jorge
- Hematology and Oncology Department, Albert Einstein Medical Center, Philadelphia, PA, USA
| | - Sorab Gupta
- Hematology and Oncology Department, Albert Einstein Medical Center, Philadelphia, PA, USA
| | - Gabor Varadi
- Hematology and Oncology Department, Albert Einstein Medical Center, Philadelphia, PA, USA
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Abstract
PURPOSE OF REVIEW The pediatric transplant patient population is growing as the number of solid organ transplants and indications for hematopoietic stem cell transplant increase. Understanding cutaneous sequelae of pediatric transplant and treatment strategies to manage these outcomes is vital to the care of these patients. RECENT FINDINGS Important work in the past year enhances our understanding of the cutaneous implications of pediatric transplantation, including further work in areas of malignancy, infection, and graft versus host disease as well as newly reported risks. SUMMARY This review highlights recent developments in the recognition and management of dermatological complications of pediatric transplant that will be useful for the practicing pediatrician or dermatologist.
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Liu Z, Zhao J, Zhang R, Han G, Zhang C, Liu B, Zhang Z, Han MY, Gao X. Cross-Platform Cancer Cell Identification Using Telomerase-Specific Spherical Nucleic Acids. ACS NANO 2018; 12:3629-3637. [PMID: 29595962 DOI: 10.1021/acsnano.8b00743] [Citation(s) in RCA: 63] [Impact Index Per Article: 9.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/08/2023]
Abstract
Distinguishing tumor cells from normal cells holds the key to precision diagnosis and effective intervention of cancers. The fundamental difficulties, however, are the heterogeneity of tumor cells and the lack of truly specific and ideally universal cancer biomarkers. Here, we report a concept of tumor cell detection, bypassing the specific genotypic and phenotypic features of different tumor cell types and directly going toward the hallmark of cancer, uncontrollable growth. Combining spherical nucleic acids (SNAs) with exquisitely engineered molecular beacons (SNA beacons, dubbed SNAB technology) is capable of identifying tumor cells from normal cells based on the molecular phenotype of telomerase activity, largely bypassing the heterogeneity problem of cancers. Owing to the cell-entry capability of SNAs, the SNAB probe readily achieves tumor cell detection across multiple platforms, ranging from solution-based assay, to single cell imaging and in vivo solid tumor imaging (unlike PCR that is restricted to cell lysates). We envision the SNAB technology will impact cancer diagnosis, therapeutic response assessment, and image-guided surgery.
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Affiliation(s)
- Zhengjie Liu
- CAS Center for Excellence in Nanoscience, Institute of Intelligent Machines , Chinese Academy of Sciences , Hefei , Anhui 230031 , China
- Department of Chemistry , University of Science and Technology of China , Hefei , Anhui 230026 , China
| | - Jun Zhao
- CAS Center for Excellence in Nanoscience, Institute of Intelligent Machines , Chinese Academy of Sciences , Hefei , Anhui 230031 , China
| | - Ruilong Zhang
- School of Chemistry and Chemical Engineering , Anhui University , Hefei , Anhui 230601 , China
| | - Guangmei Han
- CAS Center for Excellence in Nanoscience, Institute of Intelligent Machines , Chinese Academy of Sciences , Hefei , Anhui 230031 , China
- Department of Chemistry , University of Science and Technology of China , Hefei , Anhui 230026 , China
| | - Cheng Zhang
- CAS Center for Excellence in Nanoscience, Institute of Intelligent Machines , Chinese Academy of Sciences , Hefei , Anhui 230031 , China
| | - Bianhua Liu
- CAS Center for Excellence in Nanoscience, Institute of Intelligent Machines , Chinese Academy of Sciences , Hefei , Anhui 230031 , China
| | - Zhongping Zhang
- CAS Center for Excellence in Nanoscience, Institute of Intelligent Machines , Chinese Academy of Sciences , Hefei , Anhui 230031 , China
- School of Chemistry and Chemical Engineering , Anhui University , Hefei , Anhui 230601 , China
| | - Ming-Yong Han
- CAS Center for Excellence in Nanoscience, Institute of Intelligent Machines , Chinese Academy of Sciences , Hefei , Anhui 230031 , China
- Institute of Materials Research and Engineering , A-STAR , 3 Research Link , Singapore 117602
| | - Xiaohu Gao
- Department of Bioengineering , University of Washington , Seattle , Washington 98195 , United States
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Costa MHG, de Soure AM, Cabral JMS, Ferreira FC, da Silva CL. Hematopoietic Niche - Exploring Biomimetic Cues to Improve the Functionality of Hematopoietic Stem/Progenitor Cells. Biotechnol J 2017; 13. [PMID: 29178199 DOI: 10.1002/biot.201700088] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/31/2017] [Revised: 10/27/2017] [Indexed: 12/19/2022]
Abstract
The adult bone marrow (BM) niche is a complex entity where a homeostatic hematopoietic system is maintained through a dynamic crosstalk between different cellular and non-cellular players. Signaling mechanisms triggered by cell-cell, cell-extracellular matrix (ECM), cell-cytokine interactions, and local microenvironment parameters are involved in controlling quiescence, self-renewal, differentiation, and migration of hematopoietic stem/progenitor cells (HSPC). A promising strategy to more efficiently expand HSPC numbers and tune their properties ex vivo is to mimic the hematopoietic niche through integration of adjuvant stromal cells, soluble cues, and/or biomaterial-based approaches in HSPC culture systems. Particularly, mesenchymal stem/stromal cells (MSC), through their paracrine activity or direct contact with HSPC, are thought to be a relevant niche player, positioning HSPC-MSC co-culture as a valuable platform to support the ex vivo expansion of hematopoietic progenitors. To improve the clinical outcome of hematopoietic cell transplantation (HCT), namely when the available HSPC are present in a limited number such is the case of HSPC collected from umbilical cord blood (UCB), ex vivo expansion of HSPC is required without eliminating the long-term repopulating capacity of more primitive HSC. Here, we will focus on depicting the characteristics of co-culture systems, as well as other bioengineering approaches to improve the functionality of HSPC ex vivo.
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Affiliation(s)
- Marta H G Costa
- Department of Bioengineering and iBB-Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal
| | - António M de Soure
- Department of Bioengineering and iBB-Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal
| | - Joaquim M S Cabral
- Department of Bioengineering and iBB-Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal.,The Discoveries Centre for Regenerative and Precision Medicine, Lisbon Campus, Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal
| | - Frederico Castelo Ferreira
- Department of Bioengineering and iBB-Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal.,The Discoveries Centre for Regenerative and Precision Medicine, Lisbon Campus, Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal
| | - Cláudia L da Silva
- Department of Bioengineering and iBB-Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal.,The Discoveries Centre for Regenerative and Precision Medicine, Lisbon Campus, Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal
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13
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Baron F, Ruggeri A, Nagler A. Methods of ex vivo expansion of human cord blood cells: challenges, successes and clinical implications. Expert Rev Hematol 2016; 9:297-314. [PMID: 26635058 DOI: 10.1586/17474086.2016.1128321] [Citation(s) in RCA: 31] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/05/2023]
Abstract
More than 40,000 unrelated cord blood transplantations (UCBT) have been performed worldwide as treatment for patients with malignant or non-malignant life threatening hematologic disorders. However, low absolute numbers of hematopoietic stem and progenitor cells (HSPCs) within a single cord blood unit has remained a limiting factor for this transplantation modality, particularly in adult recipients. Further, because UCB contains low numbers of mostly naïve T cells, immune recovery after UCBT is slow, predisposing patients to severe infections. Other causes of UCBT failure has included graft-versus-host disease (GVHD) and relapse of the underlying disease. In this article, we first review the current landscape of cord blood engineering aimed at improving engraftment. This includes approaches of UCB-HSPCs expansion and methods aimed at improving UCB-HSCPs homing. We then discuss recent approaches of cord blood engineering developed to prevent infection [generation of multivirus-specific cytotoxic T cells (VSTs) from UCB], relapse [transduction of UCB-T cells with tumor-specific chimeric receptor antigens (CARs)] and GVHD (expansion of regulatory T cells from UCB). Although many of these techniques of UCB engineering remain currently technically challenging and expensive, they are likely to revolutionize the field of UCBT in the next decades.
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Affiliation(s)
- Frédéric Baron
- a Division of Hematology, Department of Medicine , University and CHU of Liège , Liège , Belgium.,b GIGA-I3, Section of Hematology , University of Liège , Liège , Belgium
| | - Annalisa Ruggeri
- c Eurocord Hospital Saint Louis, AP-HP , Paris , France.,d Hospital Saint Antoine , Service d'Hématologie et Thérapie Cellulaire, AP-HP , Paris , France.,e Cord Blood Committee, Cellular Therapy and Immunobiology Working Party , EBMT , Leiden , Netherlands
| | - Arnon Nagler
- f Division of Hematology and Bone Marrow Transplantation , The Chaim Sheba Medical Center, Tel-Hashomer , Ramat-Gan , Israel.,g EBMT Paris Office , Hospital Saint Antoine , Paris , France.,h Université Pierre et Marie Curie , Paris , France.,i Tel Aviv University (TAU) , Tel Aviv , Israel
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14
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Ashbridge B, Zehir A, Lubin M, Barker JN, Moore MAS. Evaluation of Initial Telomere Length and Changes after Transplantation in Adult Double-Unit Cord Blood Transplant Recipients. Biol Blood Marrow Transplant 2015; 21:1334-6. [PMID: 25865647 DOI: 10.1016/j.bbmt.2015.04.006] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/13/2014] [Accepted: 04/06/2015] [Indexed: 10/23/2022]
Abstract
Cord blood (CB) leukocytes have inherent telomere length (TL) variation, and CB hematopoietic stem cells (HSC) can maintain high telomerase levels preventing telomere attrition in vitro. We evaluated TL changes in 13 adult double-unit CB transplant (CBT) recipients. In the 26 units, we observed a marked variation in CB TL at thaw (median, 9.99 kilobases [kb]; range, 6.85 to 13.5). All 13 patients engrafted. Of 11 engrafting with 1 unit, there was no correlation between unit dominance and TL (mean dominant unit TL, 8.84 kb ± 1.76; mean nonengrafting unit TL, 10.3 kb ± 1.81; P = .77). Serial measurements of TL up to 1 year after CBT demonstrated an overall mean 3.04 kb ± .16 TL decrease with only 1 patient exhibiting telomere maintenance. In summary, initial TL does not predict CB unit dominance. Moreover, our analysis suggests neonatal hematopoiesis makes a transition to an HSC characterized by changes in average TL and potentially low telomerase asymmetric cell division in adult CBT recipients. Further investigation of alterations in telomere length and its clinical implications after transplantation of this observation are indicated.
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Affiliation(s)
- Beth Ashbridge
- Cell Biology Program, Sloan-Kettering Institute, New York, New York
| | - Ahmet Zehir
- Cell Biology Program, Sloan-Kettering Institute, New York, New York
| | - Marissa Lubin
- Department of Medicine, Adult Bone Marrow Transplantation Service, Memorial Sloan-Kettering Cancer Center, New York, New York
| | - Juliet N Barker
- Department of Medicine, Adult Bone Marrow Transplantation Service, Memorial Sloan-Kettering Cancer Center, New York, New York; Department of Medicine, Weill Cornell Medical College, New York, New York.
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15
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Bari S, Seah KKH, Poon Z, Cheung AMS, Fan X, Ong SY, Li S, Koh LP, Hwang WYK. Expansion and homing of umbilical cord blood hematopoietic stem and progenitor cells for clinical transplantation. Biol Blood Marrow Transplant 2014; 21:1008-19. [PMID: 25555449 DOI: 10.1016/j.bbmt.2014.12.022] [Citation(s) in RCA: 42] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/19/2014] [Accepted: 12/22/2014] [Indexed: 12/26/2022]
Abstract
The successful expansion of hematopoietic stem and progenitor cells (HSPCs) from umbilical cord blood (UCB) for transplantation could revolutionize clinical practice by improving transplantation-related outcomes and making available UCB units that have suboptimal cell doses for transplantation. New cytokine combinations appear able to promote HSPC growth with minimal differentiation into mature precursors and new agents, such as insulin-like growth factor-binding protein 2, are being used in clinical trials. Molecules that simulate the HSPC niche, such as Notch ligand, have also shown promise. Further improvements have been made with the use of mesenchymal stromal cells, which have made possible UCB expansion without a potentially deleterious prior CD34/CD133 cell selection step. Chemical molecules, such as copper chelators, nicotinamide, and aryl hydrocarbon antagonists, have shown excellent outcomes in clinical studies. The use of bioreactors could further add to HSPC studies in future. Drugs that could improve HSPC homing also appear to have potential in improving engraftment times in UCB transplantation. Technologies to expand HSPC from UCB and to enhance the homing of these cells appear to have attained the goal of accelerating hematopoietic recovery. Further discoveries and clinical studies are likely to make the goal of true HSPC expansion a reality for many applications in future.
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Affiliation(s)
- Sudipto Bari
- Department of Hematology, Singapore General Hospital, Singapore; Department of Pharmacy, National University of Singapore, Singapore
| | | | - Zhiyong Poon
- BioSystems and Micromechanics, Singapore-MIT Alliance for Research and Technology, Singapore
| | | | - Xiubo Fan
- Department of Clinical Research, Singapore General Hospital, Singapore
| | - Shin-Yeu Ong
- Department of Hematology, Singapore General Hospital, Singapore
| | - Shang Li
- Cancer and Stem Cell Biology, Duke-NUS Graduate Medical School, Singapore
| | - Liang Piu Koh
- Department of Hematology-Oncology, National University Cancer Institute, Singapore
| | - William Ying Khee Hwang
- Department of Hematology, Singapore General Hospital, Singapore; Cancer and Stem Cell Biology, Duke-NUS Graduate Medical School, Singapore; Singapore Cord Blood Bank, Singapore.
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16
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D'Addio F, Trevisani A, Ben Nasr M, Bassi R, El Essawy B, Abdi R, Secchi A, Fiorina P. Harnessing the immunological properties of stem cells as a therapeutic option for diabetic nephropathy. Acta Diabetol 2014; 51:897-904. [PMID: 24894496 DOI: 10.1007/s00592-014-0603-1] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/29/2014] [Accepted: 05/17/2014] [Indexed: 12/27/2022]
Abstract
Diabetic nephropathy is the leading and possibly the most devastating complication of diabetes, with a prevalence ranging from 25 to 40 % in diabetic individuals, and as such represents an important challenge for public health worldwide. As a major cause of end-stage renal disease, diabetic nephropathy also accounts for a large proportion of deaths in diabetic individuals. To date, therapeutic options for overt diabetic nephropathy include medical interventions to reduce blood glucose levels and to control blood pressure and proteinuria. Recent evidence suggests a strong role for inflammation in the development and progression of diabetic nephropathy. Various immune cells, cytokines and chemokines have been implicated in the onset of diabetic nephropathy, while immune-related transcription factors and adhesion molecules have been correlated with the establishment of a renal proinflammatory microenvironment. Both inflammation and immune activation may promote severe distress in the kidney, with subsequent increased local fibrosis, ultimately leading to the development of end-stage renal disease. Stem cells are undifferentiated cells capable of regenerating virtually any organ or tissue and bearing important immunoregulatory and anti-inflammatory properties. Due to the aforementioned considerations, significant interest has been ignited with regard to the use of stem cells as novel therapeutics for diabetic nephropathy. Here, we will be examining in detail how anti-inflammatory properties of different populations of stem cells may offer novel therapy for the treatment of diabetic nephropathy.
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17
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Park SK, Won JH. Usefulness of umbilical cord blood cells in era of hematopoiesis research. Int J Stem Cells 2014; 2:90-6. [PMID: 24855526 DOI: 10.15283/ijsc.2009.2.2.90] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 10/19/2009] [Indexed: 12/31/2022] Open
Abstract
Although worldwide experience with umbilical cord blood (UCB) transplantation is still relatively limited, clinical experience with UCB transplantation is encouraging. The use of UCB for hematopoietic stem cell transplantation (HSCT) has advantages and disadvantages. Among the advantages are rapid availability, ability to more rapidly schedule the transplant as the UCB units are stored and ready for use, the apparent reduced need for an exact human leukocyte antigen (HLA) match, and induction of a less severe graft versus host disease (GVHD) compared with bone marrow. The major limitation of reduced numbers of hematopoietic stem cells (HSC) in UCB is being addressed by basic research. It is promising that potential improvements in engraftment efficiency without increased stem cell numbers or actual increased stem cell numbers through dual UCB transplant or ex-vivo expansion might lead to improved treatment approaches. However, its therapeutic potential extends beyond the hematopoietic component suggesting regenerative potential in solid organs as well. Many different stem and progenitor cell populations have been postulated with potential ranging from embryonic like to lineage-committed progenitor cells. UCB derived MSCs have the differentiation capacity and also the therapeutic potential with regard to regenerative medicine, stromal support, immune modulation and gene therapy. Therefore, further advances are eagerly anticipated.
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Affiliation(s)
- Seong-Kyu Park
- Department of Internal Medicine, Soonchunhyang University College of Medicine, Seoul, Korea
| | - Jong-Ho Won
- Department of Internal Medicine, Soonchunhyang University College of Medicine, Seoul, Korea
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18
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Differential sensitivity of telomerase from human hematopoietic stem cells and leukemic cell lines to mild hyperthermia. Cell Biochem Biophys 2014; 69:681-91. [PMID: 24590263 DOI: 10.1007/s12013-014-9853-3] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/25/2022]
Abstract
We have investigated the effects of hyperthermia (HT) on cell proliferation and telomerase activity of human hematopoietic stem cells (HSCs) and compared with human leukemic cell lines (TF-1, K562 and HL-60). The cells were exposed to HT at 42 and 43 °C up to 120 min. The cells were incubated at 37 °C for 96 h. Then the cells were collected and assayed for cell proliferation, viability, telomerase activity, and terminal restriction fragment (TRF) lengths. The enzyme activity from HSCs was decreased up to 68.6 at 42 and 85.1 % at 43 °C for 120 min. This inhibition in leukemic cells was up to 28.9 and 53.6 % in TF-1; 53 and 63.9 % in K562; 45.2 and 61.1 % in HL-60 cells. The treated cells showed TRF lengths about 5.3 kb for control HL-60 cells, 5.0 kb for HL-60 cells treated at 42 and 4.5 kb at 43 °C for 120 min. In HSCs, the TRF length was about 4.5 kb for untreated cells and 4.0-4.5 kb for treated cells at 42 and 43 °C for 120 min. The time response curves indicated that, inhibition of the enzyme activity in leukemic cells was dependent to the time of exposure to HT. But in HSCs, the inhibition was reached to steady state at 15 min exposure to 43 °C heat stress. TRF length was constant at treated two types of cells, which implies that in cells subjected to mild HT no telomere shortening was observed.
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19
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Thomay K, Schienke A, Vajen B, Modlich U, Schambach A, Hofmann W, Schlegelberger B, Göhring G. Chromosomal Instability and Telomere Shortening in Long-Term Culture of Hematopoietic Stem Cells: Insights from a Cell Culture Model of RPS14 Haploinsufficiency. Cytogenet Genome Res 2014; 142:14-20. [DOI: 10.1159/000356096] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 08/27/2013] [Indexed: 11/19/2022] Open
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20
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Wnt1 Accelerates an Ex Vivo Expansion of Human Cord Blood CD34(+)CD38(-) Cells. Stem Cells Int 2013; 2013:909812. [PMID: 24023545 PMCID: PMC3760094 DOI: 10.1155/2013/909812] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/19/2013] [Revised: 07/09/2013] [Accepted: 07/12/2013] [Indexed: 12/20/2022] Open
Abstract
Cord blood hematopoietic stem cells (CB-HSCs) transplantation has been increasing gradually with facing the limitation of insufficient quantity of HSCs in each CB unit. Therefore, efficient expansion methods which can maintain stem cell characteristics are needed. In this study, umbilical CB-CD34+ cells were cultured in two different cytokine cocktails: 4 factors (4F = Flt3-L, SCF, IL-6, and TPO) and 5 factors (5F = Wnt1 + 4F) in both serum and serum-free media. The data revealed that the best condition to accelerate an expansion of CD34+CD38− cells was serum-free culture condition supplemented with 5F (5F KSR). This condition yielded 24.3 ± 2.1 folds increase of CD34+CD38− cells. The expanded cells exhibited CD34+ CD38− CD133+ CD71low CD33low CD3− CD19− markers, expressed nanog, oct3/4, c-myc, and sox2 genes, and maintained differentiation potential into lymphoid, erythroid and myeloid lineages. The achievement of CD34+CD38− cells expansion may overcome an insufficient quantity of the cells leading to the improvement of the stem cell transplantation. Altogether, our findings highlight the role of Wnt1 and the new culture condition in stimulating hematopoietic stem/progenitor cells expansion which may offer a new therapeutic avenue for cord blood transplantation, regenerative medicine, stem cell bank applications, and other clinical applications in the future.
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21
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Iskander A, Knight RA, Zhang ZG, Ewing JR, Shankar A, Varma NRS, Bagher-Ebadian H, Ali MM, Arbab AS, Janic B. Intravenous administration of human umbilical cord blood-derived AC133+ endothelial progenitor cells in rat stroke model reduces infarct volume: magnetic resonance imaging and histological findings. Stem Cells Transl Med 2013; 2:703-14. [PMID: 23934909 DOI: 10.5966/sctm.2013-0066] [Citation(s) in RCA: 43] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022] Open
Abstract
Endothelial progenitor cells (EPCs) hold enormous therapeutic potential for ischemic vascular diseases. Previous studies have indicated that stem/progenitor cells derived from human umbilical cord blood (hUCB) improve functional recovery in stroke models. Here, we examined the effect of hUCB AC133+ EPCs on stroke development and resolution in a middle cerebral artery occlusion (MCAo) rat model. Since the success of cell therapies strongly depends on the ability to monitor in vivo the migration of transplanted cells, we also assessed the capacity of magnetic resonance imaging (MRI) to track in vivo the magnetically labeled cells that were administered. Animals were subjected to transient MCAo and 24 hours later injected intravenously with 10(7) hUCB AC133+ EPCs. MRI performed at days 1, 7, and 14 after the insult showed accumulation of transplanted cells in stroke-affected hemispheres and revealed that stroke volume decreased at a significantly higher rate in cell-treated animals. Immunohistochemistry analysis of brain tissues localized the administered cells in the stroke-affected hemispheres only and indicated that these cells may have significantly affected the magnitude of endogenous proliferation, angiogenesis, and neurogenesis. We conclude that transplanted cells selectively migrated to the ischemic brain parenchyma, where they exerted a therapeutic effect on the extent of tissue damage, regeneration, and time course of stroke resolution.
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Affiliation(s)
- Asm Iskander
- Department of Radiology, Henry Ford Hospital, Detroit, MI, USA
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22
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Karlsson C, Baudet A, Miharada N, Soneji S, Gupta R, Magnusson M, Enver T, Karlsson G, Larsson J. Identification of the chemokine CCL28 as a growth and survival factor for human hematopoietic stem and progenitor cells. Blood 2013; 121:3838-42, S1-15. [PMID: 23509159 DOI: 10.1182/blood-2013-02-481192] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022] Open
Abstract
In an attempt to discover novel growth factors for hematopoietic stem and progenitor cells (HSPCs), we have assessed cytokine responses of cord blood (CB)-derived CD34(+) cells in a high-content growth factor screen. We identify the immunoregulatory chemokine (C-C motif) ligand 28 (CCL28) as a novel growth factor that directly stimulates proliferation of primitive hematopoietic cells from different ontogenetic origins. CCL28 enhances the functional progenitor cell content of cultured cells by stimulating cell cycling and induces gene expression changes associated with survival. Importantly, addition of CCL28 to cultures of purified putative hematopoietic stem cells (HSCs) significantly increases the ability of the cells to long-term repopulate immunodeficient mice compared with equivalent input numbers of fresh cells. Together, our findings identify CCL28 as a potent growth-promoting factor with the ability to support the in vitro and in vivo functional properties of cultured human hematopoietic cells.
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Affiliation(s)
- Christine Karlsson
- Molecular Medicine and Gene Therapy, Lund Stem Cell Center, Lund University, Lund, Sweden
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23
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Improved ex vivo expansion of adult hematopoietic stem cells by overcoming CUL4-mediated degradation of HOXB4. Blood 2013; 121:4082-9. [PMID: 23520338 DOI: 10.1182/blood-2012-09-455204] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/29/2023] Open
Abstract
Direct transduction of the homeobox (HOX) protein HOXB4 promotes the proliferation of hematopoietic stem cells (HSCs) without induction of leukemogenesis, but requires frequent administration to overcome its short protein half-life (∼1 hour). We demonstrate here that HOXB4 protein levels are post-translationally regulated by the CUL4 ubiquitin ligase, and define the degradation signal sequence (degron) of HOXB4 required for CUL4-mediated destruction. Additional HOX paralogs share the conserved degron in the homeodomain and are also subject to CUL4-mediated degradation, indicating that CUL4 likely controls the stability of all HOX proteins. Moreover, we engineered a degradation-resistant HOXB4 that conferred a growth advantage over wild-type HOXB4 in myeloid progenitor cells. Direct transduction of recombinant degradation-resistant HOXB4 protein to human adult HSCs significantly enhanced their maintenance in a more primitive state both in vitro and in transplanted NOD/SCID/IL2R-γ(null) mice compared with transduction with wild-type HOXB4 protein. Our studies demonstrate the feasibility of engineering a stable HOXB4 variant to overcome a major technical hurdle in the ex vivo expansion of adult HSCs and early progenitors for human therapeutic use.
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24
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Deezagi A, Abedi-Tashi M. Studying the enucleation process, DNA breakdown and telomerase activity of the K562 cell lines during erythroid differentiation in vitro. In Vitro Cell Dev Biol Anim 2013; 49:122-33. [PMID: 23288413 DOI: 10.1007/s11626-012-9574-0] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/03/2012] [Accepted: 11/26/2012] [Indexed: 11/30/2022]
Abstract
During erythropoiesis, some organelles such as mitochondria and nucleus are lost by autophagy and enucleation processes in the presence of macrophages in vivo. In vitro production of erythrocytes has raised many questions about the mechanism of enucleation. The aim of this work was to study the DNA breakdown, enucleation, hemoglobin synthesis and telomerase activity of K562 cells during erythroid differentiation. For these purposes, K562 cells were induced to differentiate by erythropoietin + rhGM-CSF, DMSO, and sodium butyrate separately up to 14 d. In different time intervals, hemoglobin synthesis was evaluated by benzidine staining and RT-PCR for γ-globin gene expression. DNA breakdown was analyzed by 4',6-diamidino-2-phenylindole (DAPI) staining, DNA ladder electrophoresis and comet assay. The telomerase activity was evaluated by TRAP assay. Our result indicated that, sodium butyrate and DMSO inhibited K562 cell growth about 50-60% in comparison to untreated control cells. The percentage of benzidine-positive cells was about 45% in the presence of sodium butyrate after 10 d. Densitometric analysis of RT-PCR and calculated data indicated a 1.5-fold increase in relative γ-globin gene expression at 96 h, in the presence of 1 mM sodium butyrate in comparison with untreated cells. DAPI staining did not reveal any evidence of internal lysis of the nucleus during erythroid differentiation at first wk, but this was obvious in the second wk. DNA laddering pattern was not observed in differentiated cells during 14 d. In comet assay, the percentage of DNA in tail, tail length, and tail moment were significantly different between untreated and treated cells (p < 0.05). Telomerase activity was inhibited up to 90.3% during erythroid differentiation of these cells.
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Affiliation(s)
- Abdolkhaleg Deezagi
- Department of Biochemistry, National Institute of Genetic Engineering and Biotechnology, Tehran, Iran.
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25
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Ge J, Cai H, Li Q, Du Z, Tan WS. Effects of telomerase activity and apoptosis on ex vivo expansion of cord blood CD34(+) cells. Cell Prolif 2012; 46:38-44. [PMID: 23240888 DOI: 10.1111/cpr.12006] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/06/2012] [Revised: 08/02/2012] [Accepted: 09/15/2012] [Indexed: 01/26/2023] Open
Abstract
OBJECTIVE Ex vivo expansion of CD34(+) cells has become critically important in order to obtain sufficient haematopoietic stem cells for clinical application. Among major regulators involved in ex vivo expansion, telomerase activity and apoptosis have been revealed to be closely linked to cell cycle progression. However, all exact roles remain to be elucidated. Here, change in telomerase activity and level of apoptosis in cord blood (CB) CD34(+) cells were evaluated together with specific cell population growth rate during ex vivo culture. MATERIALS AND METHODS CD34(+) cells isolated from human CB were expanded ex vivo over a 28-day period. Besides monitoring cell proliferation kinetics of the CD34(+) cells, changes in telomerase activity and apoptotic levels were investigated. Several relevant genes were quantified by qRT-PCR during the culture period. RESULTS Significant elevation of telomerase activity had close relationship to activation of CB CD34(+) cell expansion. Peak apoptotic level was accompanied by a remarkable decline in cell-specific growth rate, and apoptotic level of differentiated CD34(-) population was significantly higher than that of the CD34(+) population. CONCLUSION Although telomerase activity was activated during the culture, expansion of CB CD34(+) cells seemed to be more susceptible to apoptotic suppression when cultured ex vivo, which implied that apoptosis may serve as a rate-limiting factor involved in controlling expansion efficiency.
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Affiliation(s)
- J Ge
- State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, China
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26
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Kawakami Y, Ii M, Alev C, Kawamoto A, Matsumoto T, Kuroda R, Shoji T, Fukui T, Masuda H, Akimaru H, Mifune Y, Kuroda T, Horii M, Yokoyama A, Kurosaka M, Asahara T. Local Transplantation of Ex Vivo Expanded Bone Marrow-Derived CD34-Positive Cells Accelerates Fracture Healing. Cell Transplant 2012; 21:2689-709. [DOI: 10.3727/096368912x654920] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
Transplantation of bone marrow (BM) CD34+ cells, an endothelial/hematopoietic progenitor-enriched cell population, has shown therapeutic efficiency in the treatment of ischemic diseases enhancing neovascularization. However, the number of CD34+ cells obtained from bone marrow is not sufficient for routine clinical application. To overcome this issue, we developed a more efficient and clinically applicable CD34+ cell expansion method. Seven-day ex vivo expansion culture of BM CD34+ cells with a cocktail of five growth factors containing VEGF, SCF, IL-6, Flt-3 ligand, and TPO resulted in reproducible more than 20-fold increase in cell number. The favorable effect of the local transplantation of culture expanded (cEx)-BM CD34+ cells on rat unhealing fractures was equivalent or higher than that of nonexpanded (fresh) BM CD34+ cells exhibiting sufficient therapeutic outcome with frequent vasculogenic/osteogenic differentiation of transplanted cEx-BM CD34+ cells and fresh BM CD34+ cells as well as intrinsic enhancement of angiogenesis/osteogenesis at the treated fracture sites. Specifically, cEx-BM CD34+ cell treatment demonstrated the best blood flow recovery at fracture sites compared with the nonexpanded BM CD34+ cells. In vitro, cEx-BM CD34+ cells showed higher colony/tube-forming capacity than nonexpanded BM CD34+ cells. Both cells demonstrated differentiation potential into osteoblasts. Since fresh BM CD34+ cells can be easily collected from fracture sites at the time of primary operation and stored for future use, autologous cEx-BM CD34+ cell transplantation would be not only a simple but also a promising therapeutic strategy for unhealing fractures in the field of orthopedic trauma surgery.
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Affiliation(s)
- Yohei Kawakami
- Group of Vascular Regeneration, Institute of Biomedical Research and Innovation, Kobe, Hyogo, Japan
- Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, Kobe, Hyogo, Japan
| | - Masaaki Ii
- Department of Pharmacology, Osaka Medical College, Takatsuki, Osaka, Japan
| | - Cantas Alev
- Group of Vascular Regeneration, Institute of Biomedical Research and Innovation, Kobe, Hyogo, Japan
- Laboratory for Early Embryogenesis, RIKEN Center for Developmental Biology, Kobe, Hyogo, Japan
| | - Atsuhiko Kawamoto
- Group of Vascular Regeneration, Institute of Biomedical Research and Innovation, Kobe, Hyogo, Japan
| | - Tomoyuki Matsumoto
- Group of Vascular Regeneration, Institute of Biomedical Research and Innovation, Kobe, Hyogo, Japan
- Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, Kobe, Hyogo, Japan
| | - Ryosuke Kuroda
- Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, Kobe, Hyogo, Japan
| | - Taro Shoji
- Group of Vascular Regeneration, Institute of Biomedical Research and Innovation, Kobe, Hyogo, Japan
- Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, Kobe, Hyogo, Japan
| | - Tomoaki Fukui
- Group of Vascular Regeneration, Institute of Biomedical Research and Innovation, Kobe, Hyogo, Japan
- Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, Kobe, Hyogo, Japan
| | - Haruchika Masuda
- Department of Regenerative Medicine Science, Tokai University School of Medicine, Isehara, Kanagawa, Japan
| | - Hiroshi Akimaru
- Group of Vascular Regeneration, Institute of Biomedical Research and Innovation, Kobe, Hyogo, Japan
| | - Yutaka Mifune
- Group of Vascular Regeneration, Institute of Biomedical Research and Innovation, Kobe, Hyogo, Japan
- Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, Kobe, Hyogo, Japan
| | - Tomoya Kuroda
- Group of Vascular Regeneration, Institute of Biomedical Research and Innovation, Kobe, Hyogo, Japan
- Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, Kobe, Hyogo, Japan
| | - Miki Horii
- Group of Vascular Regeneration, Institute of Biomedical Research and Innovation, Kobe, Hyogo, Japan
| | - Ayumi Yokoyama
- Group of Vascular Regeneration, Institute of Biomedical Research and Innovation, Kobe, Hyogo, Japan
| | - Masahiro Kurosaka
- Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, Kobe, Hyogo, Japan
| | - Takayuki Asahara
- Group of Vascular Regeneration, Institute of Biomedical Research and Innovation, Kobe, Hyogo, Japan
- Department of Regenerative Medicine Science, Tokai University School of Medicine, Isehara, Kanagawa, Japan
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Janic B, Arbab AS. Cord blood endothelial progenitor cells as therapeutic and imaging probes. ACTA ACUST UNITED AC 2012; 4:477-490. [PMID: 23227114 DOI: 10.2217/iim.12.35] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/25/2022]
Abstract
Numerous studies demonstrated that neovascularization processes associated with severe tissue ischemia commonly found in conditions such as cardiovascular disorders and tumor growth occur via angiogenic and vasculogenic mechanisms. Over the past decade, it has been demonstrated that endothelial progenitor cells (EPCs) play a significant role in neo-angiogenic and neovasculogenic processes. Due to their ability to self-renew, circulate, home to the ischemic sites and differentiate into mature endothelial cells, EPCs derived from various sources hold enormous potential to be used as therapeutic agents in pro- or anti-angiogenic strategies for the treatment of ischemic and tumor conditions, respectively. However, the development of EPC-based therapies requires accompanying, noninvasive imaging protocol for in vivo tracking of transplanted cells. Hence, this review focuses on cord blood-derived EPCs and their role in neovascularization with emphasis on the potential use of EPCs as a therapeutic and imaging probe.
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Affiliation(s)
- Branislava Janic
- Cellular & Molecular Imaging Laboratory, Department of Radiology, Henry Ford Hospital, 1 Ford Place, 2F, Box 82, Detroit, MI 48202, USA
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MRI tracking of FePro labeled fresh and cryopreserved long term in vitro expanded human cord blood AC133+ endothelial progenitor cells in rat glioma. PLoS One 2012; 7:e37577. [PMID: 22662174 PMCID: PMC3360770 DOI: 10.1371/journal.pone.0037577] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/07/2011] [Accepted: 04/24/2012] [Indexed: 01/31/2023] Open
Abstract
BACKGROUND Endothelial progenitors cells (EPCs) are important for the development of cell therapies for various diseases. However, the major obstacles in developing such therapies are low quantities of EPCs that can be generated from the patient and the lack of adequate non-invasive imaging approach for in vivo monitoring of transplanted cells. The objective of this project was to determine the ability of cord blood (CB) AC133+ EPCs to differentiate, in vitro and in vivo, toward mature endothelial cells (ECs) after long term in vitro expansion and cryopreservation and to use magnetic resonance imaging (MRI) to assess the in vivo migratory potential of ex vivo expanded and cryopreserved CB AC133+ EPCs in an orthotopic glioma rat model. MATERIALS, METHODS AND RESULTS The primary CB AC133+ EPC culture contained mainly EPCs and long term in vitro conditions facilitated the maintenance of these cells in a state of commitment toward endothelial lineage. At days 15-20 and 25-30 of the primary culture, the cells were labeled with FePro and cryopreserved for a few weeks. Cryopreserved cells were thawed and in vitro differentiated or i.v. administered to glioma bearing rats. Different groups of rats also received long-term cultured, magnetically labeled fresh EPCs and both groups of animals underwent MRI 7 days after i.v. administration of EPCs. Fluorescent microscopy showed that in vitro differentiation of EPCs was not affected by FePro labeling and cryopreservation. MRI analysis demonstrated that in vivo accumulation of previously cryopreserved transplanted cells resulted in significantly higher R2 and R2* values indicating a higher rate of migration and incorporation into tumor neovascularization of previously cryopreserved CB AC133+ EPCs to glioma sites, compared to non-cryopreserved cells. CONCLUSION Magnetically labeled CB EPCs can be in vitro expanded and cryopreserved for future use as MRI probes for monitoring the migration and incorporation to the sites of neovascularization.
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Ex vivo expansion of umbilical cord blood: where are we? Int J Hematol 2012; 95:371-9. [PMID: 22438185 DOI: 10.1007/s12185-012-1053-6] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/04/2012] [Revised: 02/29/2012] [Accepted: 03/07/2012] [Indexed: 10/28/2022]
Abstract
Since the first successful clinical use of umbilical cord blood (UCB) in 1988, UCB grafts have been used for over 20,000 patients with both malignant and non-malignant diseases. UCB has several practical advantages over other transplantable graft sources. For example, the ease of procurement, the absence of donor risks, the reduced risk of transmissible infections, and the availability for immediate use make UCB an appealing graft choice. However, UCB grafts suffer from a few limitations related to the limited cell dose available for transplantation in each UCB unit and to defects in UCB stem cell homing. These limitations lead to increased post-transplant complications. In this review, we focus on the issue of limited cell dose in UCB units and discuss the possible approaches to overcome this limitation. We also summarize the various cellular pathways that have been explored to expand UCB units.
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Abstract
Cord blood (CB) is an important alternative source of hematopoietic stem cells (HSCs) for transplantation today. The principal drawbacks of cord blood transplantation are the limited number of hematopoietic stem cells and a long time to engraftment. Several promising approaches for engraftment enhancement are under intensive investigation. Such are transplantation with two cord blood units, co transplantation of cord blood and haploidentical HSCs and different methods for expansion of cord blood hematopoietic stem cells. In addition there are several ways for improving of homing of HSCs such as co- infusion of CB hematopoietic stem cells and mesenchymal stem cells, administration of parathyroid hormone (PTH), intra- bone transplantation and targeting the CXCR4/SDF1 system. These strategies are expected to increase the availability of transplantation to adults, for whom the chance to find a cord blood suitable for a single unit transplant is small. Recent advances in elucidation of the molecular mechanisms responsible for the proliferation and self-renewal of hematopoietic stem cells may bring further improvement of the outcomes of cord blood transplantation. This review summarizes the recent progress in the field of cord blood derived hematopoietic stem cells. It presents the strategies applied and points out directions for the future.
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31
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Gunetti M, Noghero A, Molla F, Staszewsky LI, de Angelis N, Soldo A, Russo I, Errichiello E, Frasson C, Rustichelli D, Ferrero I, Gualandris A, Berger M, Geuna M, Scacciatella P, Basso G, Marra S, Bussolino F, Latini R, Fagioli F. Ex vivo-expanded bone marrow CD34(+) for acute myocardial infarction treatment: in vitro and in vivo studies. Cytotherapy 2011; 13:1140-52. [PMID: 21846293 DOI: 10.3109/14653249.2011.597559] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022]
Abstract
BACKGROUND AIMS Bone marrow (BM)-derived cells appear to be a promising therapeutic source for the treatment of acute myocardial infarction (AMI). However, the quantity and quality of the cells to be used, along with the appropriate time of administration, still need to be defined. We thus investigated the use of BM CD34(+)-derived cells as cells suitable for a cell therapy protocol (CTP) in the treatment of experimental AMI. METHODS The need for a large number of cells was satisfied by the use of a previously established protocol allowing the expansion of human CD34(+) cells isolated from neonatal and adult hematopoietic tissues. We evaluated gene expression, endothelial differentiation potential and cytokine release by BM-derived cells during in vitro culture. Basal and expanded CD34(+) cells were used as a delivery product in a murine AMI model consisting of a coronary artery ligation (CAL). Cardiac function recovery was evaluated after injecting basal or expanded cells. RESULTS Gene expression analysis of in vitro-expanded cells revealed that endothelial markers were up-regulated during culture. Moreover, expanded cells generated a CD14(+) subpopulation able to differentiate efficiently into VE-cadherin-expressing cells. In vivo, we observed a cardiac function recovery in mice sequentially treated with basal and expanded cells injected 4 h and 7 days after CAL, respectively. CONCLUSIONS Our data suggest that combining basal and expanded BM-derived CD34(+) cells in a specific temporal pattern of administration might represent a promising strategy for a successful cell-based therapy.
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Affiliation(s)
- Monica Gunetti
- Stem Cell Transplantation and Cellular Therapy Unit, Pediatric Onco-Hematology Division, Regina Margherita Children's Hospital, Turin, Italy.
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Abdulrazzak H, Moschidou D, Jones G, Guillot PV. Biological characteristics of stem cells from foetal, cord blood and extraembryonic tissues. J R Soc Interface 2010; 7 Suppl 6:S689-706. [PMID: 20739312 DOI: 10.1098/rsif.2010.0347.focus] [Citation(s) in RCA: 75] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022] Open
Abstract
Foetal stem cells (FSCs) can be isolated during gestation from many different tissues such as blood, liver and bone marrow as well as from a variety of extraembryonic tissues such as amniotic fluid and placenta. Strong evidence suggests that these cells differ on many biological aspects such as growth kinetics, morphology, immunophenotype, differentiation potential and engraftment capacity in vivo. Despite these differences, FSCs appear to be more primitive and have greater multi-potentiality than their adult counterparts. For example, foetal blood haemopoietic stem cells proliferate more rapidly than those found in cord blood or adult bone marrow. These features have led to FSCs being investigated for pre- and post-natal cell therapy and regenerative medicine applications. The cells have been used in pre-clinical studies to treat a wide range of diseases such as skeletal dysplasia, diaphragmatic hernia and respiratory failure, white matter damage, renal pathologies as well as cancers. Their intermediate state between adult and embryonic stem cells also makes them an ideal candidate for reprogramming to the pluripotent status.
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Affiliation(s)
- Hassan Abdulrazzak
- Institute of Reproductive and Developmental Biology, Imperial College London, London W12 0NN, UK
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Maritz MF, Napier CE, Wen VW, MacKenzie KL. Targeting telomerase in hematologic malignancy. Future Oncol 2010; 6:769-89. [PMID: 20465390 DOI: 10.2217/fon.10.42] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022] Open
Abstract
Over the past two decades, it has become increasingly apparent that telomerase-mediated telomere maintenance plays a crucial role in hematopoiesis. Supporting evidence is underscored by recent findings of mutations in genes involved in telomerase-mediated telomere maintenance that contribute to the pathogenesis of bone marrow failure syndromes. More recently described telomere-independent functions of telomerase are also likely to contribute to both normal hematopoiesis and hematologic diseases. The high levels of telomerase detected in aggressive leukemias have fueled fervent investigation into diverse approaches to targeting telomerase in hematologic malignancies. Successful preclinical investigations that employed genetic strategies, oligonucleotides, small-molecule inhibitors and immunotherapy have resulted in a rapid translation to clinical trials. Further investigation of telomere-independent functions of telomerase and detailed preclinical studies of telomerase inhibition in both normal and malignant hematopoiesis will be invaluable for refining treatments to effectively and safely exploit telomerase as a therapeutic target in hematologic malignancies.
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Affiliation(s)
- Michelle F Maritz
- Children's Cancer Institute Australia for Medical Research, Lowy Cancer Research Centre, New South Wales, Australia
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Bordeaux-Rego P, Luzo A, Costa FF, Olalla Saad ST, Crosara-Alberto DP. Both interleukin-3 and interleukin-6 are necessary for better ex vivo expansion of CD133+ cells from umbilical cord blood. Stem Cells Dev 2010; 19:413-22. [PMID: 19656071 DOI: 10.1089/scd.2009.0098] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/29/2023] Open
Abstract
Umbilical cord blood (UCB), an ideal source for transplantable hematopoietic stem cells (HSC), is readily available and is rich in progenitor cells. Identification of conditions favoring UCB-HSC ex vivo expansion and of repopulating potential remains a major challenge in hematology. CD133+ cells constitute an earlier, less-differentiated HSC group with a potentially higher engraftment capacity. The presence of SCF, Flt3-L, and TPO are essential for CD133+ and/or CD34+ cells ex vivo expansion; however, IL-3 and IL-6 influence has not yet been clearly established. We investigated this influence on CD133+ cells from UCB ex vivo expansion and the effect of these cytokines upon cell phenotype. Immediately after isolation an 85% of CD133+ cell purity was obtained, diminishing after 4 and 8 days of ex vivo expansion. CD133+ fold-increase was higher using IMDM with SCF, Flt3-L, and TPO (BM)+IL-3 or BM+IL-3+IL-6 on day 8 (13.83- and 17.47-fold increase, respectively). BM+IL-6 presented no significant difference from BM alone. We demonstrated that 5.1% of the CD133+ cells expressed IL-6 receptor (IL-6R) after isolation. After 4 and 8 days in culture, the percentage of CD133+ cells that expressed IL-6R was as follows: BM alone (9.8% and 22.02%, respectively); BM+IL-3 (8.33% and 16.74%); BM+IL-6 (9.2% and 17.67%); and BM+IL-3+IL-6 (12.5% and 61.20%). Cell cycle analysis revealed quiescent cells after isolation, 95.5% CD133+ cells in the G0/G1 phase. Regardless of culture period or cytokine incubation, CD133+ cell cycle altered to 70% of CD133+ in the G0/G1 phase. Colony-forming unit (CFU) doubled in BM+IL-3+IL-6 after 8 days of incubation compared with BM group. SOX-2 and NANOG-relative gene expression was detected on day 0 after isolation. BM+IL-6 prevented the decrease in NANOG and SOX-2 gene expression level compared to BM+IL-3 or BM+IL-3+IL-6 incubated cells. Our results indicated that UCB-isolated CD133+ cells were better ex vivo expanded in the presence of SCF, Flt3-L, TPO, IL-3+IL-6. IL-3 probably promotes higher CD133+ cell expansion and IL-6 maintains immature phenotype.
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Affiliation(s)
- Pedro Bordeaux-Rego
- Center of Haematology and Hemotherapy, University of Campinas, Campinas, São Paulo Brazil
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35
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Delaney C, Ratajczak MZ, Laughlin MJ. Strategies to enhance umbilical cord blood stem cell engraftment in adult patients. Expert Rev Hematol 2010; 3:273-83. [PMID: 20835351 PMCID: PMC2935587 DOI: 10.1586/ehm.10.24] [Citation(s) in RCA: 67] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023]
Abstract
Umbilical cord blood (UCB) has been used successfully as a source of hematopoietic stem cells (HSCs) for allogeneic transplantation in children and adults in the treatment of hematologic diseases. However, compared with marrow or mobilized peripheral blood stem cell grafts from adult donors, significant delays in the rates and kinetics of neutrophil and platelet engraftment are noted after UCB transplant. These differences relate in part to the reduced numbers of HSCs in UCB grafts. To improve the rates and kinetics of engraftment of UCB HSC, several strategies have been proposed, including ex vivo expansion of UCB HSCs, addition of third-party mesenchymal cells, intrabone delivery of HSCs, modulation of CD26 expression, and infusion of two UCB grafts. This article will focus on ex vivo expansion of UCB HSCs and strategies to enhance UCB homing as potential solutions to overcome the problem of low stem cell numbers in a UCB graft.
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Affiliation(s)
- Colleen Delaney
- Fred Hutchinson Cancer Research Center, Mailstop D2-100, 1100 Fairview Ave N, PO Box, 9024, Seattle, WA 98109, USA
| | - Mariusz Z Ratajczak
- Stem Cell Institute James Graham Brown Cancer Center, 500 South Floyd Street, Louisville, KY 40202, USA
| | - Mary J Laughlin
- Associate Professor of Medicine & Pathology, Dr Donald & Ruth Weber Goodman Professor of Innovative Cancer Therapeutics, Case Western Reserve University, 10900 Euclid Avenue, WRB 2-125, Cleveland, OH 44106-7284, USA
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36
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Andrade PZ, dos Santos F, Almeida-Porada G, da Silva CL, S Cabral JMS. Systematic delineation of optimal cytokine concentrations to expand hematopoietic stem/progenitor cells in co-culture with mesenchymal stem cells. MOLECULAR BIOSYSTEMS 2010; 6:1207-15. [PMID: 20424784 DOI: 10.1039/b922637k] [Citation(s) in RCA: 34] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/21/2022]
Abstract
The major obstacle to the widespread use of umbilical cord blood (UCB) in hematopoietic stem/progenitor (HSC) cell therapy is the low cell dose available. A cytokine cocktail for the ex vivo expansion of UCB HSC, in co-culture with a bone marrow (BM) mesenchymal stem cells (MSC)-derived stromal layer was optimized using an experimental design approach. Proliferation of total cells (TNC), stem/progenitor cells (CD34(+)) and colony-forming units (CFU) was assessed after 7 days in culture, while sole and interactive effects of each cytokine on HSC expansion were statistically determined using a two-level Face-Centered Cube Design. The optimal cytokine cocktail obtained for HSC-MSC co-cultures was composed by SCF, Flt-3L and TPO (60, 55 and 50 ng mL(-1), respectively), resulting in 33-fold expansion in TNC, 17-fold in CD34(+) cells, 3-fold in CD34(+)CD90(+) cells and 21-fold in CFU-MIX. More importantly, these short-term expanded cells preserved their telomere length and extensively generated cobblestone area-forming cells (CAFCs) in vitro. The statistical tools used herein contributed for the rational delineation of the cytokine concentration range, in a cost-effective way, while systematically addressing complex cytokine-to-cytokine interactions, for the efficient HSC expansion towards the generation of clinically significant cell numbers for transplantation.
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Affiliation(s)
- Pedro Z Andrade
- IBB-Institute for Biotechnology and Bioengineering, Centre for Biological and Chemical Engineering, Instituto Superior Técnico, Lisboa, Portugal
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37
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Abstract
A marked increase in the utilization of umbilical cord blood (UCB) transplantation has been observed in recent years; however, the use of UCB as a hematopoietic stem cell (HSC) source is limited primarily by the number of progenitor cells contained in the graft. Graft failure, delayed engraftment and profound delay in immune reconstitution lead to significant morbidity and mortality in adults. The lack of cells available for post transplant therapies, such as donor lymphocyte infusions, has also been considered to be a disadvantage of UCB. To improve outcomes and extend applicability of UCB transplantation, one potential solution is ex vivo expansion of UCB. Investigators have used several methods, including liquid suspension culture with various cytokines and expansion factors, co-culture with stromal elements and continuous perfusion systems. Techniques combining ex vivo expanded and unmanipulated UCB are being explored to optimize the initial engraftment kinetics as well as the long-term durability. The optimal expansion conditions are still not known; however, recent studies suggest that expanded UCB is safe. It is hoped that by ex vivo expansion of UCB, a resulting decrease in the morbidity and mortality of UCB transplantation will be observed, and that the availability of additional cells may allow adoptive immunotherapy or gene transfer therapies in the UCB setting.
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Affiliation(s)
- S S Kelly
- Department of Pediatrics, The University of Texas MD Anderson Cancer Center, Houston, TX 77030-4009, USA.
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38
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Kirouac DC, Madlambayan GJ, Yu M, Sykes EA, Ito C, Zandstra PW. Cell-cell interaction networks regulate blood stem and progenitor cell fate. Mol Syst Biol 2009; 5:293. [PMID: 19638974 PMCID: PMC2724979 DOI: 10.1038/msb.2009.49] [Citation(s) in RCA: 91] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/20/2009] [Accepted: 06/18/2009] [Indexed: 12/18/2022] Open
Abstract
Communication networks between cells and tissues are necessary for homeostasis in multicellular organisms. Intercellular (between cell) communication networks are particularly relevant in stem cell biology, as stem cell fate decisions (self-renewal, proliferation, lineage specification) are tightly regulated based on physiological demand. We have developed a novel mathematical model of blood stem cell development incorporating cell-level kinetic parameters as functions of secreted molecule-mediated intercellular networks. By relation to quantitative cellular assays, our model is capable of predictively simulating many disparate features of both normal and malignant hematopoiesis, relating internal parameters and microenvironmental variables to measurable cell fate outcomes. Through integrated in silico and experimental analyses, we show that blood stem and progenitor cell fate is regulated by cell–cell feedback, and can be controlled non-cell autonomously by dynamically perturbing intercellular signalling. We extend this concept by demonstrating that variability in the secretion rates of the intercellular regulators is sufficient to explain heterogeneity in culture outputs, and that loss of responsiveness to cell–cell feedback signalling is both necessary and sufficient to induce leukemic transformation in silico.
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Affiliation(s)
- Daniel C Kirouac
- Institute for Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Ontario, Canada
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39
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Weisel KC, Moore MAS, Kanz L, Möhle R. Extended in vitro expansion of adult, mobilized CD34+ cells without significant cell senescence using a stromal cell coculture system with single cytokine support. Stem Cells Dev 2009; 18:229-34. [PMID: 18491948 DOI: 10.1089/scd.2008.0069] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022] Open
Abstract
The macrophage colony-stimulating factor-deficient bone marrow stromal cell line OP9, derived from osteopetrotic mice, is known to support hematopoietic stem cell (HSC) expansion as well as hematopoietic differentiation of embryonic stem cells. Coculture of HSC in the OP9 system requires cytokine support to achieve significant cell expansion. Recently, we reported extensive expansion without cell senescence of cord blood (CB)-derived HSC cocultured with OP9 stromal cells for more than 18 weeks with a single cytokine support using human thrombopoietin (TPO). In this study, we evaluated the efficiency of the OP9/TPO coculture system to sustain long-term hematopoiesis of adult, granulocyte colony-stimulating factor mobilized human peripheral blood (PB) CD34(+) cells. Maximum cell expansion was attained during the first 4 weeks of coculture. At the same time, the maximum progenitor cell expansion was demonstrated by the production of colony-forming cells and cobblestone area-forming cells. In contrast to the expansion of CB CD34(+) cells, PB CD34(+) cells showed termination of cultures after 8 weeks, independent of the cell expansion rates attained. The evaluation of cell senescence by assessing the telomere length in most cultures showed no relevant telomere shortening, despite rapid decrease in telomerase activity. Interestingly, increases in telomere length were demonstrated. In conclusion, OP9/TPO system provides extensive stem cell expansion without concomitant telomere erosion for both CB and adult CD34(+) cells. Termination of adult CD34(+) cell cocultures seems to be independent of telomere length.
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Affiliation(s)
- Katja C Weisel
- Department of Hematology, Oncology, and Immunology, University of Tübingen, Medical Center, Tübingen, Germany.
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40
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Howe M, Zhao J, Bodenburg Y, McGuckin CP, Forraz N, Tilton RG, Urban RJ, Denner L. Oct-4A isoform is expressed in human cord blood-derived CD133 stem cells and differentiated progeny. Cell Prolif 2009; 42:265-75. [PMID: 19438894 DOI: 10.1111/j.1365-2184.2009.00593.x] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022] Open
Abstract
OBJECTIVES This study aims to establish whether the pluripotent embryonic stem cell marker and nuclear transcription factor Oct-4A isoform is expressed in human umbilical cord blood CD133 stem cells (CD133 cells) and their differentiated progeny. MATERIALS AND METHODS CD133 cells were examined for expression of the embryonic stem cell marker Oct-4A by reverse transcription-polymerase chain reaction using primers specific for the coding region of the Oct-4A isoform. Immunocytochemistry and flow cytometry were performed using an antibody raised to a peptide from the unique amino-terminal domain of the Oct-4A isoform, that does not exist in the Oct-4B isoform. Furthermore, specificity was confirmed by pre-adsorption of the antibody with the peptide immunogen. Differentiation was determined before and after expansion in culture, by flow cytometry for haematopoietic stem cell and differentiation markers. For many studies, after 7 days of culture CD133-positive and CD133-negative cells were separated by flow cytometry for additional analyses. Multilineage haematopoietic proliferative potential was determined using colony-forming assays. RESULTS Freshly isolated CD133 cells expressed Oct-4A mRNA and protein. The cells proliferated rapidly in culture producing only a small proportion of CD133-positive cells and a much larger proportion of non-self-renewing CD133-negative cells. Proliferation was also associated with loss of other adult stem cell markers, gain of differentiated haematopoietic markers, and maintenance of potential to generate haematopoietic lineages. Oct-4A mRNA and protein were expressed throughout these changes. CONCLUSIONS Oct-4A, which is associated with self-renewal in embryonic stem cells, neither defines nor confers self-renewal to CD133 stem cells.
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Affiliation(s)
- M Howe
- Stark Diabetes Center, Galveston, Texas, USA.
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Li X, Qu Y, Mao M, Yu F, Li Q, Hua Y, Mu D. Expression of human telomerase reverse transcriptase in bone marrow CD34+ cells from patients with beta-thalassemia major. Transfusion 2008; 48:1627-33. [PMID: 18466174 DOI: 10.1111/j.1537-2995.2008.01724.x] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
Abstract
BACKGROUND Normal stem cells usually express a low level of telomerase activity that serves to stabilize the chromosomes during cell division and helps prevent cell senescence. Human telomerase reverse transcriptase (hTERT) is a rate-limiting enzyme that dictates the activity of human telomerase and thus decides the life span of cells. The expression of hTERT and its roles in beta-thalassemia major are unclear, however. STUDY DESIGN AND METHODS hTERT mRNA expression in bone marrow (BM) CD34+ cells from 25 children with beta-thalassemia major and 15 control subjects was investigated using real-time reverse transcription polymerase chain reaction (RT-PCR) analysis. The serum erythropoietin (sEPO) and hemoglobin (Hb) levels in peripheral blood were also determined. The relationship between hTERT and sEPO as well as Hb was then examined. RESULTS It was found that hTERT mRNA expression was significantly up regulated in BM CD34+ cells from patients with beta-thalassemia major. Furthermore, a significantly positive correlation was found between hTERT mRNA and sEPO (r = 0.771, p < 0.001). A significantly inverse correlation, however, was found between hTERT mRNA and Hb concentration (r = -0.929, p < 0.001). CONCLUSION Our findings suggest that severe anemia with low Hb concentration might up regulate hTERT expression of BM CD34+ cells and sEPO levels in patients with beta-thalassemia major.
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Affiliation(s)
- Xihong Li
- Department of Pediatrics, West China Second University Hospital, Sichuan University, Sichuan, China
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Transplantation of human hematopoietic repopulating cells: mechanisms of regeneration and differentiation using human???mouse xenografts. Curr Opin Organ Transplant 2008; 13:44-52. [DOI: 10.1097/mot.0b013e3282f42486] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/29/2023]
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Angiopoietin-like 5 and IGFBP2 stimulate ex vivo expansion of human cord blood hematopoietic stem cells as assayed by NOD/SCID transplantation. Blood 2008; 111:3415-23. [PMID: 18202223 DOI: 10.1182/blood-2007-11-122119] [Citation(s) in RCA: 194] [Impact Index Per Article: 11.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/16/2023] Open
Abstract
Hematopoietic stem cells (HSCs) are the basis of bone marrow transplantation and are attractive target cells for hematopoietic gene therapy, but these important clinical applications have been severely hampered by difficulties in ex vivo expansion of HSCs. In particular, the use of cord blood for adult transplantation is greatly limited by the number of HSCs. Previously we identified angiopoietin-like proteins and IGF-binding protein 2 (IGFBP2) as new hormones that, together with other factors, can expand mouse bone marrow HSCs in culture. Here, we measure the activity of multipotent human severe combined immunodeficient (SCID)-repopulating cells (SRCs) by transplantation into the nonobese diabetic SCID (NOD/SCID) mice; secondary transplantation was performed to evaluate the self-renewal potential of SRCs. A serum-free medium containing SCF, TPO, and FGF-1 or Flt3-L cannot significantly support expansion of the SRCs present in human cord blood CD133+ cells. Addition of either angiopoietin-like 5 or IGF-binding protein 2 to the cultures led to a sizable expansion of HSC numbers, as assayed by NOD/SCID transplantation. A serum-free culture containing SCF, TPO, FGF-1, angiopoietin-like 5, and IGFBP2 supports an approximately 20-fold net expansion of repopulating human cord blood HSCs, a number potentially applicable to several clinical processes including HSC transplantation.
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Martin J, Helm K, Ruegg P, Varella-Garcia M, Burnham E, Majka S. Adult lung side population cells have mesenchymal stem cell potential. Cytotherapy 2008; 10:140-51. [DOI: 10.1080/14653240801895296] [Citation(s) in RCA: 69] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023]
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Zimmermann S, Martens UM. Telomeres, senescence, and hematopoietic stem cells. Cell Tissue Res 2007; 331:79-90. [PMID: 17960423 DOI: 10.1007/s00441-007-0469-4] [Citation(s) in RCA: 41] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/13/2007] [Accepted: 07/10/2007] [Indexed: 01/01/2023]
Abstract
The replicative lifespan of normal somatic cells is restricted by the erosion of telomeres, which are protective caps at the ends of linear chromosomes. The loss of telomeres induces antiproliferative signals that eventually lead to cellular senescence. The enzyme complex telomerase can maintain telomeres, but its expression is confined to highly proliferative cells such as stem cells and tumor cells. The immense regenerative capacity of the hematopoietic system is provided by a distinct type of adult stem cell: hematopoietic stem cells (HSCs). Although blood cells have to be produced continuously throughout life, the HSC pool seems not to be spared by aging processes. Indeed, limited expression of telomerase is not sufficient to prevent telomere shortening in these cells, which is thought ultimately to limit their proliferative capacity. In this review, we discuss the relevance of telomere maintenance for the hematopoietic stem cell compartment and consider potential functions of telomerase in this context. We also present possible clinical applications of telomere manipulation in HSCs and new insights affecting the aging of the hematopoietic stem cell pool and replicative exhaustion.
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Affiliation(s)
- Stefan Zimmermann
- Department of Hematology/Oncology, Freiburg University Medical Center, Hugstetterstrasse 55, 79106, Freiburg, Germany.
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Drummond MW, Balabanov S, Holyoake TL, Brummendorf TH. Concise review: Telomere biology in normal and leukemic hematopoietic stem cells. Stem Cells 2007; 25:1853-61. [PMID: 17510216 DOI: 10.1634/stemcells.2007-0057] [Citation(s) in RCA: 44] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
The measurement of telomere length can give an insight into the replicative history of the cells in question. Much of the observed telomere loss occurs at the stem and progenitor cell level, even though these populations express the enzyme telomerase. Telomerase-transfected hematopoietic stem cells (HSC), although able to maintain telomere length, are still limited in terms of ability to undergo sequential transplantation, and other factors require to be addressed to achieve optimal levels of stem cell expansion. Unchecked telomere loss by HSC, meanwhile, would appear to play a significant role in the pathogenesis of bone marrow failure, as observed in the condition dyskeratosis congenita. This heterogeneous inherited condition appears to exhibit telomerase dysfunction as a common final pathogenic mechanism. Although less well-established for acquired marrow failure syndromes, mutations in key telomerase components have been described. The identification of the leukemic stem cell (LSC), along with the desire to target this population with anti-leukemia therapy, demands that telomerase biology be fully understood in this cell compartment. Future studies using primary selected LSC-rich samples are required. A better understanding of telomerase regulation in this population may allow effective targeting of the telomerase enzyme complex using small molecule inhibitors or additional novel approaches. Disclosure of potential conflicts of interest is found at the end of this article.
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MESH Headings
- Acute Disease
- Animals
- Cell Proliferation
- DNA Replication/physiology
- DNA, Neoplasm/physiology
- Hematopoietic Stem Cell Transplantation
- Hematopoietic Stem Cells/enzymology
- Hematopoietic Stem Cells/pathology
- Hematopoietic Stem Cells/physiology
- Humans
- Leukemia/enzymology
- Leukemia/genetics
- Leukemia/pathology
- Leukemia/physiopathology
- Leukemia, Myelogenous, Chronic, BCR-ABL Positive/enzymology
- Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics
- Leukemia, Myelogenous, Chronic, BCR-ABL Positive/physiopathology
- Leukemia, Myeloid/enzymology
- Leukemia, Myeloid/genetics
- Leukemia, Myeloid/physiopathology
- Mice
- Mice, Knockout
- Models, Biological
- Neural Tube Defects/enzymology
- Neural Tube Defects/genetics
- Neural Tube Defects/physiopathology
- Telomerase/genetics
- Telomerase/metabolism
- Telomerase/physiology
- Telomere/metabolism
- Telomere/physiology
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Weisel KC, Yildirim S, Schweikle E, Kanz L, Möhle R. Effect of FLT3 Inhibition on Normal Hematopoietic Progenitor Cells. Ann N Y Acad Sci 2007; 1106:190-6. [PMID: 17442779 DOI: 10.1196/annals.1392.020] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022]
Abstract
Ligand-mediated activation of the FMS-like tyrosine kinase-3 (FLT3) receptor is important for normal proliferation of primitive hematopoietic cells. FLT3 expression in the bone marrow is restricted to CD34(+) cells and a subset of dendritic precursors. FLT3, as a member of the type III RTK subfamily, is closely related to c-kit, c-FMS, and PDGFalpha/beta and is an unspecific target of tyrosine kinase inhibitors, such as imatinib. Activating mutations of FLT3 play an important role in leukemogenesis and their presence is associated with poor prognosis in acute myeloid leukemia (AML). Targeting the mutation by inhibiting the tyrosine kinase activity of FLT3 is a promising therapeutic option in the treatment of AML patients. CEP-701 (Lestaurtinib), an indocarbazole derivate, is an FLT3 tyrosine kinase inhibitor. In this study, we investigated the effect of FLT3 kinase inhibition on normal hematopoietic stem and progenitor cells in vitro. FLT3 inhibition in normal CD34(+) cells resulted in a dose-dependent inhibitory effect in cell expansion. In contrast, progenitor cell function remained nearly unaffected. Blocking the FLT3 ligand by a neutralizing antibody partially restored the effects of FLT3 inhibition. These findings might explain hematotoxicity of tyrosine kinase inhibitors such as imatinib.
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Affiliation(s)
- Katja C Weisel
- University of Tübingen, Medical Center, Department of Hematology, Oncology and Immunology, Otfried-Müller-Strasse 10, 72076 Tübingen, Germany.
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Moore MAS, Dorn DC, Schuringa JJ, Chung KY, Morrone G. Constitutive activation of Flt3 and STAT5A enhances self-renewal and alters differentiation of hematopoietic stem cells. Exp Hematol 2007; 35:105-16. [PMID: 17379095 DOI: 10.1016/j.exphem.2007.01.018] [Citation(s) in RCA: 47] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/13/2023]
Abstract
OBJECTIVE To model human leukemogenesis by transduction of human hematopoietic stem cells (HSC) with genes associated with leukemia and expressed in leukemic stem cells. METHODS Constitutive activation of Flt3 (Flt3-ITD) has been reported in 25 to 30% of patients with acute myeloid leukemia (AML). Retroviral vectors expressing constitutively activated Flt3 and STAT5A were used to transduce human cord blood CD34(+) cells and HSC cell self-renewal and differentiation were evaluated. RESULTS We have demonstrated that retroviral transduction of Flt3 mutations into CD34(+) cells enhanced HSC self-renewal as measured in vitro in competitive stromal coculture and limiting-dilution week-2 cobblestone (CAFC) assays. Enhanced erythropoiesis and decreased myelopoiesis were noted together with strong activation of STAT5A. Consequently, transduction studies were undertaken with a constitutively active mutant of STAT5A (STAT5A[1( *)6]) and here also a marked, selective expansion of transduced CD34(+) cells was noted, with a massive increase in self-renewing CAFC detectable at both 2 and 5 weeks of stromal coculture. Differentiation was biased to erythropoiesis, including erythropoietin independence, with myeloid maturation inhibition. The observed phenotypic changes correlated with differential gene expression, with a number of genes differentially regulated by both the Flt3 and STAT5A mutants. These included upregulation of genes involved in erythropoiesis and downregulation of genes involved in myelopoiesis. The phenotype of week-2 self-renewing CAFC also characterized primary Flt3-ITD(+) AML bone marrow samples. Isolation of leukemic stem cells (LSC) with a CD34(+), CD38(-), HLA-DR(-) phenotype was undertaken with Flt3-ITD(+) AML samples resulting in co-purification of early CAFC. Gene expression of LSC relative to the bulk leukemic population revealed upregulation of homeobox genes (HOXA9, HOXA5) implicated in leukemogenesis, and hepatic leukemia factor (HLF) involved in stem cell proliferation. CONCLUSION Myeloid leukemogenesis is a multi-stage process that can involve constitutively activated receptors and downstream pathways involving STAT5, HOX genes, and HLF.
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Affiliation(s)
- Malcolm A S Moore
- Moore Laboratory, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA.
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Madlambayan GJ, Rogers I, Purpura KA, Ito C, Yu M, Kirouac D, Casper RF, Zandstra PW. Clinically relevant expansion of hematopoietic stem cells with conserved function in a single-use, closed-system bioprocess. Biol Blood Marrow Transplant 2007; 12:1020-30. [PMID: 17084368 DOI: 10.1016/j.bbmt.2006.07.005] [Citation(s) in RCA: 40] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/24/2006] [Accepted: 07/07/2006] [Indexed: 01/27/2023]
Abstract
The clinical potential of umbilical cord blood-derived stem and progenitor cells has been demonstrated in various animal and human transplantation studies. However, the need for increased numbers of appropriate umbilical cord blood-derived cells continues to limit the development and success of these therapies. Ex vivo expansion has been widely studied as a method to overcome this limitation. We describe the use of a clinically relevant single-use, closed-system bioprocess capable of generating greater numbers of hematopoietic stem and progenitor cells that maintain in vivo and in vitro developmental potential. In addition to expanded numbers of CD34+ cells, CD34(+)CD38(-) cells, colony-forming cells, and long-term culture-initiating cells, the bioprocess generated > or =3.3-fold more long-term nonobese diabetic/severe combined immunodeficient repopulating cells (quantitatively determined using limiting dilution analysis) than present at input. Interestingly, these cells were also capable of multilineage engraftment and were shown to maintain their engraftment potency on a per long-term nonobese diabetic/severe combined immunodeficient repopulating cell basis compared with input noncultured cells. The developmental capacity of bioprocess-generated cells was further demonstrated by their ability to repopulate secondary nonobese diabetic/severe combined immunodeficient recipients. In vitro lineage analysis confirmed that bioprocess-generated cells could differentiate into myeloid and natural killer, B, and T cell lymphoid lineages. This in-depth analysis describes a bioprocess that generates human hematopoietic stem and progenitor cells with conserved hematopoietic activity, establishes analysis criteria for in vitro hematopoietic stem cell expansion studies, and serves as a foundation to test the therapeutic utility of cultured hematopoietic stem cells in large animals and humans.
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Affiliation(s)
- Gerard J Madlambayan
- Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Ontario, Canada
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Schuller CE, Jankowski K, Mackenzie KL. Telomere length of cord blood-derived CD34(+) progenitors predicts erythroid proliferative potential. Leukemia 2007; 21:983-91. [PMID: 17344914 DOI: 10.1038/sj.leu.2404631] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/28/2023]
Abstract
Excessive telomere shortening has been demonstrated in inherited and acquired blood disorders, including aplastic anemia and myelodysplastic syndromes. It is possible that replicative exhaustion, owing to critical telomere shortening in hematopoietic progenitor cells (HPCs), contributes to the development of cytopenias in these disorders. However to date, a direct link between the telomere length (TL) of human HPCs and their proliferative potential has not been demonstrated. In the present investigation, the TL and level of telomerase enzyme activity (TA) detected in cord blood (CB)-derived HPCs was found to predict erythroid expansion (P<0.01 and P=0.01 respectively). These results were corroborated by a correlation between proliferation of erythroid cells and telomere loss (P=0.01). In contrast, no correlations were found between initial TL, telomere loss or TA and the expansion of other myeloid lineage-committed cells. There was also no correlation between TL or TA and the number of clonogenic progenitors, including primitive progenitors derived from long-term culture. Our investigations revealed upregulation of telomerase to tumor cell levels in CD34- cells undergoing erythroid differentiation. Together, these results provide new insight into the regulation of TL and TA during myeloid cell expansion and demonstrate that TL is an important determinant of CB-derived erythroid cell proliferation.
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Affiliation(s)
- C E Schuller
- Stem Cell Biology Program, Children's Cancer Institute Australia for Medical Research, Randwick, New South Wales, Australia
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