|
1
|
Liu D, Zheng W, Pan S, Liu Z. Concise review: current trends on applications of stem cells in diabetic nephropathy. Cell Death Dis 2020; 11:1000. [PMID: 33221823 PMCID: PMC7680458 DOI: 10.1038/s41419-020-03206-1] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/01/2020] [Revised: 10/27/2020] [Accepted: 10/27/2020] [Indexed: 12/15/2022]
Abstract
Diabetic nephropathy, with high prevalence, is the main cause of renal failure in diabetic patients. The strategies for treating DN are limited with not only high cost but an unsatisfied effect. Therefore, the effective treatment of DN needs to be explored urgently. In recent years, due to their self-renewal ability and multi-directional differentiation potential, stem cells have exerted therapeutic effects in many diseases, such as graft-versus-host disease, autoimmune diseases, pancreatic diseases, and even acute kidney injury. With the development of stem cell technology, stem cell-based regenerative medicine has been tried to be applied to the treatment of DN. Related stem cells include embryonic stem cells, induced pluripotent stem cells, mesenchymal cells, and endothelial progenitor cells. Undoubtedly, stem cell transplantation has achieved certain results in the treatment of DN animal models. However, stem cell therapy still remains certain thorny issues during treatment. For instance, poor engraftment and limited differentiation of stem cells caused by the diabetic microenvironment, differentiation into unwanted cell lineages, and malignant transformation or genetic aberrations of stem cells. At present, various researches on the therapeutic effects of stem cells in DN with different opinions are reported and the specific mechanism of stem cells is still unclear. We review here the potential mechanism of stem cells as new therapeutic agents in the treatment of DN. Also, we review recent findings and updated information about not only the utilization of stem cells on DN in both preclinical and clinical trials but limitations and future expectations of stem cell-based therapy for DN.
Collapse
Affiliation(s)
- Dongwei Liu
- Department of Nephrology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, 450052, P.R. China.,Research Institute of Nephrology, Zhengzhou University, Zhengzhou, 450052, P.R. China.,Key Laboratory of Precision Diagnosis and Treatment for Chronic Kidney Disease in Henan Province, Zhengzhou, 450052, P.R. China.,Core Unit of National Clinical Medical Research Center of Kidney Disease, Zhengzhou, 450052, P.R. China
| | - Wen Zheng
- Department of Nephrology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, 450052, P.R. China.,Research Institute of Nephrology, Zhengzhou University, Zhengzhou, 450052, P.R. China.,Key Laboratory of Precision Diagnosis and Treatment for Chronic Kidney Disease in Henan Province, Zhengzhou, 450052, P.R. China.,Core Unit of National Clinical Medical Research Center of Kidney Disease, Zhengzhou, 450052, P.R. China
| | - Shaokang Pan
- Department of Nephrology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, 450052, P.R. China.,Research Institute of Nephrology, Zhengzhou University, Zhengzhou, 450052, P.R. China.,Key Laboratory of Precision Diagnosis and Treatment for Chronic Kidney Disease in Henan Province, Zhengzhou, 450052, P.R. China.,Core Unit of National Clinical Medical Research Center of Kidney Disease, Zhengzhou, 450052, P.R. China
| | - Zhangsuo Liu
- Department of Nephrology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, 450052, P.R. China. .,Research Institute of Nephrology, Zhengzhou University, Zhengzhou, 450052, P.R. China. .,Key Laboratory of Precision Diagnosis and Treatment for Chronic Kidney Disease in Henan Province, Zhengzhou, 450052, P.R. China. .,Core Unit of National Clinical Medical Research Center of Kidney Disease, Zhengzhou, 450052, P.R. China.
| |
Collapse
|
|
2
|
Stuelsatz P, Keire P, Yablonka-Reuveni Z. Isolation, Culture, and Immunostaining of Skeletal Muscle Myofibers from Wildtype and Nestin-GFP Mice as a Means to Analyze Satellite Cell. Methods Mol Biol 2017; 1556:51-102. [PMID: 28247345 DOI: 10.1007/978-1-4939-6771-1_4] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 06/06/2023]
Abstract
Multinucleated myofibers, the functional contractile units of adult skeletal muscle, harbor mononuclear Pax7+ myogenic progenitors on their surface between the myofiber basal lamina and plasmalemma. These progenitors, known as satellite cells, are the primary myogenic stem cells in adult muscle. This chapter describes our laboratory protocols for isolating, culturing, and immunostaining intact myofibers from mouse skeletal muscle as a means for studying satellite cell dynamics. The first protocol discusses myofiber isolation from the flexor digitorum brevis (FDB) muscle. These short myofibers are plated in dishes coated with PureCol collagen (formerly known as Vitrogen) and maintained in a mitogen-poor medium (± supplemental growth factors). Employing such conditions, satellite cells remain at the surface of the parent myofiber while synchronously undergoing a limited number of proliferative cycles and rapidly differentiate. The second protocol discusses the isolation of longer myofibers from the extensor digitorum longus (EDL) muscle. These EDL myofibers are routinely plated individually as adherent myofibers in wells coated with Matrigel and maintained in a mitogen-rich medium, conditions in which satellite cells migrate away from the parent myofiber, proliferate extensively, and generate numerous differentiating progeny. Alternatively, these EDL myofibers can be plated as non-adherent myofibers in uncoated wells and maintained in a mitogen-poor medium (± supplemental growth factors), conditions that retain satellite cell progeny at the myofiber niche similar to the FDB myofiber cultures. However, the adherent myofiber format is our preferred choice for monitoring satellite cells in freshly isolated (Time 0) myofibers. We conclude this chapter by promoting the Nestin-GFP transgenic mouse as an efficient tool for direct analysis of satellite cells in isolated myofibers. While satellite cells have been often detected by their expression of the Pax7 protein or the Myf5nLacZ knockin reporter (approaches that are also detailed herein), the Nestin-GFP reporter distinctively permits quantification of satellite cells in live myofibers, which enables linking initial Time 0 numbers and subsequent performance upon culturing. We additionally point out to the implementation of the Nestin-GFP transgene for monitoring other selective cell lineages as illustrated by GFP expression in capillaries, endothelial tubes and neuronal cells. Myofibers from other types of muscles, such as diaphragm, masseter, and extraocular, can also be isolated and analyzed using protocols described herein. Collectively, this chapter provides essential tools for studying satellite cells in their native position and their interplay with the parent myofiber.
Collapse
MESH Headings
- Animals
- Biomarkers
- Cell Culture Techniques
- Cell Differentiation
- Cell Separation/methods
- Genes, Reporter
- Green Fluorescent Proteins/genetics
- Green Fluorescent Proteins/metabolism
- Immunophenotyping/methods
- Mice
- Mice, Transgenic
- Microscopy, Fluorescence
- Muscle Fibers, Skeletal/cytology
- Muscle Fibers, Skeletal/metabolism
- Muscle Fibers, Skeletal/ultrastructure
- Muscle, Skeletal/cytology
- Nestin/genetics
- Nestin/metabolism
- Phenotype
- Primary Cell Culture
- Satellite Cells, Skeletal Muscle/cytology
- Satellite Cells, Skeletal Muscle/metabolism
- Satellite Cells, Skeletal Muscle/ultrastructure
Collapse
Affiliation(s)
- Pascal Stuelsatz
- Department of Biological Structure, School of Medicine, University of Washington, Health Sciences Building, Room G520, 1959 NE Pacific Street, Box 357420, Seattle, WA, 98195, USA
| | - Paul Keire
- Department of Biological Structure, School of Medicine, University of Washington, Health Sciences Building, Room G520, 1959 NE Pacific Street, Box 357420, Seattle, WA, 98195, USA
| | - Zipora Yablonka-Reuveni
- Department of Biological Structure, School of Medicine, University of Washington, Health Sciences Building, Room G520, 1959 NE Pacific Street, Box 357420, Seattle, WA, 98195, USA.
| |
Collapse
|
|
3
|
Duan FF, Liu JH, March JC. Engineered commensal bacteria reprogram intestinal cells into glucose-responsive insulin-secreting cells for the treatment of diabetes. Diabetes 2015; 64:1794-803. [PMID: 25626737 PMCID: PMC4407861 DOI: 10.2337/db14-0635] [Citation(s) in RCA: 149] [Impact Index Per Article: 14.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/22/2014] [Accepted: 12/10/2014] [Indexed: 12/25/2022]
Abstract
The inactive full-length form of GLP-1(1-37) stimulates conversion of both rat and human intestinal epithelial cells into insulin-secreting cells. We investigated whether oral administration of human commensal bacteria engineered to secrete GLP-1(1-37) could ameliorate hyperglycemia in a rat model of diabetes by reprogramming intestinal cells into glucose-responsive insulin-secreting cells. Diabetic rats were fed daily with human lactobacilli engineered to secrete GLP-1(1-37). Diabetic rats fed GLP-1-secreting bacteria showed significant increases in insulin levels and, additionally, were significantly more glucose tolerant than those fed the parent bacterial strain. These rats developed insulin-producing cells within the upper intestine in numbers sufficient to replace ∼25-33% of the insulin capacity of nondiabetic healthy rats. Intestinal tissues in rats with reprogrammed cells expressed MafA, PDX-1, and FoxA2. HNF-6 expression was observed only in crypt epithelia expressing insulin and not in epithelia located higher on the villous axis. Staining for other cell markers in rats treated with GLP-1(1-37)-secreting bacteria suggested that normal function was not inhibited by the close physical proximity of reprogrammed cells. These results provide evidence of the potential for a safe and effective nonabsorbed oral treatment for diabetes and support the concept of engineered commensal bacterial signaling to mediate enteric cell function in vivo.
Collapse
Affiliation(s)
- Franklin F Duan
- Department of Biological and Environmental Engineering, Cornell University, Ithaca, NY
| | - Joy H Liu
- Department of Biological and Environmental Engineering, Cornell University, Ithaca, NY
| | - John C March
- Department of Biological and Environmental Engineering, Cornell University, Ithaca, NY
| |
Collapse
|
|
4
|
Shi Q, Luo S, Jia H, Feng L, Lu X, Zhou L, Cai J. Wnt/β-catenin signaling may be involved with the maturation, but not the differentiation, of insulin-producing cells. Biomed Pharmacother 2013; 67:745-50. [PMID: 23680408 DOI: 10.1016/j.biopha.2013.04.002] [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] [Received: 03/12/2013] [Accepted: 04/08/2013] [Indexed: 10/26/2022] Open
Abstract
Wnt/β-catenin signaling (WNT) has widespread roles during stem cell differentiation. Whether WNT suppresses or promotes insulin-producing cell (IPC) differentiation and function is still not known. In this study, we investigated the role of WNT signaling during human adipose-derived stem cell (hADSC) differentiation into IPCs. Western blot analysis revealed that several key components of WNT were dynamically regulated in a 12-day IPC differentiation assay. Specifically, protein levels of Wnt1, β-catenin, and GSK3β steadily increased from day 0 to day 9 and rapidly decreased by day 12 of differentiation. Similarly, endonuclear β-catenin levels peaked at day 9 and then, fell to pre-differentiation levels. The expression of two WNT pathway targets, TCF-1 and cyclin D1, closely followed the same pattern of regulation, confirming that WNT signaling was transiently activated during IPC differentiation. Interestingly, the inhibition of WNT signaling did not block IPC differentiation; instead, it resulted in the upregulation of IPC-specific markers, including PDX-1, insulin, IRS-1, and IRS-2. Notably, another IPC marker, glucokinase, remained downregulated since it is a direct target of WNT signaling. Next, we examined the effect of maintaining active WNT signaling from day 9 to day 12 of IPC differentiation. Differentiating cells were treated with Wnt1 on day 9, when WNT signaling is typically turned off, and subjected to gene expression analysis on day 12. Remarkably, Wnt1 treatment resulted in reduced expression of IPC-specific markers. Taken together, these data indicate that WNT may not be necessary for IPC differentiation but may be involved in IPC maturation.
Collapse
Affiliation(s)
- Qiping Shi
- The First Affiliated Hospital, Jinan University, Guangzhou 510632, China.
| | | | | | | | | | | | | |
Collapse
|
|
5
|
Viñas JL, Ventayol M, Brüne B, Jung M, Sola A, Pi F, Mastora C, Hotter G. miRNA let-7e modulates the Wnt pathway and early nephrogenic markers in mouse embryonic stem cell differentiation. PLoS One 2013; 8:e60937. [PMID: 23593353 PMCID: PMC3622609 DOI: 10.1371/journal.pone.0060937] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/19/2012] [Accepted: 03/05/2013] [Indexed: 11/23/2022] Open
Abstract
This study indicates that embryonic stem cells [ESCs] cultured with retinoic acid and activin A significantly upregulate the miRNA let-7e. This specific miRNA modulates the Wnt pathway and the expression of early nephrogenic markers under these differentiation conditions. The differentiation markers WT1, Pax2 and Wnt4 were downregulated when miRNA let-7e was silenced, thus indicating the role of miRNA let-7e in the differentiation process. PKCβ, GSK3β phosphorylation (GSK3βP) and β-catenin expression was reduced in differentiated cells and reversed by miRNA let-7e silencing. Addition of a PKCβ inhibitor to the miRNA let-7e silenced cells abolished let-7e-derived effects in differentiation markers, and reversed the increase in GSK3βP and β-catenin, thus indicating that miRNA let-7e is involved in differentiation via the modulation of GSK3β phosphorylation and β-catenin production.
Collapse
Affiliation(s)
- Jose Luis Viñas
- Departament of Experimental Pathology, Instituto de Investigaciones Biomédicas de Barcelona (IIBB-CSIC, IDIBAPS), Barcelona, Spain.
| | | | | | | | | | | | | | | |
Collapse
|
|
6
|
Ma Y, Gu J, Li C, Wei X, Tang F, Shi G, Jiang J, Kuang Y, Li J, Wang Z, Xie X, Jin Y. Human foreskin fibroblast produces interleukin-6 to support derivation and self-renewal of mouse embryonic stem cells. Stem Cell Res Ther 2012; 3:29. [PMID: 22849865 PMCID: PMC3580467 DOI: 10.1186/scrt120] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/08/2011] [Accepted: 07/31/2012] [Indexed: 02/06/2023] Open
Abstract
Introduction Embryonic stem cells (ESCs) provide an attractive cell source for basic research and disease treatment. Currently, the common culture system for mouse ESC requires mouse embryonic fibroblast (MEF) as a feeder layer supplemented with leukemia inhibitory factor (LIF). The drawbacks associated with MEF and the cost of LIF have motivated exploration of new feeder cell types to maintain self-renewal of mouse ESCs without the need of exogenous LIF. However, why these feeder cells could maintain ESCs at the undifferentiated state independent of exogenous LIF is unclear. Methods We derived mouse ESC lines using human foreskin fibroblast (HFF) in the absence of exogenous LIF. We also examined the dependence of HFF on the JAK-Stat3 pathway to maintain ESC identities and explored the potential molecular basis for HFF to support self-renewal of ESCs. Results HFF supported mouse ESC self-renewal superiorly to MEFs. Using the HFF system, multiple lines of mouse ESCs were successfully derived without addition of exogenous LIF and any small molecular inhibitors. These ESCs had capacities to self-renew for a long period of time and to differentiate into various cell types of the three germ layers both in vitro and in vivo. Moreover, the ESCs participated in embryonic development and contributed to germ cell lineages in the chimeric mouse. At a molecular level, HFF was dependent on the JAK-Stat3 pathway to maintain ESC self-renewal. The high level of interleukin-6 (IL-6) produced by HFF might be responsible for the exogenous LIF-independent effect. Conclusion This study describes an efficient, convenient and economic system to establish and maintain mouse ESC lines, and provides insights into the functional difference in the support of ESC culture between MEF and HFF.
Collapse
|
|
7
|
Abstract
Diseases related to the pancreas are of highest importance in public health. It is anticipated that a detailed understanding of the molecular events that govern the embryonic development of this organ will have an immediate impact on clinical research relating to this issue. One major aim is the reconstruction of embryonic development in vitro with appropriate precursor cells, a second strategy is aimed at understanding the transdifferentiation of non-pancreatic into pancreatic tissue, and a third avenue is defined by the stimulation of the intrinsic ability of the pancreas to regenerate. Recent progress in developmental biology with respect to these different topics is reviewed in the present article. In addition, we also address evolutionary aspects of pancreas development, emphasizing the role of the South African clawed frog, Xenopus laevis, as an additional useful model system to study the molecular control of pancreas development.
Collapse
Affiliation(s)
- Tomas Pieler
- Georg-August-Universität Göttingen, Zentrum Biochemie und Molekulare Zellbiologie, Abteilung Entwicklungsbiochemie, Justus von Liebig Weg 11, 37077 Göttingen, Germany.
| | | |
Collapse
|
|
8
|
Turner AEB, Flynn LE. Design and characterization of tissue-specific extracellular matrix-derived microcarriers. Tissue Eng Part C Methods 2011; 18:186-97. [PMID: 21981618 DOI: 10.1089/ten.tec.2011.0246] [Citation(s) in RCA: 61] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/27/2023] Open
Abstract
The three-dimensional (3D) extracellular matrix (ECM) environment plays a critical role in mediating normal cellular behavior and tissue organization. While commercially available microcarriers have shown promise, limited research has been conducted on the design of tissue-specific, custom-fabricated microcarriers, engineered to mimic the composition of the native ECM of cells or tissues of interest. Moving toward this goal, methods were developed to fabricate microcarriers from decellularized adipose tissue (DAT) via minimally-cytotoxic protocols. Characterization by microscopy confirmed the production of stable spherical microcarriers, with a microporous surface topography and porous interior. The mean diameter of the DAT microcarriers was 934±51 μm, while the porosity was estimated as 29%±4% using liquid displacement. Stability and swelling behavior over 4 weeks indicated that the DAT microcarriers were effectively stabilized with the photochemical crosslinking agent rose bengal, with total protein release in a simulated physiological environment remaining below 10 μg/mL at all time points. Preliminary cell culture studies with human adipose-derived stem cells (ASCs) in a spinner flask system indicated enhanced cell attachment and proliferation of ASCs on DAT microcarriers over 14 days, as compared with gelatin control microcarriers fabricated using similar methods. Testing confirmed injectability of the DAT microcarriers, further supporting the clinical potential of the approach for localized cell delivery and small volume augmentation in plastic and reconstructive surgery. Overall, tissue-specific microcarriers prepared from solubilized DAT were found to be highly supportive of human ASCs cultured in a 3D dynamic environment.
Collapse
Affiliation(s)
- Allison E B Turner
- Department of Chemical Engineering, Queen's University, Kingston, Ontario, Canada
| | | |
Collapse
|
|
9
|
Cheng H, Zhang YC, Wolfe S, Valencia V, Qian K, Shen L, Tang YL, Hsu WH, Atkinson MA, Phillips MI. Combinatorial treatment of bone marrow stem cells and stromal cell-derived factor 1 improves glycemia and insulin production in diabetic mice. Mol Cell Endocrinol 2011; 345:88-96. [PMID: 21801807 PMCID: PMC3171644 DOI: 10.1016/j.mce.2011.07.024] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/11/2011] [Revised: 07/06/2011] [Accepted: 07/12/2011] [Indexed: 01/16/2023]
Abstract
Transdifferentiation of stem cells into insulin-producing cells for the treatment of diabetes have shown promising but inconsistent results. We examined the potential for attracting bone marrow stem cells (BMSCs) to the pancreas using a chemokine, stromal cell-derived factor 1 (SDF-1). SDF-1 treatment markedly increased the number of GFP labeled BMSCs in the pancreas, but surprisingly, the majority was observed in liver. The liver cells had typical pancreatic endocrine cell gene expression including insulin I, insulin II, PDX-1, somatostatin, and glucagon. Combined treatment with SDF-1 and BMSC transplant reduced hyperglycemia and prolonged the long-term survival of diabetic mice, and a sub group had complete normoglycemia (<150 mg/dl), restored blood insulin levels, and normal glucose tolerance. Our results suggest that SDF-1 could potentially be used to improve the homing of stem cells and β-cell regeneration. The mechanism appears to involve an increase in insulin producing cells mainly in the liver.
Collapse
Affiliation(s)
- H Cheng
- Department of Comparative Biomedical Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, 70803, USA.
| | | | | | | | | | | | | | | | | | | |
Collapse
|
|
10
|
Mfopou JK, Chen B, Mateizel I, Sermon K, Bouwens L. Noggin, retinoids, and fibroblast growth factor regulate hepatic or pancreatic fate of human embryonic stem cells. Gastroenterology 2010; 138:2233-45, 2245.e1-14. [PMID: 20206178 DOI: 10.1053/j.gastro.2010.02.056] [Citation(s) in RCA: 123] [Impact Index Per Article: 8.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/22/2009] [Revised: 01/27/2010] [Accepted: 02/23/2010] [Indexed: 01/30/2023]
Abstract
BACKGROUND & AIMS New sources of beta cells are needed to develop cell therapies for patients with diabetes. An in vitro, sequential method has been developed to derive pancreatic progenitors, but this technique has not been used for other cell lines. We investigated whether definitive endoderm derived from human embryonic stem (hES) cells might be used to create beta cells. METHODS Five hES cell lines were induced to form pancreatic progenitors and analyzed for pancreas markers. Cells were incubated with a bone morphogenetic protein (BMP) antagonist, retinoids, a Hedgehog antagonist, or fibroblast growth factor (FGF) and phenotypes were analyzed. RESULTS Four hES cell lines sequentially generated definitive endoderm, primitive gut, and posterior foregut equivalents, as described previously. However, functional hepatocytes, rather than pancreas progenitors, developed. Consistent with liver development, FGF and BMP signaling pathways were involved in this process; their inhibition disrupted hepatocyte differentiation. During early stages of development, exposure of cells to noggin and retinoid acid, followed by FGF10, generated pancreatic cells (PDX1+; 50%-80%) that coexpressed FOXA2, HNF6, and SOX9. CONCLUSIONS These findings demonstrate the combined functions of endogenous BMP and supplemented FGF in inducing differentiation of hepatocytes from hES cells and the ability to shift developmental pathways from hepatic to pancreatic cell differentiation. Although additional signals appear to be required for full specification of PDX1(+) early pancreatic progenitors (via PTF1a and NKX6.1 coexpression), these findings indicate the signaling pathways required for differentiation of bipotential progenitors.
Collapse
Affiliation(s)
- Josué Kunjom Mfopou
- Cell Differentiation Unit, Diabetes Research Centre, Vrije Universiteit Brussel, Brussels, Belgium
| | | | | | | | | |
Collapse
|
|
11
|
Nikolic B, Faintuch S, Goldberg SN, Kuo MD, Cardella JF. Stem Cell Therapy: A Primer for Interventionalists and Imagers. J Vasc Interv Radiol 2009; 20:999-1012. [DOI: 10.1016/j.jvir.2009.04.075] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/16/2009] [Revised: 04/24/2009] [Accepted: 04/28/2009] [Indexed: 02/06/2023] Open
|
|
12
|
Pax2 overexpression in embryoid bodies induces upregulation of integrin alpha8 and aquaporin-1. In Vitro Cell Dev Biol Anim 2008; 45:62-8. [PMID: 19037705 DOI: 10.1007/s11626-008-9151-8] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/24/2008] [Accepted: 09/24/2008] [Indexed: 10/21/2022]
Abstract
The transcription factor Pax2 is essential for kidney development in mice, and overexpression of Pax2 in chick embryos leads to ectopic formation of nephric structures. We have generated embryonic stem (ES) cell lines that repress Pax2 expression in a tetracycline-dependent manner. In the absence of tetracycline, embryoid bodies derived from these cell lines expressed Pax2 and subsequently integrin alpha8 and aquaporin-1 (Aqp1), both of which are possibly involved in kidney development. Considering the slow induction kinetics, our data suggest that Pax2 and additional factors that are induced in embryoid bodies synergistically regulate the two targets. The ES cell lines with inducible Pax2 expression will also be useful for dissecting genetic cascades functioning in a variety of organ development.
Collapse
|
|
13
|
Schroeder IS, Rolletschek A, Blyszczuk P, Kania G, Wobus AM. Differentiation of mouse embryonic stem cells to insulin-producing cells. Nat Protoc 2007; 1:495-507. [PMID: 17406275 DOI: 10.1038/nprot.2006.71] [Citation(s) in RCA: 81] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/12/2023]
Abstract
Here, we describe a basic protocol for the in vitro differentiation of mouse embryonic stem (ES) cells into insulin-producing cells. The three-step protocol comprises (i) the formation of embryoid bodies, (ii) the spontaneous differentiation of embryoid bodies into progenitor cells of ecto-, meso- and endodermal lineages, and (iii) the induction of differentiation of early progenitors into the pancreatic lineage. Differentiated cells can be obtained within approximately 33 d. Differentiation induction by growth and extracellular-matrix factors, including laminin, nicotinamide and insulin, leads to the formation of ES-derived progeny that resembles cells committed to the pancreatic lineage. During differentiation, transcript levels of genes expressed in early pancreatic cells are upregulated. Continued differentiation results in the development of C-peptide/insulin-positive islet-like clusters that release insulin upon glucose stimulation. Differentiated ES cells that overexpress the pancreatic developmental control gene Pax4 develop insulin-secretory granules and reveal functional properties with respect to the pancreas-specific ATP-modulated K+ channel and the normalization of glycemia of streptozotocin-treated diabetic mice.
Collapse
Affiliation(s)
- Insa S Schroeder
- In Vitro Differentiation Group, Leibniz-Institute of Plant Genetics and Crop Plant Research (IPK) Gatersleben, Corrensstr.3, D-06466 Germany
| | | | | | | | | |
Collapse
|
|
14
|
Sensken S, Waclawczyk S, Knaupp AS, Trapp T, Enczmann J, Wernet P, Kogler G. In vitro differentiation of human cord blood-derived unrestricted somatic stem cells towards an endodermal pathway. Cytotherapy 2007; 9:362-78. [PMID: 17573612 DOI: 10.1080/14653240701320254] [Citation(s) in RCA: 31] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/08/2023]
Abstract
BACKGROUND Pluripotent unrestricted somatic stem cells (USSC) from UC blood can differentiate into hepatic cells in the in utero sheep model, resulting in 20% human albumin-producing parenchymal hepatic cells without cell fusion or tumor-formation events. Additionally, we have shown in vitro differentiation of USSC by hepatocyte growth factor and oncostatin M induction, causing changes in the gene expression towards the endodermal lineage. Positive glycogen synthase expression and a positive periodic acid-schiff reaction demonstrated a functional production of polysaccharides in the cells. METHODS We describe the in vitro differentiation of USSC towards an endodermal pathway using different matrices, growth factors and organic substances. Also, co-cultures of USSC with primary cells of endodermal tissue were prepared to mimic the biologic niche. We investigated the effect of direct co-culture of USSC with primary rat hepatocytes or with sheep tissue of endodermal origin. Direct co-cultures were set up to ensure cell-cell contacts. For co-cultures without cell-cell contacts, transwell inlays with 1-microm membranes were used to separate the cells. Furthermore, the effect of endodermally conditioned medium was investigated. Changes in the gene expression patterns were analyzed by RT-PCR. RESULTS We have shown that USSC can differentiate in vitro into an endodermal-like cell with a phenotype similar to hepatic cells. Differentiation of USSC with growth factors, retinoic acid, matrigel matrix and different co-cultures led to an increased expression of albumin and also to the detection of GSC, SOX 17, Cyp2B6, Cyp3A4, Gys2, HNF4a, ISL-1 and Nkx6.1. In addition, functional albumin secretion was observed. DISCUSSION Although the differentiation assays demonstrated here produce only an immature hepatocyte-like cell, endodermaly differentiated USSC might be a useful alternative for cell replacement in the future.
Collapse
Affiliation(s)
- S Sensken
- Institute for Transplantation Diagnostics and Cell Therapeutics, University of Duesseldorf Medical School, Duesseldorf, Germany
| | | | | | | | | | | | | |
Collapse
|
|
15
|
Zhou QJ, Xiang LX, Shao JZ, Hu RZ, Lu YL, Yao H, Dai LC. In vitro differentiation of hepatic progenitor cells from mouse embryonic stem cells induced by sodium butyrate. J Cell Biochem 2007; 100:29-42. [PMID: 16888815 DOI: 10.1002/jcb.20970] [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: 12/17/2022]
Abstract
Recently it was shown that embryonic stem (ES) cells could differentiate into hepatocytes both in vitro and in vivo, however, prospective hepatic progenitor cells have not yet been isolated and characterized from ES cells. Here we presented a novel 4-step procedure for the differentiation of mouse ES cells into hepatic progenitor cells and then hepatocytes. The differentiated hepatocytes were identified by morphological, biochemical, and functional analyses. The hepatic progenitor cells were isolated from the cultures after the withdrawal of sodium butyrate, which was characterized by scant cytoplasm, ovoid nuclei, the ability of rapid proliferation, expression of a series of hepatic progenitor cell markers, and the potential of differentiation into hepatocytes and bile duct-like cells under the proper conditions that favor hepatocyte and bile epithelial differentiation. The differentiation of hepatocytes from hepatic progenitor cells was characterized by a number of hepatic cell markers including albumin secretion, upregulated transcription of glucose-6-phosphatase and tyrosine aminotransferase, and functional phenotypes such as glycogen storage. The results from our experiments demonstrated that ES cells could differentiate into a novel bipotential hepatic progenitor cell and mature into hepatocytes with typical morphological, phenotypic and functional characteristics, which provides an useful model for the studies of key events during early liver development and a potential source of transplantable cells for cell-replacement therapies.
Collapse
Affiliation(s)
- Qing-Jun Zhou
- College of Life Science, Zhejiang University, Hangzhou 310012, PR China
| | | | | | | | | | | | | |
Collapse
|
|
16
|
Goicoa S, Alvarez S, Ricordi C, Inverardi L, Domínguez-Bendala J. Sodium butyrate activates genes of early pancreatic development in embryonic stem cells. CLONING AND STEM CELLS 2006; 8:140-9. [PMID: 17009890 DOI: 10.1089/clo.2006.8.140] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/05/2023]
Abstract
Embryonic stem (ES) cells can differentiate into any tissue, including pancreatic islet cell types. Protocols for the efficient generation of these cells in vitro could have therapeutic applications for type I diabetes. Here we describe a simple method for the differentiation of mouse ES cells into epithelial cells with a gene expression profile consistent with that expected of early pancreatic progenitors (PP). It is based on the addition of sodium butyrate, an agent known to induce chromatin rearrangements. Variations on the length of exposure to butyrate result in the generation of hepatocytes or PP-like cells. qRT-PCR indicates that butyrate induces mesendoderm/definitive endoderm, but not neuroectoderm differentiation. PPlike cells show a strong upregulation of Ipf1/Pdx1, p48, Isl-1 and Nkx6.1, but not Ngn3, NeuroD/ Beta2 or Pax4. PP-like cells also express the epithelial marker E-cadherin. Taken together, our observations suggest that butyrate stimulates early events of pancreatic specification, prior to the onset of endocrine differentiation. These findings are discussed in the context of the development of protocols for the in vitro differentiation of islets.
Collapse
Affiliation(s)
- Stacey Goicoa
- Diabetes Research Institute, University of Miami Leonard M. Miller School of Medicine, Miami, Florida 33136, USA
| | | | | | | | | |
Collapse
|
|
17
|
Wiese C, Rolletschek A, Kania G, Navarrete-Santos A, Anisimov SV, Steinfarz B, Tarasov KV, Brugh SA, Zahanich I, Rüschenschmidt C, Beck H, Blyszczuk P, Czyz J, Heubach JF, Ravens U, Horstmann O, St-Onge L, Braun T, Brüstle O, Boheler KR, Wobus AM. Signals from embryonic fibroblasts induce adult intestinal epithelial cells to form nestin-positive cells with proliferation and multilineage differentiation capacity in vitro. Stem Cells 2006; 24:2085-97. [PMID: 16741226 DOI: 10.1634/stemcells.2006-0008] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/17/2022]
Abstract
The intestinal epithelium has one of the greatest regenerative capacities in the body; however, neither stem nor progenitor cells have been successfully cultivated from the intestine. In this study, we applied an "artificial niche" of mouse embryonic fibroblasts to derive multipotent cells from the intestinal epithelium. Cocultivation of adult mouse and human intestinal epithelium with fibroblast feeder cells led to the generation of a novel type of nestin-positive cells (intestinal epithelium-derived nestin-positive cells [INPs]). Transcriptome analyses demonstrated that mouse embryonic fibroblasts expressed relatively high levels of Wnt/bone morphogenetic protein (BMP) transcripts, and the formation of INPs was specifically associated with an increase in Lef1, Wnt4, Wnt5a, and Wnt/BMP-responsive factors, but a decrease of BMP4 transcript abundance. In vitro, INPs showed a high but finite proliferative capacity and readily differentiated into cells expressing neural, pancreatic, and hepatic transcripts and proteins; however, these derivatives did not show functional properties. In vivo, INPs failed to form chimeras following injection into mouse blastocysts but integrated into hippocampal brain slice cultures in situ. We conclude that the use of embryonic fibroblasts seems to reprogram adult intestinal epithelial cells by modulation of Wnt/BMP signaling to a cell type with a more primitive embryonic-like stage of development that has a high degree of flexibility and plasticity.
Collapse
Affiliation(s)
- Cornelia Wiese
- In Vitro Differentiation Group, Institute of Plant Genetics and Crop Plant Research, IPK, Corrensstrasse 3, D-06466 Gatersleben, Germany
| | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | |
Collapse
|
|
18
|
Miszta-Lane H, Mirbolooki M, James Shapiro AM, Lakey JRT. Stem cell sources for clinical islet transplantation in type 1 diabetes: Embryonic and adult stem cells. Med Hypotheses 2006; 67:909-13. [PMID: 16762516 DOI: 10.1016/j.mehy.2006.03.036] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/26/2006] [Revised: 03/28/2006] [Accepted: 03/29/2006] [Indexed: 12/20/2022]
Abstract
Lifelong immunosuppressive therapy and inadequate sources of transplantable islets have led the islet transplantation benefits to less than 0.5% of type 1 diabetics. Whereas the potential risk of infection by animal endogenous viruses limits the uses of islet xeno-transplantation, deriving islets from stem cells seems to be able to overcome the current problems of islet shortages and immune compatibility. Both embryonic (derived from the inner cell mass of blastocysts) and adult stem cells (derived from adult tissues) have shown controversial results in secreting insulin in vitro and normalizing hyperglycemia in vivo. ESCs research is thought to have much greater developmental potential than adult stem cells; however it is still in the basic research phase. Existing ESC lines are not believed to be identical or ideal for generating islets or beta-cells and additional ESC lines have to be established. Research with ESCs derived from humans is controversial because it requires the destruction of a human embryo and/or therapeutic cloning, which some believe is a slippery slope to reproductive cloning. On the other hand, adult stem cells are already in some degree specialized, recipients may receive their own stem cells. They are flexible but they have shown mixed degree of availability. Adult stem cells are not pluripotent. They may not exist for all organs. They are difficult to purify and they cannot be maintained well outside the body. In order to draw the future avenues in this field, existent discrepancies between the results need to be clarified. In this study, we will review the different aspects and challenges of using embryonic or adult stem cells in clinical islet transplantation for the treatment of type 1 diabetes.
Collapse
Affiliation(s)
- Helena Miszta-Lane
- Clinical Islet Transplantation Program, Department of Surgery, Faculty of Medicine and Dentistry, University of Alberta, 1074 Dentistry/Pharmacy Centre, Edmonton, Alta., Canada T6G 2N8
| | | | | | | |
Collapse
|
|
19
|
Pelz O, Wu M, Nikolova T, Kamprad M, Ackermann M, Egger D, Emmrich F, Wobus AM, Cross M. Duplex polymerase chain reaction quantification of human cells in a murine background. Stem Cells 2005; 23:828-33. [PMID: 15917478 DOI: 10.1634/stemcells.2004-0206] [Citation(s) in RCA: 17] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/17/2022]
Abstract
Studies of the regenerative potential of human stem cells commonly involve their transplantation into immune-deficient mice or in vitro coculture with mouse cells. The optimal use of such models requires the detection and quantification of relatively low numbers of human cells in a murine background. We report here a duplex polymerase chain reaction (PCR) approach involving the coamplification of human-and mouse-specific repetitive sequences. The determination of product ratios compensates against variations in sample quality and enables quantitation from >50% down to 0.01% human-in-mouse from a single reaction. Product ratios are determined by standard electrophoresis of end-stage PCR reactions followed by image analysis techniques using freely available software, with no requirement for real-time PCR. The approach has been used to analyze tissue from mice transplanted with human cells and cocultures between differentiating mouse embryonal stem cells and human umbilical cord blood cells.
Collapse
Affiliation(s)
- Oliver Pelz
- Laboratory of Molecular Medicine, Interdisiplinary Centre for Clinical Research, University of Leipzig Faculty of Medicine, Germany
| | | | | | | | | | | | | | | | | |
Collapse
|
|
20
|
Kania G, Blyszczuk P, Jochheim A, Ott M, Wobus AM. Generation of glycogen- and albumin-producing hepatocyte-like cells from embryonic stem cells. Biol Chem 2005; 385:943-53. [PMID: 15551869 DOI: 10.1515/bc.2004.123] [Citation(s) in RCA: 53] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/12/2023]
Abstract
We present a novel two-step protocol for the differentiation of embryonic stem (ES) cells into the hepatic lineage. Differentiated hepatocyte-like cells express genes and proteins characteristic for endodermal and hepatic cells and acquire a functional hepatic phenotype as demonstrated by albumin secretion and glycogen storage. During differentiation, alpha-fetoprotein, albumin, transthyretin, alpha-1-antitrypsin, cytochrome P450 subunits 2b9 and 2b13 and tyrosine aminotransferase transcripts are upregulated. Quantitative RT-PCR data revealed a fetal hepatic phenotype corresponding to day 13-14 of liver development. Terminally differentiated hepatocyte-like cells show a bi-nucleated, cuboidal morphology labeled by albumin, alpha-1-antitrypsin, liver amylase, dipeptidyl peptidase IV, c-met and cytokeratin 18. ES-derived intermediate cell types transiently and partially co-express nestin with albumin and alpha-fetoprotein, respectively, but not cytokeratin 19. This finding suggests an ES-derived potential hepatic progenitor cell type, which is partially nestin-, albumin- and alpha-fetoprotein-positive, but cytokeratin 19-negative.
Collapse
Affiliation(s)
- Gabriela Kania
- In vitro Differentiation Group, IPK Gatersleben, D-06466 Gatersleben, Germany
| | | | | | | | | |
Collapse
|
|
21
|
Liu YY, Brent GA. Thyroid hormone-dependent gene expression in differentiated embryonic stem cells and embryonal carcinoma cells: identification of novel thyroid hormone target genes by deoxyribonucleic acid microarray analysis. Endocrinology 2005; 146:776-83. [PMID: 15550503 DOI: 10.1210/en.2004-1177] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/27/2022]
Abstract
T3 is required for normal early development, but relatively few T3-responsive target genes have been identified. In general, in vitro stem cell differentiation techniques stimulate a wide range of developmental programs, including thyroid hormone receptor (TR) pathways. We developed several in vitro stem cell models to more specifically identify TR-mediated gene expression in early development. We found that embryonic carcinoma (EC) cells have reduced T3 nuclear binding capacity and only modestly express the known T3 target genes, neurogranin (RC3) and Ca2+/calmodulin-dependent protein kinase IV (CaMKIV), in response to T3. Full T3 induction in transient transfection of EC cells was restored with cotransfection of a TR expression vector. We, therefore, performed gene expression profiles in wild-type embryonic stem (ES) cells compared with expression in cells with deficient (EC) or mutant TR (TRalpha P398H mutant ES cells), to identify T3 target genes. T3 stimulation of wild-type ES cells altered mRNA expression of 610 known genes (26% of those studied), although only approximately 60 genes (1%) met criteria for direct T3 stimulation based on the magnitude of induction and requirement for the presence of TR. We selected five candidate T3 target genes, neurexophilin 2, spermatid perinuclear RNA-binding protein (SPNR), kallikrein-binding protein (KBP), prostate-specific membrane antigen (PSMA), and synaptotagmin II, for more detailed study. T3 responsiveness of these genes was evaluated in both in vitro endogenous gene expression and in vivo mouse model systems. These genes identified in a novel stem cell system, including those induced and repressed in response to T3, may mediate thyroid hormone actions in early development.
Collapse
Affiliation(s)
- Yan-Yun Liu
- Molecular Endocrinology Laboratory, VA Greater Los Angeles Healthcare System, Building 114, Room 230, 11301 Wilshire Boulevard, Los Angeles, California 90073, USA
| | | |
Collapse
|
|
22
|
Shefer G, Wleklinski-Lee M, Yablonka-Reuveni Z. Skeletal muscle satellite cells can spontaneously enter an alternative mesenchymal pathway. J Cell Sci 2004; 117:5393-404. [PMID: 15466890 DOI: 10.1242/jcs.01419] [Citation(s) in RCA: 235] [Impact Index Per Article: 11.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
We show that muscle satellite cells, traditionally considered as committed myogenic precursors, are comprised of Pax7-expressing progenitors that preserve a mesenchymal repertoire extending beyond a mere myogenic potential. Mouse satellite cells from freshly isolated single myofibers, cultured individually in serum-rich growth medium, produced myogenic and non-myogenic clones. Only the myogenic clones expressed muscle-specific transcription factors and formed myotubes. Pax7 was initially expressed in all clones, but subsequently was associated only with the myogenic clones. Some cells in the non-myogenic clones expressed alpha-smooth muscle actin and nestin whereas others differentiated into mature adipocytes. This type of cell composition mirrors characteristics of mesenchymal stem cell progeny. Overall, individual myofibers persistently gave rise to both clonal phenotypes, but the ratio of myogenic to non-myogenic clones randomly varied among fibers. This randomness indicates that clonal dichotomy reflects satellite cell suppleness rather than pre-fated cell heterogeneity. We conclude that satellite cells possess mesenchymal plasticity, being able to commit either to myogenesis or to a mesenchymal alternative differentiation (MAD) program.
Collapse
Affiliation(s)
- Gabi Shefer
- Department of Biological Structure, School of Medicine, University of Washington, Seattle, WA 98195, USA
| | | | | |
Collapse
|
|
23
|
Abstract
CONTEXT Curative therapy for diabetes mellitus mainly implies replacement of functional insulin-producing pancreatic beta cells, with pancreas or islet-cell transplants. However, shortage of donor organs spurs research into alternative means of generating beta cells from islet expansion, encapsulated islet xenografts, human islet cell-lines, and stem cells. Stem-cell therapy here implies the replacement of diseased or lost cells from progeny of pluripotent or multipotent cells. Both embryonic stem cells (derived from the inner cell mass of a blastocyst) and adult stem cells (found in the postnatal organism) have been used to generate surrogate beta cells or otherwise restore beta-cell functioning. STARTING POINT Recently, Andreas Lechner and colleagues failed to see transdifferentiation into pancreatic beta cells after transplantation of bone-marrow cells into mice (Diabetes 2004; 53: 616-23). Last year, Jayaraj Rajagopal and colleagues failed to derive beta cells from embryonic stem cells (Science 2003; 299: 363). However, others have seen such effects. WHERE NEXT? As in every emerging field in biology, early reports seem confusing and conflicting. Embryonic and adult stem cells are potential sources for beta-cell replacement and merit further scientific investigation. Discrepancies between different results need to be reconciled. Fundamental processes in determining the differentiation pathways of stem cells remain to be elucidated, so that rigorous and reliable differentiation protocols can be established. Encouraging studies in rodent models may ultimately set the stage for large-animal studies and translational investigation.
Collapse
Affiliation(s)
- Mehboob A Hussain
- Liver and Stem Cell Research Laboratory, Division of Digestive Diseases, Department of Internal Medicine, Beth Israel Medical Center, Albert Einstein College of Medicine, New York, NY 10003, USA.
| | | |
Collapse
|
|
24
|
Yao ZX, Qin ML, Liu JJ, Chen XS, Zhou DS. In vitro cultivation of human fetal pancreatic ductal stem cells and their differentiation into insulin-producing cells. World J Gastroenterol 2004; 10:1452-6. [PMID: 15133852 PMCID: PMC4656283 DOI: 10.3748/wjg.v10.i10.1452] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
AIM: To isolate, culture and identify the human fetal pancreatic ductal stem cells in vitro, and to observe the potency of these multipotential cells differentiation into insulin-producing cells.
METHODS: The human fetal pancreas was digested by 1 g/L collagease type IV and then 2.5 g/L trypsin was used to isolate the pancreatic ductal stem cells, followed by culture in serum-free, glucose-free DMEM media with some additional chemical substrates in vitro (according to the different stage). The cells were induced by glucose-free (control), 5 mmol/L, 17.8 mmol/L and 25 mmol/L glucose, respectively. The cell types of differentiated cells were identified using immunocytochemical staining.
RESULTS: The shape of human fetal pancreatic ductal stem cells cultured in vitro was firstly fusiform in the first 2 wk, and became monolayer and cobblestone pattern after another 3 to 4 wk. After induced and differentiated by the glucose of different concentrations for another 1 to 2 wk, the cells formed the pancreatic islet-like structures. The identification and potency of these cells were then identified by using the pancreatic ductal stem cell marker, cytokeratin-19 (CK-19), pancreatic β cell marker, insulin and pancreatic α cell marker, glucagons with immunocytochemical staining. At the end of the second week, 95.2% of the cells were positive for CK-19 immunoreactivity. Up to 22.7% of the cells induced by glucose were positive for insulin immunoreactivity, and less than 3.8% of the cells were positive for glucagon immunoreactivity in pancreatic islet-like structures. The positive ratio of immunoreactive staining was dependent on the concentration of glucose, and it was observed that the 17.8 mmol/L glucose stimulated effectively to produce insulin- and glucagons-producing cells.
CONCLUSION: The human fetal pancreatic ductal stem cells are capable of proliferation in vitro. These cells have multidifferentiation potential and can be induced by glucose and differentiated into insulin-producing cells in vitro.
Collapse
Affiliation(s)
- Zhong-Xiang Yao
- Department of Histology and Embryology, The Third Military Medical University, Chongqing 400038, China.
| | | | | | | | | |
Collapse
|