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Intoh A, Watanabe-Susaki K, Kato T, Kiritani H, Kurisaki A. EPHA2 is a novel cell surface marker of OCT4-positive undifferentiated cells during the differentiation of mouse and human pluripotent stem cells. Stem Cells Transl Med 2024; 13:763-775. [PMID: 38811016 PMCID: PMC11328934 DOI: 10.1093/stcltm/szae036] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/22/2023] [Accepted: 04/14/2024] [Indexed: 05/31/2024] Open
Abstract
Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) possess the intrinsic ability to differentiate into diverse cellular lineages, marking them as potent instruments in regenerative medicine. Nonetheless, the proclivity of these stem cells to generate teratomas post-transplantation presents a formidable obstacle to their therapeutic utility. In previous studies, we identified an array of cell surface proteins specifically expressed in the pluripotent state, as revealed through proteomic analysis. Here we focused on EPHA2, a protein found to be abundantly present on the surface of undifferentiated mouse ESCs and is diminished upon differentiation. Knock-down of Epha2 led to the spontaneous differentiation of mouse ESCs, underscoring a pivotal role of EPHA2 in maintaining an undifferentiated cell state. Further investigations revealed a strong correlation between EPHA2 and OCT4 expression during the differentiation of both mouse and human PSCs. Notably, removing EPHA2+ cells from mouse ESC-derived hepatic lineage reduced tumor formation after transplanting them into immune-deficient mice. Similarly, in human iPSCs, a larger proportion of EPHA2+ cells correlated with higher OCT4 expression, reflecting the pattern observed in mouse ESCs. Conclusively, EPHA2 emerges as a potential marker for selecting undifferentiated stem cells, providing a valuable method to decrease tumorigenesis risks after stem-cell transplantation in regenerative treatments.
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Affiliation(s)
- Atsushi Intoh
- Division of Biological Science, Nara Institute of Science and Technology, Nara, 630-0192, Japan
- Organ Development Research Laboratory, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, 305-8560, Japan
| | - Kanako Watanabe-Susaki
- Organ Development Research Laboratory, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, 305-8560, Japan
| | - Taku Kato
- Division of Biological Science, Nara Institute of Science and Technology, Nara, 630-0192, Japan
| | - Hibiki Kiritani
- Division of Biological Science, Nara Institute of Science and Technology, Nara, 630-0192, Japan
| | - Akira Kurisaki
- Division of Biological Science, Nara Institute of Science and Technology, Nara, 630-0192, Japan
- Organ Development Research Laboratory, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, 305-8560, Japan
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Wang G, Spassieva SD, Bieberich E. Ceramide and S1P Signaling in Embryonic Stem Cell Differentiation. Methods Mol Biol 2018; 1697:153-171. [PMID: 28540559 PMCID: PMC5815858 DOI: 10.1007/7651_2017_43] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022]
Abstract
Bioactive sphingolipids are important regulators for stem cell survival and differentiation. Most recently, we have coined the term "morphogenetic lipids" for sphingolipids that regulate stem cells during embryonic and postnatal development. The sphingolipid ceramide and its derivative, sphingosine-1-phosphate (S1P), can act synergistically as well as antagonistically on embryonic stem (ES) cell differentiation. We show here simple as well as state-of-the-art methods to analyze sphingolipids in differentiating ES cells and discuss new protocols to use ceramide and S1P analogs for the guided differentiation of mouse ES cells toward neuronal and glial lineage.
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Affiliation(s)
- Guanghu Wang
- Department of Neuroscience and Regenerative Medicine, Medical College of Georgia, Augusta University, Augusta, GA, USA
| | - Stefka D Spassieva
- Department of Molecular and Cellular Medicine, Texas A&M Medical Health Sciences Center, Bryan, TX, USA
| | - Erhard Bieberich
- Department of Neuroscience and Regenerative Medicine, Medical College of Georgia, Augusta University, Augusta, GA, USA.
- Department of Neuroscience and Regenerative Medicine, Medical College of Georgia, Augusta University, 1120 15th Street Room CA4012, Augusta, GA, 30912, USA.
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3
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Kang SJ, Park YI, Hwang SR, Yi H, Tham N, Ku HO, Song JY, Kang HG. Hepatic population derived from human pluripotent stem cells is effectively increased by selective removal of undifferentiated stem cells using YM155. Stem Cell Res Ther 2017; 8:78. [PMID: 28412976 PMCID: PMC5392904 DOI: 10.1186/s13287-017-0517-2] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/03/2016] [Revised: 02/09/2017] [Accepted: 02/21/2017] [Indexed: 01/29/2023] Open
Abstract
Background Pluripotent stem cells (PSCs) such as embryonic stem cells and induced pluripotent stem cells are promising target cells for cell regenerative medicine together with recently advanced technology of in-vitro differentiation. However, residual undifferentiated stem cells (USCs) during in-vitro differentiation are considered a potential risk for development of cancer cells and nonspecific lineage cell types. In this study we observed that USCs still exist during hepatic differentiation, consequently resulting in poor quality of the hepatic population and forming teratoma in vivo. Therefore, we hypothesized that effectively removing USCs from in-vitro differentiation could improve the quality of the hepatic population and guarantee safety from risk of teratoma formation. Methods Human PSCs were differentiated to hepatocytes via four steps. YM155, a known BIRC5 inhibitor, was applied for removing the residual USCs on the hepatic differentiation. After YM155 treatment, hepatocyte development was evaluated by measuring gene expression, immunostaining and hepatic functions at each stage of differentiation, and forming teratomas were confirmed by cell transplantation with or without YM155. Results The selected concentrations of YM155 removed USCs (NANOG+ and OCT4+) in a dose-dependent manner. As a result, expression of endodermal markers (SOX17, FOXA2 and CXCR4) at stage II of differentiation and hepatic markers (ALB, AFP and HNF4A) at stage III was up-regulated by YM155 treatment as well as the hepatic population (ALB+), and functions (ALB/urea secretion and CYP450 enzyme activity) were enhanced at the final stage of differentiation (stage IV). Furthermore, we demonstrated that NANOG and OCT4 expression remaining until stage III (day 15 of differentiation) completely disappeared when treated with YM155 and teratoma formation was effectively prevented by YM155 pretreatment in the in-vitro study. Conclusions We suggest that the removal of USCs using YM155 could improve the quantity and quality of induced hepatocytes and eliminate the potential risk of teratoma formation. Electronic supplementary material The online version of this article (doi:10.1186/s13287-017-0517-2) contains supplementary material, which is available to authorized users.
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Affiliation(s)
- Seok-Jin Kang
- Vet Drugs and Biologics Division, Animal and Plant Quarantine Agency, 177, Hyeoksin 8-ro, Gimcheon-si, Gyeongsangbuk-do, 39660, Republic of Korea
| | - Young-Il Park
- Vet Drugs and Biologics Division, Animal and Plant Quarantine Agency, 177, Hyeoksin 8-ro, Gimcheon-si, Gyeongsangbuk-do, 39660, Republic of Korea
| | - So-Ryeon Hwang
- Vet Drugs and Biologics Division, Animal and Plant Quarantine Agency, 177, Hyeoksin 8-ro, Gimcheon-si, Gyeongsangbuk-do, 39660, Republic of Korea
| | - Hee Yi
- Vet Drugs and Biologics Division, Animal and Plant Quarantine Agency, 177, Hyeoksin 8-ro, Gimcheon-si, Gyeongsangbuk-do, 39660, Republic of Korea
| | - Nga Tham
- Vet Drugs and Biologics Division, Animal and Plant Quarantine Agency, 177, Hyeoksin 8-ro, Gimcheon-si, Gyeongsangbuk-do, 39660, Republic of Korea
| | - Hyun-Ok Ku
- Vet Drugs and Biologics Division, Animal and Plant Quarantine Agency, 177, Hyeoksin 8-ro, Gimcheon-si, Gyeongsangbuk-do, 39660, Republic of Korea
| | - Jae-Young Song
- Vet Drugs and Biologics Division, Animal and Plant Quarantine Agency, 177, Hyeoksin 8-ro, Gimcheon-si, Gyeongsangbuk-do, 39660, Republic of Korea
| | - Hwan-Goo Kang
- Vet Drugs and Biologics Division, Animal and Plant Quarantine Agency, 177, Hyeoksin 8-ro, Gimcheon-si, Gyeongsangbuk-do, 39660, Republic of Korea.
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Sciancalepore AG, Portone A, Moffa M, Persano L, De Luca M, Paiano A, Sallustio F, Schena FP, Bucci C, Pisignano D. Micropatterning control of tubular commitment in human adult renal stem cells. Biomaterials 2016; 94:57-69. [PMID: 27105437 DOI: 10.1016/j.biomaterials.2016.03.042] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/27/2016] [Revised: 03/19/2016] [Accepted: 03/28/2016] [Indexed: 12/12/2022]
Abstract
The treatment of renal injury by autologous, patient-specific adult stem cells is still an unmet need. Unsolved issues remain the spatial integration of stem cells into damaged areas of the organ, the commitment in the required cell type and the development of improved bioengineered devices. In this respect, biomaterials and architectures have to be specialized to control stem cell differentiation. Here, we perform an extensive study on micropatterned extracellular matrix proteins, which constitute a simple and non-invasive approach to drive the differentiation of adult renal progenitor/stem cells (ARPCs) from human donors. ARPCs are interfaced with fibronectin (FN) micropatterns, in the absence of exogenous chemicals or cellular reprogramming. We obtain the differentiation towards tubular cells of ARPCs cultured in basal medium conditions, the tubular commitment thus being specifically induced by micropatterned substrates. We characterize the stability of the tubular differentiation as well as the induction of a polarized phenotype in micropatterned ARPCs. Thus, the developed cues, driving the functional commitment of ARPCs, offer a route to recreate the microenvironment of the stem cell niche in vitro, that may serve, in perspective, for the development of ARPC-based bioengineered devices.
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Affiliation(s)
- Anna G Sciancalepore
- Istituto Nanoscienze-CNR, Euromediterranean Center for Nanomaterial Modelling and Technology (ECMT), via Arnesano, 73100 Lecce, Italy; Center for Biomolecular Nanotechnologies @UNILE, Istituto Italiano di Tecnologia, via Barsanti, 73010 Arnesano, Lecce, Italy.
| | - Alberto Portone
- Istituto Nanoscienze-CNR, Euromediterranean Center for Nanomaterial Modelling and Technology (ECMT), via Arnesano, 73100 Lecce, Italy; Dipartimento di Matematica e Fisica "Ennio De Giorgi", Università del Salento, via Arnesano, 73100 Lecce, Italy
| | - Maria Moffa
- Istituto Nanoscienze-CNR, Euromediterranean Center for Nanomaterial Modelling and Technology (ECMT), via Arnesano, 73100 Lecce, Italy; Center for Biomolecular Nanotechnologies @UNILE, Istituto Italiano di Tecnologia, via Barsanti, 73010 Arnesano, Lecce, Italy
| | - Luana Persano
- Istituto Nanoscienze-CNR, Euromediterranean Center for Nanomaterial Modelling and Technology (ECMT), via Arnesano, 73100 Lecce, Italy
| | - Maria De Luca
- Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali (DiSTeBA), Università del Salento, via Provinciale Monteroni, 73100 Lecce, Italy
| | - Aurora Paiano
- Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali (DiSTeBA), Università del Salento, via Provinciale Monteroni, 73100 Lecce, Italy
| | - Fabio Sallustio
- Nephrology, Dialysis and Transplantation Unit, Department of Emergency and Organ Transplantation, University of Bari, 70124 Bari, Italy; Centro Addestramento Ricerca Scientifica in Oncologia (C.A.R.S.O.) Consortium, Strada Prov. le Valenzano-Casamassima, 70010 Valenzano, Italy
| | - Francesco P Schena
- Nephrology, Dialysis and Transplantation Unit, Department of Emergency and Organ Transplantation, University of Bari, 70124 Bari, Italy; Centro Addestramento Ricerca Scientifica in Oncologia (C.A.R.S.O.) Consortium, Strada Prov. le Valenzano-Casamassima, 70010 Valenzano, Italy
| | - Cecilia Bucci
- Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali (DiSTeBA), Università del Salento, via Provinciale Monteroni, 73100 Lecce, Italy
| | - Dario Pisignano
- Istituto Nanoscienze-CNR, Euromediterranean Center for Nanomaterial Modelling and Technology (ECMT), via Arnesano, 73100 Lecce, Italy; Center for Biomolecular Nanotechnologies @UNILE, Istituto Italiano di Tecnologia, via Barsanti, 73010 Arnesano, Lecce, Italy; Dipartimento di Matematica e Fisica "Ennio De Giorgi", Università del Salento, via Arnesano, 73100 Lecce, Italy.
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5
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Tomizawa M, Shinozaki F, Motoyoshi Y, Sugiyama T, Yamamoto S, Ishige N. Hepatocyte selection medium eliminating induced pluripotent stem cells among primary human hepatocytes. World J Methodol 2015; 5:108-114. [PMID: 26413482 PMCID: PMC4572022 DOI: 10.5662/wjm.v5.i3.108] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/10/2015] [Revised: 05/22/2015] [Accepted: 08/30/2015] [Indexed: 02/06/2023] Open
Abstract
Hepatic insufficiency is a fatal liver disease with a significant decrease in functioning hepatocytes. If hepatocytes could be generated from human induced pluripotent stem (hiPS) cells and transplanted into patients with hepatic insufficiency, the disease may become curable. However, a major limitation to this therapeutic strategy is due to the tumorigenicity of hiPS cells and their ability to form cancer. Current methods for eliminating unwanted hiPS cells use genetic manipulation or reagents that are potentially hazardous for hepatocytes; therefore, revised methods are necessary and anticipated. Glucose and arginine are essential cell culture medium ingredients for the survival of most cells, including hiPS cells. However, hepatocytes can produce its own glucose and arginine through galactokinase and ornithine transcarbamylase, respectively. Therefore, it was hypothesized that unwanted hiPS cells could be eliminated in a medium without glucose and arginine, and supplemented with galactose and ornithine instead. This modified medium has been established as hepatocyte selection medium (HSM). So far, attempts to generate a pure colony of mature hepatocytes from hiPS cells have not been successful. After establishment of co-culture in HSM, primary human hepatocytes survive while hiPS cells die within three days. Our latest results regarding a modification of HSM will be introduced in this manuscript.
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Navarro-Tableros V, Herrera Sanchez MB, Figliolini F, Romagnoli R, Tetta C, Camussi G. Recellularization of rat liver scaffolds by human liver stem cells. Tissue Eng Part A 2015; 21:1929-39. [PMID: 25794768 DOI: 10.1089/ten.tea.2014.0573] [Citation(s) in RCA: 52] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
In the present study, rat liver acellular scaffolds were used as biological support to guide the differentiation of human liver stem-like cells (HLSC) to hepatocytes. Once recellularized, the scaffolds were maintained for 21 days in different culture conditions to evaluate hepatocyte differentiation. HLSC lost the embryonic markers (alpha-fetoprotein, nestin, nanog, sox2, Musashi1, Oct 3/4, and pax2), increased the expression of albumin, and acquired the expression of lactate dehydrogenase and three subtypes of cytochrome P450. The presence of urea nitrogen in the culture medium confirmed their metabolic activity. In addition, cells attached to tubular remnant matrix structures expressed cytokeratin 19, CD31, and vimentin. The rat extracellular matrix (ECM) provides not only a favorable environment for differentiation of HLSC in functional hepatocytes (hepatocyte like) but also promoted the generation of some epithelial-like and endothelial-like cells. When fibroblast growth factor-epidermal growth factor or HLSC-derived conditioned medium was added to the perfusate, an improvement of survival rate was observed. The conditioned medium from HLSC potentiated also the metabolic activity of hepatocyte-like cells repopulating the acellular liver. In conclusion, HLSC have the potential, in association with the natural ECM, to generate in vitro a functional "humanized liver-like tissue."
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Affiliation(s)
- Victor Navarro-Tableros
- 1Translational Center for Regenerative Medicine and Molecular Biotechnology Center, University of Torino, Torino, Italy
| | - Maria Beatriz Herrera Sanchez
- 1Translational Center for Regenerative Medicine and Molecular Biotechnology Center, University of Torino, Torino, Italy
| | - Federico Figliolini
- 1Translational Center for Regenerative Medicine and Molecular Biotechnology Center, University of Torino, Torino, Italy
| | - Renato Romagnoli
- 2Liver Transplantation Center, University of Torino, Torino, Italy
| | - Ciro Tetta
- 3EMEA LA Medical Board, Fresenius Medical Care, Bad Homburg, Germany
| | - Giovanni Camussi
- 4Department of Medical Sciences, University of Torino, Torino, Italy
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7
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Zare M, Soleimani M, Mohammadian M, Akbarzadeh A, Havasi P, Zarghami N. Efficient biotechnological approach for lentiviral transduction of induced pluripotent stem cells. ARTIFICIAL CELLS NANOMEDICINE AND BIOTECHNOLOGY 2014; 44:743-8. [PMID: 25420755 DOI: 10.3109/21691401.2014.982804] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/13/2022]
Abstract
Induced pluripotent stem (iPS) cells are generated from differentiated adult somatic cells by reprogramming them. Unlimited self-renewal, and the potential to differentiate into any cell type, make iPS cells very promising candidates for basic and clinical research. Furthermore, iPS cells can be genetically manipulated for use as therapeutic tools. DNA can be introduced into iPS cells, using lentiviral vectors, which represent a helpful choice for efficient transduction and stable integration of transgenes. In this study, we compare two methods of lentiviral transduction of iPS cells, namely, the suspension method and the hanging drop method. In contrast to the conventional suspension method, in the hanging drop method, embryoid body (EB) formation and transduction occur concurrently. The iPS cells were cultured to form EBs, and then transduced with lentiviruses, using the conventional suspension method and the hanging drop method, to express miR-128 and green fluorescent protein (GFP). The number of transduced cells were assessed by fluorescent microscopy and flow cytometry. MTT assay and real-time PCR were performed to determine the cell viability and transgene expression, respectively. Morphologically, GFP+ cells were more detectable in the hanging drop method, and this finding was quantified by flow cytometric analysis. According to the results of the MTT assay, cell viability was considerably higher in the hanging drop method, and real-time PCR represented a higher relative expression of miR-128 in the iPS cells introduced with lentiviruses in drops. Altogether, it seems that lentiviral transduction of challenging iPS cells using the hanging drop method offers a suitable and sufficient strategy in their gene transfer, with less toxicity than the conventional suspension method.
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Affiliation(s)
- Mehrak Zare
- a Department of Clinical Biochemistry and Laboratory , Faculty of Medicine, Tabriz University of Medical Sciences , Tabriz , Iran.,b Department of Cellular Biology , Stem Cell Technology Research Center , Tehran , Iran.,c Neurosciences Research Center, Tabriz University of Medical Sciences , Tabriz , Iran
| | - Masoud Soleimani
- d Department of Haematology , School of Medicine, Tarbiat Modares University , Tehran , Iran
| | - Mozhdeh Mohammadian
- e Amol Faculty of Paramedical Sciences, Mazandaran University of Medical Sciences , Sari , Iran
| | - Abolfazl Akbarzadeh
- f Department of Medical Nanotechnology , Faculty of Advanced Medical Science, Tabriz University of Medical Sciences , Tabriz , Iran
| | - Parvaneh Havasi
- b Department of Cellular Biology , Stem Cell Technology Research Center , Tehran , Iran
| | - Nosratollah Zarghami
- a Department of Clinical Biochemistry and Laboratory , Faculty of Medicine, Tabriz University of Medical Sciences , Tabriz , Iran.,c Neurosciences Research Center, Tabriz University of Medical Sciences , Tabriz , Iran
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8
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Abstract
Radiation injury to skin results in a variety of deterministic effects including inflammatory reactions and cell depletion leading to distinct clinical symptoms following a defined time pattern. Therapeutic approaches are still limited, a complete restitution of affected areas is so far impossible. In the last few years increasing experimental knowledge about acquisition and administration of autologous stem cells also in the field of radiation injuries has been obtained. Evidence reviewed in this article shows that the beneficial effects of stem cell transplantation are not necessarily due to the replacement of damaged cells by transplanted cells but most probably due in the most part to a paracrine effect. Transplanted cells secrete bioactive factors that initiate the stimulation of the host stem cells to regenerate the damaged tissues. Transplanted stem cells produce trophic factors which aid the systemic healing of the victims. Furthermore, administration of stem cell secretomes in the form of conditioned media containing microvesicles or exosomes can be as effective as administering the stem cells. This hypothesis is supported by findings that cell-free derivatives from hMSCs were useful for wound healing purposes and could circumvent the need for intact cells. Furthermore, the beneficial effect of MSC injection on reperfusion and tissue damage in a mouse model of hind limb ischemia could be attributed to paracrine mechanisms with local release of arteriogenic cytokines. Further evaluation of the paracrine potential of autologous stem cells may open new means for treatment of acute as well as chronic sequelae of cutaneous radiation injuries.
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Affiliation(s)
- M Rezvani
- Natural Biosciences SA, Lake Garden Medical Center, Kilchberg, Zurich, Switzerland,
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Kim HJ, Alam Z, Hwang JW, Hwang YH, Kim MJ, Yoon S, Byun Y, Lee DY. Optimal formation of genetically modified and functional pancreatic islet spheroids by using hanging-drop strategy. Transplant Proc 2013; 45:605-10. [PMID: 23498797 DOI: 10.1016/j.transproceed.2012.11.014] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/28/2012] [Revised: 05/26/2012] [Accepted: 11/11/2012] [Indexed: 11/17/2022]
Abstract
BACKGROUND Rejection and hypoxia are important factors causing islet loss at an early stage after pancreatic islet transplantation. Recently, islets have been dissociated into single cells for reaggregation into so-called islet spheroids. Herein, we used a hanging-drop strategy to form islet spheroids to achieve functional equivalence to intact islets. METHODS To obtain single islet cells, we dissociated islets with trypsin-EDTA digestion for 10 minutes. To obtain spheroids, we dropped various numbers of single cells (125, 250, or 500 cells/30 μL drop) onto a Petri dish, that was inverted for incubation in humidified air containing 5% CO(2) at 37 °C for 7 days. The aggregated spheroids in the droplets were harvested for further culture. RESULTS The size of the aggregated islet spheroids depended on the number of single cells (125-500 cells/30 μL droplet). Their morphology was similar to that of intact islets without any cellular damage. When treated with various concentrations of glucose to evaluate responsiveness, their glucose-mediated stimulation index value was similar to that of intact islets, an observation that was attributed to strong cell-to-cell interactions in islet spheroids. However, islet spheroids aggregated in general culture dishes showed abnormal glucose responsiveness owing to weak cell-to-cell interactions. Cell-to-cell interactions in islet spheroids were confirmed with an anti-connexin-36 monoclonal antibody. Finally, nonviral poly(ethylene imine)-mediated interleukin-10 cytokine gene delivered beforehand into dissociated single cells before formation of islet spheroids increased the gene transfection efficacy and interleukin-10 secretion from islet spheroids >4-fold compared with intact islets. CONCLUSION These results demonstrated the potential application of genetically modified, functional islet spheroids with of controlled size and morphology using an hanging-drop technique.
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Affiliation(s)
- H J Kim
- Department of Bioengineering, College of Engineering, and Institute for Bioengineering and Biopharmaceutical Research, Hanyang University, Seoul, Republic of Korea
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Pagni G, Kaigler D, Rasperini G, Avila-Ortiz G, Bartel R, Giannobile W. Bone repair cells for craniofacial regeneration. Adv Drug Deliv Rev 2012; 64:1310-9. [PMID: 22433781 DOI: 10.1016/j.addr.2012.03.005] [Citation(s) in RCA: 56] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/17/2011] [Revised: 03/02/2012] [Accepted: 03/05/2012] [Indexed: 12/17/2022]
Abstract
Reconstruction of complex craniofacial deformities is a clinical challenge in situations of injury, congenital defects or disease. The use of cell-based therapies represents one of the most advanced methods for enhancing the regenerative response for craniofacial wound healing. Both somatic and stem cells have been adopted in the treatment of complex osseous defects and advances have been made in finding the most adequate scaffold for the delivery of cell therapies in human regenerative medicine. As an example of such approaches for clinical application for craniofacial regeneration, Ixmyelocel-T or bone repair cells are a source of bone marrow derived stem and progenitor cells. They are produced through the use of single pass perfusion bioreactors for CD90+ mesenchymal stem cells and CD14+ monocyte/macrophage progenitor cells. The application of ixmyelocel-T has shown potential in the regeneration of muscular, vascular, nervous and osseous tissue. The purpose of this manuscript is to highlight cell therapies used to repair bony and soft tissue defects in the oral and craniofacial complex. The field at this point remains at an early stage, however this review will provide insights into the progress being made using cell therapies for eventual development into clinical practice.
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Abstract
Recent studies show that bioactive lipids are important regulators for stem cell survival and differentiation. The sphingolipid ceramide and its derivative, sphingosine-1-phosphate (S1P), can act synergistically on embryonic stem (ES) cell differentiation. We show here simple methods to analyze sphingolipids in differentiating ES cells and to use ceramide and S1P analogs for the guided differentiation of mouse ES cells toward neuronal and glial lineage.
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Affiliation(s)
- Erhard Bieberich
- Program in Developmental Neurobiology, Institute of Molecular Medicine and Genetics, Medical College of Georgia, Georgia Health Sciences University, Augusta, GA, USA.
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12
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Ward HH, Romero E, Welford A, Pickett G, Bacallao R, Gattone VH, Ness SA, Wandinger-Ness A, Roitbak T. Adult human CD133/1(+) kidney cells isolated from papilla integrate into developing kidney tubules. BIOCHIMICA ET BIOPHYSICA ACTA 2011; 1812:1344-57. [PMID: 21255643 PMCID: PMC3166446 DOI: 10.1016/j.bbadis.2011.01.010] [Citation(s) in RCA: 55] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Subscribe] [Scholar Register] [Received: 09/02/2010] [Revised: 12/09/2010] [Accepted: 01/11/2011] [Indexed: 01/19/2023]
Abstract
Approximately 60,000 patients in the United States are waiting for a kidney transplant due to genetic, immunologic and environmentally caused kidney failure. Adult human renal stem cells could offer opportunities for autologous transplant and repair of damaged organs. Current data suggest that there are multiple progenitor types in the kidney with distinct localizations. In the present study, we characterize cells derived from human kidney papilla and show their capacity for tubulogenesis. In situ, nestin(+) and CD133/1(+) cells were found extensively intercalated between tubular epithelia in the loops of Henle of renal papilla, but not of the cortex. Populations of primary cells from the renal cortex and renal papilla were isolated by enzymatic digestion from human kidneys unsuited for transplant and immuno-enriched for CD133/1(+) cells. Isolated CD133/1(+) papillary cells were positive for nestin, as well as several human embryonic stem cell markers (SSEA4, Nanog, SOX2, and OCT4/POU5F1) and could be triggered to adopt tubular epithelial and neuronal-like phenotypes. Isolated papillary cells exhibited morphologic plasticity upon modulation of culture conditions and inhibition of asymmetric cell division. Labeled papillary cells readily associated with cortical tubular epithelia in co-culture and 3-dimensional collagen gel cultures. Heterologous organ culture demonstrated that CD133/1(+) progenitors from the papilla and cortex became integrated into developing kidney tubules. Tubular epithelia did not participate in tubulogenesis. Human renal papilla harbor cells with the hallmarks of adult kidney stem/progenitor cells that can be amplified and phenotypically modulated in culture while retaining the capacity to form new kidney tubules. This article is part of a Special Issue entitled: Polycystic Kidney Disease.
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Affiliation(s)
- Heather H. Ward
- Department of Pathology, University of New Mexico Health Sciences Center, Albuquerque, NM 87131, USA
- Department of Medicine and Division of Nephrology, Indiana University School of Medicine, Indianapolis, IN 46202, USA
| | - Elsa Romero
- Department of Pathology, University of New Mexico Health Sciences Center, Albuquerque, NM 87131, USA
| | - Angela Welford
- Department of Pathology, University of New Mexico Health Sciences Center, Albuquerque, NM 87131, USA
| | - Gavin Pickett
- Cancer Center, University of New Mexico Health Sciences Center, Albuquerque, NM 87131, USA
| | - Robert Bacallao
- Department of Medicine and Division of Nephrology, Indiana University School of Medicine, Indianapolis, IN 46202, USA
| | - Vincent H. Gattone
- Department of Anatomy and Cell Biology, Indiana University School of Medicine, Indianapolis, IN 46202, USA
| | - Scott A. Ness
- Cancer Center, University of New Mexico Health Sciences Center, Albuquerque, NM 87131, USA
- Department of Molecular Genetics and Microbiology, University of New Mexico Health Sciences Center, Albuquerque, NM 87131, USA
| | - Angela Wandinger-Ness
- Department of Pathology, University of New Mexico Health Sciences Center, Albuquerque, NM 87131, USA
- Cancer Center, University of New Mexico Health Sciences Center, Albuquerque, NM 87131, USA
| | - Tamara Roitbak
- Department of Neurosurgery, University of New Mexico Health Sciences Center, Albuquerque, NM 87131, USA
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13
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Bieberich E. There is more to a lipid than just being a fat: sphingolipid-guided differentiation of oligodendroglial lineage from embryonic stem cells. Neurochem Res 2011; 36:1601-11. [PMID: 21136155 PMCID: PMC3717256 DOI: 10.1007/s11064-010-0338-5] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 11/19/2010] [Indexed: 12/16/2022]
Abstract
Dr. Robert K. Yu's research showed for the first time that the composition of glycosphingolipids is tightly regulated during embryo development. Studies in our group showed that the glycosphingolipid precursor ceramide is also critical for stem cell differentiation and apoptosis. Our new studies suggest that ceramide and its derivative, sphingosine-1-phosphate (S1P), act synergistically on embryonic stem (ES) cell differentiation. When using neural precursor cells (NPCs) derived from ES cells for transplantation, residual pluripotent stem (rPS) cells pose a significant risk of tumor formation after stem cell transplantation. We show here that rPS cells did not express the S1P receptor S1P1, which left them vulnerable to ceramide or ceramide analog (N-oleoyl serinol or S18)-induced apoptosis. In contrast, ES cell-derived NPCs expressed S1P1 and were protected in the presence of S1P or its pro-drug analog FTY720. Consistent with previous studies, FTY720-treated NPCs differentiated predominantly toward oligodendroglial lineage as tested by the expression of the oligodendrocyte precursor cell (OPC) markers Olig2 and O4. As the consequence, a combined administration of S18 and FTY720 to differentiating ES cells eliminated rPS cells and promoted oligodendroglial differentiation. In addition, we show that this combination promoted differentiation of ES cell-derived NPCs toward oligodendroglial lineage in vivo after transplantation into mouse brain.
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Affiliation(s)
- Erhard Bieberich
- Program in Developmental Neurobiology, Institute of Molecular Medicine and Genetics, School of Medicine, Medical College of Georgia/Georgia Health Sciences University, 1120 15th Street Room CA4012, Augusta, GA 30912, USA.
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14
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Fu W, Wang SJ, Zhou GD, Liu W, Cao Y, Zhang WJ. Residual undifferentiated cells during differentiation of induced pluripotent stem cells in vitro and in vivo. Stem Cells Dev 2011; 21:521-9. [PMID: 21631153 DOI: 10.1089/scd.2011.0131] [Citation(s) in RCA: 35] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/12/2023] Open
Abstract
Induced pluripotent stem (iPS) cells are a potential cell source for regenerative medicine. However, the tumorigenicity of iPS cells is a big concern for clinical application. In addition to the genetic manipulation of the reprogramming process and the greater risk of tumor formation, it is unclear whether iPS cells with normal development potential are still tumorigenic. Here, we investigated 3 mouse iPS cell lines, including one line that is able to generate full-term mice via tetraploid blastocyst complementation. We found that a small number of undifferentiated iPS cells could be steadily isolated and expanded after long-term differentiation of cells in vitro or in vivo. The residual undifferentiated iPS cells could be expanded and redifferentiated, and undifferentiated pluripotent stem cells could again be isolated after further rounds of differentiation, suggesting that residual undifferentiated iPS cells could not be eliminated by extended cell differentiation. The residual undifferentiated cells could form teratomas in vivo, indicating that they are a potential tumorigenic risk during transplantation. These findings prompt us to reconsider the strategies for solving the tumorigenic problem of iPS cells, not only focusing on improving the reprogramming process.
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Affiliation(s)
- Wei Fu
- Shanghai Key Laboratory of Tissue Engineering, Department of Plastic and Reconstructive Surgery, National Tissue Engineering Center of China, Shanghai Jiao Tong University School of Medicine, Shanghai 9th People's Hospital, Shanghai, China
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15
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Kumar A, Pati NT, Sarin SK. Use of stem cells for liver diseases-current scenario. J Clin Exp Hepatol 2011; 1:17-26. [PMID: 25755306 PMCID: PMC3940313 DOI: 10.1016/s0973-6883(11)60114-x] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/01/2011] [Accepted: 06/04/2011] [Indexed: 12/12/2022] Open
Abstract
End-stage liver disease and liver failure are major health problems worldwide leading to high mortality and morbidity and high healthcare costs. Currently, orthotropic liver transplantation is the only effective treatment available to the patients of end-stage liver disease. However, a serious shortage of liver donors, high cost, and risk of organ rejection are the major obstacles to liver transplantation. Because of the ability of stem cells for differentiation into any tissue type, they have huge potential in therapy of various end-stage or degenerative diseases and traumatic injuries. Stem cell therapy has the potential to provide a valuable adjunct and alternative to liver transplantation and has immense potential in the management of end stage liver disease and liver failure. Stem cell therapy can be mediated by either a direct contribution to the functional hepatocyte population with embryonic, induced pluripotent, or adult stem cells or by promotion of endogenous regenerative processes with bone marrow-derived stem cells. Initial translational studies have been encouraging and have suggested improved liver function in advanced chronic liver disease and enhanced liver regeneration after portal vein embolization and partial hepatic resection. Stem cells infusion in cirrhotic patients has improved liver parameters and could form a viable bridge to transplantation. The present review summarizes basic of stem cell biology relevant to clinicians and an update on recent advances on the management of liver diseases using stem cells.
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Key Words
- AFP, alpha (α)-fetoprotein
- BM, bone marrow
- EPCAM, epithelial cell adhesion molecule
- ES, embryonic stem
- FSCs, fetal stem cells
- HPC, hepatic progenitor cells
- HSC, hematopoietic stem cells
- Hepatocyte transplantation
- ICAM, intercellular adhesion molecule
- MSCs, mesenchymal stem cells
- NCAM, neural cell adhesion molecule
- UCB, umbilical cord blood
- hAECs, human amniotic epithelial cells
- iPSCs, induced pluripotent stem cells
- liver transplantation
- stem cell
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Affiliation(s)
- Ashish Kumar
- Department of Hepatology, Institute of Liver and Biliary Sciences (ILBS), New Delhi, India
- Special Center for Molecular Medicine, Jawaharlal Nehru University, New Delhi, India
- Address for correspondence: Dr Ashish Kumar MD DM, Associate Professor, Department of Hepatology, Institute of Liver and Biliary Sciences (ILBS), D-1, Vasant Kunj, New Delhi-110070, India
| | - Nirupama Trehan Pati
- Department of Research, Institute of Liver and Biliary Sciences (ILBS), New Delhi, India
| | - Shiv Kumar Sarin
- Department of Hepatology, Institute of Liver and Biliary Sciences (ILBS), New Delhi, India
- Special Center for Molecular Medicine, Jawaharlal Nehru University, New Delhi, India
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16
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Stutchfield BM, Forbes SJ, Wigmore SJ. Prospects for stem cell transplantation in the treatment of hepatic disease. Liver Transpl 2010; 16:827-36. [PMID: 20583084 DOI: 10.1002/lt.22083] [Citation(s) in RCA: 59] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
Stem cell therapy has the potential to provide a valuable adjunct to the management of hepatic disease. Preclinical studies have demonstrated a range of endogenous repair processes that can be exploited through stem cell therapy. Initial translational studies have been encouraging and have suggested improved liver function in advanced chronic liver disease and enhanced liver regeneration after portal vein embolization. This article reviews the potential for stem cell therapies to enhance hepatic regeneration in acute and chronic hepatic disease and is based on a MEDLINE and PubMed search for English language articles investigating mechanisms of hepatic regeneration and delivery of cell therapies. Two main mechanisms of potential stem cell therapy delivery have emerged: (1) a direct contribution to the functional hepatocyte population with embryonic, induced pluripotent, or adult stem cells and (2) the promotion of endogenous regenerative processes with bone marrow-derived stem cells. Bioartificial hepatic support systems may be proven to be an effective method of using ex vivo differentiated hepatocytes and be indicated as a bridging therapy to definitive surgery in acute liver failure. The administration of bone marrow-derived stem cells may enhance liver regeneration in chronic liver disease after portal vein embolization and could facilitate regeneration after partial hepatic resection. Ultimately, the most appropriate hepatic disease targets for stem cell therapies will become apparent as mechanisms of stem involvement in hepatic regeneration are further elucidated.
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17
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Zhou M, Li P, Tan L, Qu S, Ying QL, Song H. Differentiation of mouse embryonic stem cells into hepatocytes induced by a combination of cytokines and sodium butyrate. J Cell Biochem 2010; 109:606-14. [PMID: 20039312 DOI: 10.1002/jcb.22442] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/08/2023]
Abstract
There is increasing evidence to suggest that embryonic stem cells (ESCs) are capable of differentiating into hepatocytes in vitro. In this study, we used a combination of cytokines and sodium butyrate in a novel three-step procedure to efficiently direct the differentiation of mouse ESCs into hepatocytes. Mouse ESCs were first differentiated into definitive endoderm cells by 3 days of treatment with Activin A. The definitive endoderm cells were then differentiated into hepatocytes by the addition of acidic fibroblast growth factor (aFGF) and sodium butyrate to the culture medium for 5 days. After 10 days of further in vitro maturation, the morphological and phenotypic markers of hepatocytes were characterized using immunohistochemistry, immunoblotting, and reverse transcription-polymerase chain reaction (RT-PCR). Furthermore, the cells were tested for functions associated with mature hepatocytes, including glycogen storage and indocyanine green uptake and release, and the ratio of hepatic differentiation was determined by counting the percentage of albumin-positive cells. In the presence of medium containing cytokines and sodium butyrate, numerous epithelial cells resembling hepatocytes were observed, and approximately 74% of the cells expressed the hepatic marker, albumin, after 18 days in culture. RT-PCR analysis and immunohistochemistry showed that these cells expressed adult liver cell markers, and had the abilities of glycogen storage and indocyanine green uptake and release. We have developed an efficient method for directing the differentiation of mouse ESCs into cells that exhibit the characteristics of mature hepatocytes. This technique will be useful for research into the molecular mechanisms underlying liver development, and could provide a source of hepatocytes for transplantation therapy and drug screening.
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Affiliation(s)
- Mingming Zhou
- Key Laboratory of Molecular Medicine, Ministry of Education, Fudan University, Shanghai 200032, China
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18
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Meyburg J, Hoffmann GF. Liver, liver cell and stem cell transplantation for the treatment of urea cycle defects. Mol Genet Metab 2010; 100 Suppl 1:S77-83. [PMID: 20156696 DOI: 10.1016/j.ymgme.2010.01.011] [Citation(s) in RCA: 43] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/07/2009] [Revised: 01/13/2010] [Accepted: 01/13/2010] [Indexed: 12/23/2022]
Abstract
Despite advances in pharmacological therapy of urea cycle disorders (UCDs), the overall long-term prognosis is poor, especially for neonatal manifestations. Transplantation of liver tissue or isolated cells appears suitable for transfer of the missing enzyme. Liver transplantation (LT) for UCDs has an excellent 5-year survival rate of approximately 90% and is the only way to completely cure the disease. However, major neurological damage can only be prevented if the operation is performed during the first months of life. Unfortunately, such early LTs have a substantial risk for peri- and postoperative complications, mostly caused by a relatively large liver graft. Liver cell transplantation (LCT) is less invasive than LT, but has still to be regarded as an experimental therapy with about 100 patients treated since its first use in 1993. UCDs are a model disease for LCT, because of the poor prognosis, mainly hepatic enzyme defects, and excellent outcome after LT. So far, 10 children underwent LCT for UCDs with very few technical complications and encouraging clinical results. A first prospective study on its use in severe neonatal UCDs has recently started. However, availability of hepatocytes is limited by the scarcity of donor livers; therefore the use of stem cells is under investigation. Several different cell types may be regarded as liver stem cells, and in vivo transformation into hepatocyte-like cells has been shown in animal studies. However, a clear proof of principle in animal models of human metabolic disease is still missing, which is the prerequisite for clinical application in humans.
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Affiliation(s)
- Jochen Meyburg
- Department of General Pediatrics, University Children's Hospital, Heidelberg, Germany.
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Ohta Y, Takenaga M, Tokura Y, Hamaguchi A, Matsumoto T, Kano K, Mugishima H, Okano H, Igarashi R. Mature adipocyte-derived cells, dedifferentiated fat cells (DFAT), promoted functional recovery from spinal cord injury-induced motor dysfunction in rats. Cell Transplant 2009; 17:877-86. [PMID: 19069631 DOI: 10.3727/096368908786576516] [Citation(s) in RCA: 64] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022] Open
Abstract
Transplantation of mature adipocyte-derived cells (dedifferentiated fat cells) led to marked functional recovery from spinal cord injury (SCI)-induced motor dysfunction in rats. When mature adipocytes were isolated from rat adipose tissue and grown in ceiling culture, transformation into fibroblast-like cells without lipid droplets occurred. These fibroblast-like cells, termed dedifferentiated fat cells (DFAT), could proliferate and could also differentiate back into adipocytes. DFAT expressed neural markers such as nestin, betaIII tubulin, and GFAP. Allografting of DFAT into SCI-induced rats led to significant recovery from hindlimb dysfunction. Grafted cells were detected at the injection site, and some of these cells expressed betaIII tubulin. DFAT expressed neurotrophic factors such as BDNF and GDNF prior to transplantation, and grafted cells were also positive for these factors. Therefore, these neurotrophic factors derived from grafted DFAT might have contributed to the promotion of functional recovery. These findings also suggest that mature adipocytes could become a new source for cell replacement therapy to treat central nervous system disorders.
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Affiliation(s)
- Yuki Ohta
- Institute of Medical Science, St. Marianna University School of Medicine, Kawasaki 216-8512, Japan.
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Matsuda R, Yoshikawa M, Kimura H, Ouji Y, Nakase H, Nishimura F, Nonaka JI, Toriumi H, Yamada S, Nishiofuku M, Moriya K, Ishizaka S, Nakamura M, Sakaki T. Cotransplantation of Mouse Embryonic Stem Cells and Bone Marrow Stromal Cells following Spinal Cord Injury Suppresses Tumor Development. Cell Transplant 2009; 18:39-54. [DOI: 10.3727/096368909788237122] [Citation(s) in RCA: 43] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/24/2022] Open
Abstract
Embryonic stem (ES) cells are a potential source for treatment of spinal cord injury (SCI). Although one of the main problems of ES cell-based cell therapy is tumor formation, there is no ideal method to suppress tumor development. In this study, we examined whether transplantation with bone marrow stromal cells (BMSCs) prevented tumor formation in SCI model mice that received ES cell-derived grafts containing both undifferentiated ES cells and neural stem cells. Embryoid bodies (EBs) formed in 4-day hanging drop cultures were treated with retinoic acid (RA) at a low concentration of 5 × 10–9 M for 4 days, in order to allow some of the ES cells to remain in an undifferentiated state. RA-treated EBs were enzymatically digested into single cells and used as ES cell-derived graft cells. Mice transplanted with ES cell-derived graft cells alone developed tumors at the grafted site and behavioral improvement ceased after day 21. In contrast, no tumor development was observed in mice cotransplanted with BMSCs, which also showed sustained behavioral improvement. In vitro results demonstrated the disappearance of SSEA-1 expression in cytochemical examinations, as well as attenuated mRNA expressions of the undifferentiated markers Oct3/4, Utf1, Nanog, Sox2, and ERas by RT-PCR in RA-treated EBs cocultured with BMSCs. In addition, MAP2-immunopositive cells appeared in the EBs cocultured with BMSCs. Furthermore, the synthesis of NGF, GDNF, and BDNF was confirmed in cultured BMSCs, while immunohistochemical examinations demonstrated the survival of BMSCs and their maintained ability of neurotrophic factor production at the grafted site for up to 5 weeks after transplantation. These results suggest that BMSCs induce undifferentiated ES cells to differentiate into a neuronal lineage by neurotrophic factor production, resulting in suppression of tumor formation. Cotransplantation of BMSCs with ES cell-derived graft cells may be useful for preventing the development of ES cell-derived tumors.
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Affiliation(s)
- Ryosuke Matsuda
- Department of Neurosurgery, Nara Medical University, Nara 634–8521, Japan
| | - Masahide Yoshikawa
- Department of Parasitology, Nara Medical University, Nara 634–8521, Japan
| | - Hajime Kimura
- Department of Neurosurgery, Nara Medical University, Nara 634–8521, Japan
| | - Yukiteru Ouji
- Department of Parasitology, Nara Medical University, Nara 634–8521, Japan
| | - Hiroyuki Nakase
- Department of Neurosurgery, Nara Medical University, Nara 634–8521, Japan
| | - Fumihiko Nishimura
- Department of Neurosurgery, Nara Medical University, Nara 634–8521, Japan
| | - Jun-Ichi Nonaka
- Department of Neurosurgery, Nara Medical University, Nara 634–8521, Japan
| | - Hayato Toriumi
- Department of Neurosurgery, Nara Medical University, Nara 634–8521, Japan
| | - Shuichi Yamada
- Department of Neurosurgery, Nara Medical University, Nara 634–8521, Japan
| | - Mariko Nishiofuku
- Department of Parasitology, Nara Medical University, Nara 634–8521, Japan
| | - Kei Moriya
- Department of Parasitology, Nara Medical University, Nara 634–8521, Japan
| | - Shigeaki Ishizaka
- Department of Parasitology, Nara Medical University, Nara 634–8521, Japan
| | | | - Toshisuke Sakaki
- Department of Neurosurgery, Nara Medical University, Nara 634–8521, Japan
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21
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Cananzi M, Atala A, De Coppi P. Stem cells derived from amniotic fluid: new potentials in regenerative medicine. Reprod Biomed Online 2009; 18 Suppl 1:17-27. [DOI: 10.1016/s1472-6483(10)60111-3] [Citation(s) in RCA: 103] [Impact Index Per Article: 6.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
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22
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Tomizawa M, Toyama Y, Ito C, Toshimori K, Iwase K, Takiguchi M, Saisho H, Yokosuka O. Hepatoblast-like cells enriched from mouse embryonic stem cells in medium without glucose, pyruvate, arginine, and tyrosine. Cell Tissue Res 2008; 333:17-27. [PMID: 18478268 DOI: 10.1007/s00441-008-0618-4] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/15/2007] [Accepted: 03/31/2008] [Indexed: 12/15/2022]
Abstract
In order to enrich hepatocytes differentiated from embryonic stem cells, we developed a novel medium. Since only hepatocytes have the activity of ornithine transcarbamylase, phenylalanine hydroxylase, galactokinase, and glycerol kinase, we expected that hepatocytes would be enriched in a medium without arginine, tyrosine, glucose, and pyruvate, but supplemented with ornithine, phenylanaline, galactose, and glycerol (hepatocyte-selection medium, HSM). Embryoid bodies were transferred onto dishes coated with gelatin in HSM after 4 days of culture. At 18 days after embryoid body formation, a single type of polygonal cell survived with an enlarged intercellular space and micorvilli. These cells were positive for indocyanine green uptake and for mRNAs of albumin, transthyretin, and alpha-feto protein, but negative for mRNAs of tyrosine aminotransferase, alpha1-antitrypsin, glucose-6-phosphatase, and phosphoenol pyruvate carboxykinase. Since cells in HSM were positive for cytokeratin (CK)8 and CK18 (hepatocyte markers) and for CK19 (a marker of bile duct epithelial cells), we concluded that they were hepatoblasts. They showed weaker expression of CCAAT/enhancer-binding protein (C/EBP)alpha than fetal liver (18.5 days of gestation) and expression of C/EBPbeta at a similar level to that of fetal liver. These data support our conclusion that HSM allows the selection of hepatoblast-like cells.
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Affiliation(s)
- Minoru Tomizawa
- Department of Medicine and Clinical Oncology, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba City, Chiba, 260-8670, Japan.
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23
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Fujimori H, Asahina K, Shimizu-Saito K, Ikeda R, Tanaka Y, Teramoto K, Morita I, Teraoka H. Vascular endothelial growth factor promotes proliferation and function of hepatocyte-like cells in embryoid bodies formed from mouse embryonic stem cells. J Hepatol 2008; 48:962-73. [PMID: 18384904 DOI: 10.1016/j.jhep.2008.01.031] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/29/2007] [Revised: 01/09/2008] [Accepted: 01/15/2008] [Indexed: 12/04/2022]
Abstract
BACKGROUND/AIMS Embryoid bodies (EBs) formed from embryonic stem cells (ESCs) differentiate into hepatocyte-like cells (HLCs), and are thus thought to be a useful cell source for drug testing and bioartificial liver. The aim of this study was to induce proliferation and function of ESC-derived HLCs in EBs using HLC-endothelial cell interaction. METHODS EBs were cultured in the presence of vascular endothelial growth factor (VEGF) and/or VEGF receptor (VEGFR) inhibitors. To reproduce HLC-endothelial cell interaction, we overexpressed VEGF in ESC-derived HLCs under the control of Cyp7a1 gene in EBs. RESULTS VEGF added to the cultured EBs increased the proliferation of ESC-derived endothelial cells, resulting in the promotion of proliferation and function of ESC-derived HLCs. In EBs, the VEGFR2 inhibitor suppressed expression of albumin and endothelial cell marker genes, whereas the inhibitor for both VEGFR1 and VEGFR2 suppressed expression of Cyp7a1 and hepatocyte growth factor (Hgf) genes. Upon exposure to VEGF, the endothelial cells in EBs increased Hgf mRNA expression. Forced VEGF expression in ESC-derived HLCs in EBs induced angiogenesis around the HLCs and resulted in an increase in the amount of HLCs. CONCLUSIONS VEGF indirectly induces the proliferation and function of ESC-derived HLCs through VEGFR1 and VEGFR2 signaling in endothelial cells.
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Affiliation(s)
- Hiroaki Fujimori
- Department of Pathological Biochemistry, Medical Research Institute, Tokyo Medical and Dental University, 2-3-10 Kandasurugadai, Chiyoda-ku, Tokyo 101-0062, Japan
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24
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Kalka C, Baumgartner I. Gene and stem cell therapy in peripheral arterial occlusive disease. Vasc Med 2008; 13:157-72. [DOI: 10.1177/1358863x08088616] [Citation(s) in RCA: 32] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/15/2022]
Abstract
Abstract Peripheral arterial occlusive disease (PAOD) is a manifestation of systemic atherosclerosis strongly associated with a high risk of cardiovascular morbidity and mortality. In a considerable proportion of patients with PAOD, revascularization either by endovascular means or by open surgery combined with best possible risk factor modification does not achieve limb salvage or relief of ischaemic rest pain. As a consequence, novel therapeutic strategies have been developed over the last two decades aiming to promote neovascularization and remodelling of collaterals. Gene and stem cell therapy are the main directions for clinical investigation concepts. For both, preclinical studies have shown promising results using a wide variety of genes encoding for growth factors and populations of adult stem cells, respectively. As a consequence, clinical trials have been performed applying gene and stem cell-based concepts. However, it has become apparent that a straightforward translation into humans is not possible. While several trials reported relief of symptoms and functional improvement, other trials did not confirm this early promise of efficacy. Ongoing clinical trials with an improved study design are needed to confirm the potential that gene and cell therapy may have and to prevent the gaps in our scientific knowledge that will jeopardize the establishment of angiogenic therapy as an additional medical treatment of PAOD. This review summarizes the experimental background and presents the current status of clinical applications and future perspectives of the therapeutic use of gene and cell therapy strategies for PAOD.
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Affiliation(s)
- C Kalka
- Division of Vascular Medicine, Swiss Cardiovascular Center, University Hospital of Bern, Switzerland
| | - Iris Baumgartner
- Division of Vascular Medicine, Swiss Cardiovascular Center, University Hospital of Bern, Switzerland
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25
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Sarić T, Frenzel LP, Hescheler J. Immunological barriers to embryonic stem cell-derived therapies. Cells Tissues Organs 2008; 188:78-90. [PMID: 18303241 DOI: 10.1159/000118784] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022] Open
Abstract
Replacement of diseased tissues with healthy cells derived from embryonic stem (ES) cells has a potential to become, in the future, a better alternative to current treatments of a number of conditions characterized by irreversible tissue injury, such as heart and liver failure, diabetes mellitus and neurodegeneration. However, several obstacles have to be overcome before this new therapeutic modality becomes part of a standard clinical practice. First of all, ethical and safety issues have to be resolved, the methodologies must be developed to enable obtaining sufficient amounts of differentiated cells, and the immune rejection of allogeneic cells must be prevented in order to ensure their long-term engraftment and function. Data on immunological properties of human and murine ES cells and their differentiated derivatives are controversial, ranging from those claiming unique immune-privileged properties for ES cells to those which refute these conclusions. This indicates that much more research is required to definitively understand the immunological features and engraftment capacity of ES cell derivatives. We review here the current state of the art in this new and exciting field of ES cell immunology and discuss the implications of these findings for the development of ES cell-based therapies.
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Affiliation(s)
- Tomo Sarić
- Center for Physiology, Medical Faculty of the University of Cologne, Cologne, Germany.
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26
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Bieberich E. Smart drugs for smarter stem cells: making SENSe (sphingolipid-enhanced neural stem cells) of ceramide. Neurosignals 2008; 16:124-39. [PMID: 18253053 DOI: 10.1159/000111558] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
Ceramide and its derivative sphingosine-1-phosphate (S1P) are important signaling sphingolipids for neural stem cell apoptosis and differentiation. Most recently, our group has shown that novel ceramide analogs can be used to eliminate teratoma (stem cell tumor)-forming cells from a neural stem cell graft. In new studies, we found that S1P promotes survival of specific neural precursor cells that undergo differentiation to cells expressing oligodendroglial markers. Our studies suggest that a combination of novel ceramide and S1P analogs eliminates tumor-forming stem cells and at the same time, triggers oligodendroglial differentiation. This review discusses recent studies on the function of ceramide and S1P for the regulation of apoptosis, differentiation, and polarity in stem cells. We will also discuss results from ongoing studies in our laboratory on the use of sphingolipids in stem cell therapy.
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Affiliation(s)
- Erhard Bieberich
- Program in Developmental Neurobiology, Institute of Molecular Medicine and Genetics, School of Medicine, Medical College of Georgia, Augusta, GA 30912, USA.
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27
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Jukes JM, Moroni L, van Blitterswijk CA, de Boer J. Critical Steps toward a Tissue-Engineered Cartilage Implant Using Embryonic Stem Cells. ACTA ACUST UNITED AC 2008. [DOI: 10.1089/ten.2006.0397] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022]
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Jukes JM, Moroni L, van Blitterswijk CA, de Boer J. Critical Steps toward a Tissue-Engineered Cartilage Implant Using Embryonic Stem Cells. Tissue Eng Part A 2008; 14:135-47. [DOI: 10.1089/ten.a.2006.0397] [Citation(s) in RCA: 46] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022] Open
Affiliation(s)
- Jojanneke M. Jukes
- Department of Tissue Regeneration, Institute for Biomedical Technology, University of Twente, Enschede, The Netherlands
| | - Lorenzo Moroni
- Department of Tissue Regeneration, Institute for Biomedical Technology, University of Twente, Enschede, The Netherlands
| | - Clemens A. van Blitterswijk
- Department of Tissue Regeneration, Institute for Biomedical Technology, University of Twente, Enschede, The Netherlands
| | - Jan de Boer
- Department of Tissue Regeneration, Institute for Biomedical Technology, University of Twente, Enschede, The Netherlands
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Min J, Shang CZ, Chen YJ, Zhang L, Liu L, Deng XG, Yang M, Chen DP, Cao J, Song EW, Chen JS. Selective enrichment of hepatocytes from mouse embryonic stem cells with a culture system containing cholestatic serum. Acta Pharmacol Sin 2007; 28:1931-7. [PMID: 18031607 DOI: 10.1111/j.1745-7254.2007.00715.x] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
AIM There is increasing evidence indicating that embryonic stem (ES) cells are capable of differentiating into hepatocyte-like cells in vitro. However, it is necessary to improve the differentiation efficiency so as to promote the clinical application. Here, we report an efficient culture system to support hepatocyte differentiation from ES cells by utilizing cholestatic serum. METHODS One week after the induction of E14 mouse ES cells into hepatocytes with sodium butyrate, cholestatic serum was added into the culture system at various concentrations and hepatocyte-like cells were induced to proliferate. The morphological and phenotypic markers of hepatocytes were characterized using light microscopy, immunocytochemistry, and RT-PCR, respectively. The function of glycogen storage of the differentiated cells was detected by Periodic acid-Schiff (PAS) reaction, and the ratio of hepatic differentiation was determined by counting the albumin and PAS-positive cells. RESULTS In the presence of conditional selective medium containing cholestatic serum, numerous epithelial cells resembling hepatocytes were observed. The RT-PCR analysis showed that undifferentiated ES cells did not express any hepatic-specific markers; however, in the presence of sodium butyrate and conditional selective medium containing cholestatic serum, hepatic differentiation markers were detected. Immunofluorescence staining showed that those ES-derived hepatocytes were alpha-fetoprotein, albumin, and cytokeratin 18 positive, with the ability of storing glycogen. Further determination of the hepatic differentiation ratio showed that the application of cholestatic serum efficiently enriched ES-derived hepatocyte-like cells by inducing lineage differentiation and enhancing lineage proliferation. CONCLUSION The conditional selective medium containing cholestatic serum is optimal to selectively enrich hepatocyte-like cells from mixed differentiated ES cells, which may provide a novel method to improve the hepatic differentiation ratio of ES cells.
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Affiliation(s)
- Jun Min
- Department of Hepatobiliary Surgery, The Second Affiliated Hospital of Sun Yat-sen University, Guangzhou 510120, China.
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Bruce SJ, Rea RW, Steptoe AL, Busslinger M, Bertram JF, Perkins AC. In vitro differentiation of murine embryonic stem cells toward a renal lineage. Differentiation 2007; 75:337-49. [PMID: 17286599 DOI: 10.1111/j.1432-0436.2006.00149.x] [Citation(s) in RCA: 81] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
Embryonic stem (ES) cells have the capacity to differentiate into all cells of the developing embryo and may provide a renewable resource for future cell replacement therapies. The addition of bone morphogenetic protein 4 (BMP4) to serum-free ES cell culture has previously been shown to induce transcription factors, signaling molecules, and cell adhesion proteins expressed during mesoderm specification of the embryo. Here, we show the dynamics of primitive streak mesoderm differentiation in ES cells is comparable between serum and serum-free embryoid body (EB) cultures, supplemented with BMP4. Furthermore, we show a delayed wave of expression of a cohort of genes (Pax2, WT1, podocalyxin, pod-1, and nephrin), which play important roles during embryonic kidney development. The paired box transcription factor, Pax2, is one of the earliest genes expressed during kidney organogenesis and is required for normal urogenital development. ES cell lines containing either a modified Pax2 promoter-lacZ or bacterial artificial chromosome-green fluorescent protein (GFP) transgene were generated, which enabled the quantitative analysis of kidney rather than neuronal Pax2 expression within EBs. Both beta-galactosidase activity and GFP expression were detected by immunohistochemical and flow cytometric analysis following 16 days of EB culture, which correlated with an increase in Pax2 transcript levels. Together, these results suggest a spontaneous kidney gene expression program develops in mature EBs grown in both serum and serum-free conditions, when supplemented with BMP4. Further, the recombinant growth factors BMP2, BMP4, and BMP7 strongly influence gene expression within mesoderm induced EBs. BMP4 promotes ventral (blood) and intermediate (kidney) mesoderm gene expression, whereas BMP2 and BMP7 promote kidney outcomes at the expense of hematopoietic commitment. This induction assay and these unique ES cell lines will be useful for the generation of mesoderm-derived cell populations with implications for future cell therapeutic/integration assays.
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Affiliation(s)
- Stephen J Bruce
- Department of Anatomy and Cell Biology School of Biomedical Sciences Monash University Melbourne, Vic., Australia
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Lyra AC, Soares MBP, da Silva LFM, Fortes MF, Silva AGP, Mota ACDA, Oliveira SA, Braga EL, de Carvalho WA, Genser B, dos Santos RR, Lyra LGC. Feasibility and safety of autologous bone marrow mononuclear cell transplantation in patients with advanced chronic liver disease. World J Gastroenterol 2007; 13:1067-73. [PMID: 17373741 PMCID: PMC4146869 DOI: 10.3748/wjg.v13.i7.1067] [Citation(s) in RCA: 78] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To evaluate the safety and feasibility of bone marrow cell (BMC) transplantation in patients with chronic liver disease on the waiting list for liver transplantation.
METHODS: Ten patients (eight males) with chronic liver disease were enrolled to receive infusion of autologous bone marrow-derived cells. Seven patients were classified as Child-Pugh B and three as Child-Pugh C. Baseline assessment included complete clinical and laboratory evaluation and abdominal MRI. Approximately 50 mL of bone marrow aspirate was prepared by centrifugation in a ficoll-hypaque gradient. At least of 100 millions of mononuclear-enriched BMCs were infused into the hepatic artery using the routine technique for arterial chemoembolization for liver tumors. Patients were followed up for adverse events up to 4 mo.
RESULTS: The median age of the patients was 52 years (range 24-70 years). All patients were discharged 48 h after BMC infusion. Two patients complained of mild pain at the bone marrow needle puncture site. No other complications or specific side effects related to the procedure were observed. Bilirubin levels were lower at 1 (2.19 ± 0.9) and 4 mo (2.10 ± 1.0) after cell transplantation that baseline levels (2.78 ± 1.2). Albumin levels 4 mo after BMC infusion (3.73 ± 0.5) were higher than baseline levels (3.47 ± 0.5). International normalized ratio (INR) decreased from 1.48 (SD = 0.23) to 1.43 (SD = 0.23) one month after cell transplantation.
CONCLUSION: BMC infusion into hepatic artery of patients with advanced chronic liver disease is safe and feasible. In addition, a decrease in mean serum bilirubin and INR levels and an increase in albumin levels are observed. Our data warrant further studies in order to evaluate the effect of BMC transplantation in patients with advanced chronic liver disease.
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Soto-Gutiérrez A, Kobayashi N, Rivas-Carrillo JD, Navarro-Alvarez N, Zhao D, Okitsu T, Noguchi H, Basma H, Tabata Y, Chen Y, Tanaka K, Narushima M, Miki A, Ueda T, Jun HS, Yoon JW, Lebkowski J, Tanaka N, Fox IJ. Reversal of mouse hepatic failure using an implanted liver-assist device containing ES cell-derived hepatocytes. Nat Biotechnol 2006; 24:1412-1419. [PMID: 17086173 DOI: 10.1038/nbt1257] [Citation(s) in RCA: 166] [Impact Index Per Article: 8.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/18/2006] [Accepted: 10/06/2006] [Indexed: 01/10/2023]
Abstract
Severe acute liver failure, even when transient, must be treated by transplantation and lifelong immune suppression. Treatment could be improved by bioartificial liver (BAL) support, but this approach is hindered by a shortage of human hepatocytes. To generate an alternative source of cells for BAL support, we differentiated mouse embryonic stem (ES) cells into hepatocytes by coculture with a combination of human liver nonparenchymal cell lines and fibroblast growth factor-2, human activin-A and hepatocyte growth factor. Functional hepatocytes were isolated using albumin promoter-based cell sorting. ES cell-derived hepatocytes expressed liver-specific genes, secreted albumin and metabolized ammonia, lidocaine and diazepam. Treatment of 90% hepatectomized mice with a subcutaneously implanted BAL seeded with ES cell-derived hepatocytes or primary hepatocytes improved liver function and prolonged survival, whereas treatment with a BAL seeded with control cells did not. After functioning in the BAL, ES cell-derived hepatocytes developed characteristics nearly identical to those of primary hepatocytes.
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Affiliation(s)
- Alejandro Soto-Gutiérrez
- Department of Surgery, Okayama University Graduate School of Medicine and Dentistry, 2-5-1 Shikata-cho, Okayama 700-8558, Japan
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Abstract
Strong evidence suggests that bone marrow-derived cells play a role in physiological and pathological blood vessel growth in the adult, both by augmenting angiogenesis through the secretion of angiogenic growth factors and by providing a rich source of progenitor cells that can differentiate into mature vascular endothelial cells. This is a true paradigm shift, since adult neovascularization processes were thought to be limited to angiogenesis. The cells that are critical to postnatal blood vessel growth – endothelial progenitor cells – may be analogous to the embryonic angioblast, in that they can circulate, proliferate and participate in the development of vascular networks by differentiating in situ, probably via the formation of cell clusters into mature endothelial cells. Therefore, initial reports have seen analogs to the process of vasculogenesis in the embryo, where the de novo synthesis of vessels occurs through the formation of blood island-like clusters, which subsequently connect and eventually form systemic vasculature. Recent work implicates precursors of endothelial cells in such processes as myocardial ischemia and infarction, limb ischemia, wound healing, atherosclerosis, endogenous endothelial repair and tumor vascularization. These new insights into the vascular biology of endothelial regeneration and repair led to the development of new cell therapeutic strategies to enhance adult neovascularization and re-endothelialization in ischemic cardiovascular diseases.
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Affiliation(s)
- Christoph Kalka
- Swiss Cardiovascular Center, Department of Vascular Medicine, Inselspital, Freiburgstrasse, 3010 Bern, Switzerland
| | - Stefano Di Santo
- Swiss Cardiovascular Center, Department of Vascular Medicine, Inselspital, University Hospital of Bern, Switzerland
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Sinha S, Wamhoff BR, Hoofnagle MH, Thomas J, Neppl RL, Deering T, Helmke BP, Bowles DK, Somlyo AV, Owens GK. Assessment of contractility of purified smooth muscle cells derived from embryonic stem cells. Stem Cells 2006; 24:1678-88. [PMID: 16601077 DOI: 10.1634/stemcells.2006-0002] [Citation(s) in RCA: 49] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/17/2022]
Abstract
The aims of this study were to develop a method for deriving purified populations of contractile smooth muscle cells (SMCs) from embryonic stem cells (ESCs) and to characterize their function. Transgenic ESC lines were generated that stably expressed a puromycin-resistance gene under the control of either a smooth muscle alpha-actin (SMalphaAlpha) or smooth muscle-myosin heavy chain (SM-MHC) promoter. Negative selection, either overnight or for 3 days, was then used to purify SMCs from embryoid bodies. Purified SMCs expressed multiple SMC markers by immunofluorescence, immunoblotting, quantitative reverse transcription-polymerase chain reaction, and flow cytometry and were designated APSCs (SMalphaAlpha-puromycin-selected cells) or MPSCs (SM-MHC-puromycin-selected cells), respectively. Both SMC lines displayed agonist-induced Ca(2+) transients, expressed functional Ca(2+) channels, and generated contractile force when aggregated within collagen gels and stimulated with vasoactive agonists, such as endothelin-1, or in response to depolarization with KCl. Importantly, subcutaneous injection of APSCs or MPSCs subjected to 18 hours of puromycin selection led to the formation of teratomas, presumably due to residual contamination by pluripotent stem cells. In contrast, APSCs or MPSCs subjected to prolonged puromycin selection for 3 days did not form teratomas in vivo. These studies describe for the first time a method for generating relatively pure populations of SMCs from ESCs which display appropriate excitation and contractile responses to vasoactive agonists. However, studies also indicate the potential for teratoma development in ESC-derived cell lines, even after prolonged differentiation, highlighting the critical requirement for efficient methods of separating differentiated cells from residual pluripotent precursors in future studies that use ESC derivatives, whether SMC or other cell types, in tissue engineering applications.
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Affiliation(s)
- Sanjay Sinha
- Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, 22908-0736, USA
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Taylor RA, Cowin PA, Cunha GR, Pera M, Trounson AO, Pedersen J, Risbridger GP. Formation of human prostate tissue from embryonic stem cells. Nat Methods 2006; 3:179-81. [PMID: 16489334 DOI: 10.1038/nmeth855] [Citation(s) in RCA: 77] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/28/2005] [Accepted: 01/17/2006] [Indexed: 11/09/2022]
Abstract
Rodent models and immortalized or genetically modified cell lines are frequently used-but have limited utility-for studying human prostate development and maturation. Using rodent mesenchyme to establish reciprocal mesenchymal-epithelial cell interactions with human embryonic stem cells (hESCs), we generated human prostate tissue expressing prostate-specific antigen (PSA) within 8-12 weeks. This human prostate model shows species-conserved signalling mechanisms that could extend to integumental, gastrointestinal and genital tissues.
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Affiliation(s)
- Renea A Taylor
- Centre for Urological Research, Monash Institute of Medical Research, Monash University, 27-31 Wright Street, Clayton, Victoria, Australia 3168
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Teramoto K, Asahina K, Kumashiro Y, Kakinuma S, Chinzei R, Shimizu-Saito K, Tanaka Y, Teraoka H, Arii S. Hepatocyte differentiation from embryonic stem cells and umbilical cord blood cells. ACTA ACUST UNITED AC 2005; 12:196-202. [PMID: 15995807 DOI: 10.1007/s00534-005-0980-5] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/28/2005] [Accepted: 03/02/2005] [Indexed: 12/17/2022]
Abstract
With the development of regeneration medicine, many researchers have attempted hepatic differentiation from nonhepatic-origin cell sources. The differentiation of embryonic stem (ES) cells into hepatocyte-like cells has been reported in several papers. Mouse ES cells have shown a potential to develop into hepatocyte-like cells in vitro on the basis of hepatic gene expression after adding several growth factors. We transplanted cultured embryoid body (EB) cells (male) into female mice. A liver specimen of the recipient was examined by immunohistochemical staining for albumin and fluorescence in situ hybridization for the Y chromosome after transplantation. Both Y chromosome- and albumin-positive cells were recognized in the recipient female liver, and were considered to be hepatocyte-like cells derived from ES cells containing the Y chromosome. Many groups, including ourselves, have studied hepatocyte-like cell differentiation from umbilical cord blood cells (UBCs). We cultured nucleated cells isolated from UBCs. Using immunostaining, ALB-positive and CK-19-positive cells were recognized in the culture. Dual staining of ALB and CK-19 demonstrated that ALB was coexpressed with CK-19, suggesting the existence of hepatic progenitors. In this review, we consider recent studies of the differentiation of hepatocytes from nonhepatic origins, especially ES cells and umbilical cord blood.
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Affiliation(s)
- Kenichi Teramoto
- Department of Hepato-Biliary-Pancreatic Surgery, Graduate School of Medicine and Dentistry, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8519, Japan
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