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Luo Y, Yu P, Liu J. The efficiency of stem cell differentiation into functional beta cells for treating insulin-requiring diabetes: Recent advances and current challenges. Endocrine 2024; 86:1-14. [PMID: 38730069 DOI: 10.1007/s12020-024-03855-8] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/14/2024] [Accepted: 04/29/2024] [Indexed: 05/12/2024]
Abstract
In recent years, the potential of stem cells (SCs) to differentiate into various types of cells, including β-cells, has led to a significant boost in development. The efficiency of this differentiation process and the functionality of the cells post-transplantation are crucial factors for the success of stem cell therapy in diabetes. Herein, this article reviews the current advances and challenges faced by stem cell differentiation into functional β-cells for diabetes treatment. In vitro, researchers have sought to enhance the differentiation efficiency of functional β-cells by mimicking the normal pancreatic development process, using gene manipulation, pharmacological and culture conditions stimulation, three-dimensional (3D) and organoid culture, or sorting for functional β-cells based on mature islet cell markers. Furthermore, in vivo studies have also looked at suitable transplantation sites, the enhancement of the transplantation microenvironment, immune modulation, and vascular function reconstruction to improve the survival rate of functional β-cells, thereby enhancing the treatment of diabetes. Despite these advancements, developing stem cells to produce functional β-cells for efficacious diabetes treatment is a continuous research endeavor requiring significant multidisciplinary collaboration, for the stem-cell-derived beta cells to evolve into an effective cellular therapy.
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Affiliation(s)
- Yunfei Luo
- Department of Metabolism and Endocrinology, The Second Affiliated Hospital, Jiangxi Medical College, Nanchang University, Nanchang, China
| | - Peng Yu
- Department of Metabolism and Endocrinology, The Second Affiliated Hospital, Jiangxi Medical College, Nanchang University, Nanchang, China
| | - Jianping Liu
- Department of Metabolism and Endocrinology, The Second Affiliated Hospital, Jiangxi Medical College, Nanchang University, Nanchang, China.
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Nihad M, Shenoy P S, Bose B. Spontaneous Efficient Differentiation of Human Pluripotent Stem Cells (hPSC) Upon Co-culture of hPSCs with Human Neonatal Foreskin Fibroblasts in 3D. Methods Mol Biol 2024. [PMID: 39316337 DOI: 10.1007/7651_2024_569] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 09/25/2024]
Abstract
Pluripotent stem cells (PSCs) form well-formed embryoid bodies (EBs) in 3D culture. These EBs are formed in culture media lacking leukemia inhibitory factor (LIF) or basic fibroblast growth factor (bFGF) in mouse and human PSCs, respectively. EBs are excellent technical tools for understanding developmental biology and inducing controlled differentiation in succeeding experimental steps. Technically speaking, EBs are spontaneously differentiated PSCs in 3D and exhibit all three lineages in a time-point/sequential manner. For example, ectoderm will form first, followed by mesoderm and endoderm. We have attempted to co-culture human neonatal foreskin-derived fibroblast cells in our laboratory with the PSCs first in 2D conditions followed by the induction of EBs (PSC+fibroblasts co-cultured) in low attachment dishes. We also performed spontaneous differentiation of such EBs (co-cultured with fibroblasts). We checked the presence of markers of various lineages, namely, ectoderm, mesoderm, and endoderm in days 6, 10, and 12 day EBs. We have also compared the fibroblast co-cultured EBs, along with control EBs (derived from only PSCs). This co-culture system mimics the natural conditions of uterine implantation and the role of the endometrial fibroblasts in the induction of further embryonic development. The fibroblast co-cultured iPSC EBs had better roundness scores than the normal iPSC EBs and had a higher expression of lineage-specific markers.
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Affiliation(s)
- Muhammad Nihad
- Stem Cells and Regenerative Medicine Centre, Yenepoya Research Centre, Yenepoya (Deemed to be University), Mangalore, Karnataka, India
| | - Sudheer Shenoy P
- Stem Cells and Regenerative Medicine Centre, Yenepoya Research Centre, Yenepoya (Deemed to be University), Mangalore, Karnataka, India
| | - Bipasha Bose
- Stem Cells and Regenerative Medicine Centre, Yenepoya Research Centre, Yenepoya (Deemed to be University), Mangalore, Karnataka, India.
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Bose B, Nihad M, P SS. Pluripotent stem cells: Basic biology or else differentiations aimed at translational research and the role of flow cytometry. Cytometry A 2023; 103:368-377. [PMID: 36918734 DOI: 10.1002/cyto.a.24726] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/21/2022] [Revised: 01/19/2023] [Accepted: 02/25/2023] [Indexed: 03/16/2023]
Abstract
Pluripotent stem cell research has revolutionized the modern era for the past 14 years with the advent of induced pluripotent stem cells. Before this time, scientists had access to human and mouse embryonic stem cells primarily for basic research and an attempt towards lineage-specific differentiations for cell therapy applications. Regarding pluripotent stem cells, expression of bonafide marker proteins such as Oct4, Nanog, Sox2, Klf4, c-Myc, and Lin28 have been considered giving a perfect readout for pluripotent stem cells and assessed using an analytical flow cytometer. In addition to the intracellular markers, surface markers such as stage-specific embryonic antigen-1 for mouse cells and SSEA-4 for human cells are needed to sort pure populations of stem cells for further downstream applications for cell therapy. The surface marker SSEA-4 is the most appropriate for obtaining pure populations of human pluripotent stem cells. When differentiated in a controlled manner using growth factors or small molecules, it is mandatory to assess the downregulation of pluripotency markers (Oct4, Nanog, Sox2, and Klf4) with subsequent up-regulation of stage-specific differentiation markers. Such assessments are done using flow cytometry. Pluripotent stem cells have a high teratoma-forming potential in vivo. Small amounts of undifferentiated PSCs might lead to dangerous teratomas upon transplantation if leftover in the pool of differentiated cells. Hence, flow cytometry is essential for sorting out PSC populations with teratoma-forming potential. The pure populations of differentiated progenitors need to be flow-sorted before differentiating them further for cell therapy applications. For example, Glycoprotein 2 is a specific cell-surface marker for pancreatic progenitors that enables one to sort the pancreatic progenitors differentiated from human PSCs. Taken together, analytical flow cytometry, and cell sorting provide indispensable tools in PSC research and cell therapy.
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Affiliation(s)
- Bipasha Bose
- Stem Cells and Regenerative Medicine Centre, Yenepoya Research Centre, Yenepoya (Deemed to be University), Mangalore, Karnataka, India
| | - Muhammad Nihad
- Stem Cells and Regenerative Medicine Centre, Yenepoya Research Centre, Yenepoya (Deemed to be University), Mangalore, Karnataka, India
| | - Sudheer Shenoy P
- Stem Cells and Regenerative Medicine Centre, Yenepoya Research Centre, Yenepoya (Deemed to be University), Mangalore, Karnataka, India
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Rao SS, Venkatesan J, Yuvarajan S, Rekha PD. Self-assembled polyelectrolyte complexes of chitosan and fucoidan for sustained growth factor release from PRP enhance proliferation and collagen deposition in diabetic mice. Drug Deliv Transl Res 2022; 12:2838-2855. [PMID: 35445942 DOI: 10.1007/s13346-022-01144-3] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 02/28/2022] [Indexed: 02/07/2023]
Abstract
Diabetic wound management is a serious health care challenge due to higher rates of relapse, expensive treatment approaches, and poor healing outcomes. Among cell-based therapies, use of platelet-rich plasma (PRP) has been shown to be effective for diabetic wounds, but its poor shelf-life limits its clinical use. Here, we demonstrate a simple but effective polymer system to increase the shelf-life of PRP by developing a polyelectrolyte complex with dropwise addition of chitosan solution containing PRP by simple mixing at room temperature. Thus, prepared chitosan-fucoidan (CF) carrier complex encapsulated more than 95% of the loaded PRP. The resulting CF/PRP colloids were spherical in shape and ensured extended PRP release up to 72 h at 37 °C. Routine characterization (FT-IR, XRD, SEM) showed the material properties. The biological assays showed that CF complexes were biocompatible while CF/PRP enhanced the proliferation of fibroblasts and keratinocytes via higher Ki67 expression and fibroblast migration. Further investigations using a diabetic mouse model demonstrated significantly higher wound contraction and histopathological observations showed increased fibroblast migration, and collagen and cytokeratin deposition in treatment groups. The results are suggestive of the efficacy of CF/PRP as a cost-effective topical formulation for the sustained delivery of growth factors in treating chronic diabetic wounds.
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Affiliation(s)
- Sneha Subramanya Rao
- Yenepoya Research Centre, Yenepoya (Deemed To Be University), Deralakatte, Mangalore, Karnataka, 575018, India
| | - Jayachandran Venkatesan
- Yenepoya Research Centre, Yenepoya (Deemed To Be University), Deralakatte, Mangalore, Karnataka, 575018, India
| | - Subramaniyan Yuvarajan
- Yenepoya Research Centre, Yenepoya (Deemed To Be University), Deralakatte, Mangalore, Karnataka, 575018, India
| | - Punchappady-Devasya Rekha
- Yenepoya Research Centre, Yenepoya (Deemed To Be University), Deralakatte, Mangalore, Karnataka, 575018, India.
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Rao SS, Prabhu A, Kudkuli J, Surya S, Rekha P. Hyaluronic acid sustains platelet stability with prolonged growth factor release and accelerates wound healing by enhancing proliferation and collagen deposition in diabetic mice. J Drug Deliv Sci Technol 2022. [DOI: 10.1016/j.jddst.2021.102898] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/20/2022]
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Nihad M, Shenoy P S, Bose B. Cell therapy research for Diabetes: Pancreatic β cell differentiation from pluripotent stem cells. Diabetes Res Clin Pract 2021; 181:109084. [PMID: 34673084 DOI: 10.1016/j.diabres.2021.109084] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/23/2021] [Revised: 09/24/2021] [Accepted: 09/28/2021] [Indexed: 12/16/2022]
Abstract
Human pluripotent stem cells (PSCs), both embryonic and induced pluripotent stem cells (iPSCs), have been differentiated into pancreatic β isletsin vitrofor more than a decade. The idea is to get enough β cells for cell transplantation for diabetics. Finding a standard cell therapy for diabetes is essential because of the logarithmic increase in the global population of people with diabetes and the insufficient availability of the human cadaveric pancreas. Moreover, with better insights into developmental biology, thein vitroβ cell differentiation protocols have depended on thein vivoβ cell organogenesis. Various protocols for pancreatic β cell differentiation have been developed. Such protocols are based on the modulation of cell signalling pathways with growth factors, small molecules, RNAi approaches, directed differentiation using transcription factors, genome editing. Growth factor free differentiation protocols, epigenetic modulations, 3D differentiation approaches, and encapsulation strategies have also been reported for better glycemic control and endocrine modulations. Here, we have reviewed various aforementionedin vitroβ cell differentiation protocols from human PSCs, their respective comparisons, challenges, past, present, and future. The literature has been reviewed primarily from PubMed from the year 2000 till date using the mentioned keywords.
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Affiliation(s)
- Muhammad Nihad
- Stem Cells and Regenerative Medicine Centre, Yenepoya Research Centre, Yenepoya (Deemed to be University), Mangalore, Pincode-575 018, Karnataka, India
| | - Sudheer Shenoy P
- Stem Cells and Regenerative Medicine Centre, Yenepoya Research Centre, Yenepoya (Deemed to be University), Mangalore, Pincode-575 018, Karnataka, India
| | - Bipasha Bose
- Stem Cells and Regenerative Medicine Centre, Yenepoya Research Centre, Yenepoya (Deemed to be University), Mangalore, Pincode-575 018, Karnataka, India.
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Abstract
PURPOSE OF REVIEW Human islet transplantation has proven to be a highly effective treatment for patients with labile type 1 diabetes mellitus, which can free patients from daily glucose monitoring and insulin injections. However, the shortage of islet donors limits its' broad application. Porcine islet xenotransplantation presents a solution to the donor shortage and recent advances in genetic modification and immunosuppressive regimens provide renewed enthusiasm for the potential of this treatment. RECENT FINDINGS Advances in genetic editing technology are leading to multigene modified porcine islet donors with alterations in expression of known xenoantigens, modifications of their complement and coagulation systems, and modifications to gain improved immunological compatibility. Recent NHP-based trials of costimulation blockade using CD154 blockade show promising improvements in islet survival, whereas results targeting CD40 are less consistent. Furthermore, trials using IL-6 receptor antagonism have yet to demonstrate improvement in glucose control and suffer from poor graft revascularization. SUMMARY This review will detail the current status of islet xenotransplantation as a potential treatment for type I diabetes mellitus, focusing on recent advances in porcine xenogeneic islet production, assessment in nonhuman primate preclinical models, the outcome of human clinical trials and review barriers to translation of xenoislets to the clinic.
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Generation of Insulin-Producing Cells from Canine Adipose Tissue-Derived Mesenchymal Stem Cells. Stem Cells Int 2020; 2020:8841865. [PMID: 33133196 PMCID: PMC7591982 DOI: 10.1155/2020/8841865] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/03/2020] [Revised: 09/30/2020] [Accepted: 10/03/2020] [Indexed: 12/18/2022] Open
Abstract
The potential of mesenchymal stem cells (MSCs) to differentiate into nonmesodermal cells such as pancreatic beta cells has been reported. New cell-based therapy using MSCs for diabetes mellitus is anticipated as an alternative treatment option to insulin injection or islet transplantation in both human and veterinary medicine. Several protocols were reported for differentiation of MSCs into insulin-producing cells (IPCs), but no studies have reported IPCs generated from canine MSCs. The purpose of this study was to generate IPCs from canine adipose tissue-derived MSCs (AT-MSCs) in vitro and to investigate the effects of IPC transplantation on diabetic mice in vivo. Culturing AT-MSCs with the differentiation protocol under a two-dimensional culture system did not produce IPCs. However, spheroid-like small clusters consisting of canine AT-MSCs and human recombinant peptide μ-pieces developed under a three-dimensional (3D) culture system were successfully differentiated into IPCs. The generated IPCs under 3D culture condition were stained with dithizone and anti-insulin antibody. Canine IPCs also showed gene expression typical for pancreatic beta cells and increased insulin secretion in response to glucose stimulation. The blood glucose levels in streptozotocin-induced diabetic mice were decreased after injection with the supernatant of canine IPCs, but the hyperglycemic states of diabetic mice were not improved after transplanting IPCs subcutaneously or intramesenterically. The histological examination showed that the transplanted small clusters of IPCs were successfully engrafted to the mice and included cells positive for insulin by immunofluorescence. Several factors, such as the transplanted cell number, the origin of AT-MSCs, and the differentiation protocol, were considered potential reasons for the inability to improve the hyperglycemic state after IPC transplantation. These findings suggest that canine AT-MSCs can be differentiated into IPCs under a 3D culture system and IPC transplantation may be a new treatment option for dogs with diabetes mellitus.
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Zhang T, Wang H, Wang T, Wei C, Jiang H, Jiang S, Yang J, Shao J, Ma L. Pax4 synergistically acts with Pdx1, Ngn3 and MafA to induce HuMSCs to differentiate into functional pancreatic β-cells. Exp Ther Med 2019; 18:2592-2598. [PMID: 31572507 PMCID: PMC6755441 DOI: 10.3892/etm.2019.7854] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/02/2018] [Accepted: 07/05/2019] [Indexed: 02/05/2023] Open
Abstract
It has been indicated that the combination of pancreatic and duodenal homeobox 1 (Pdx1), MAF bZIP transcription factor A (MafA) and neurogenin 3 (Ngn3) was able to reprogram various cell types towards pancreatic β-like cells (pβLCs). Paired box 4 (Pax4), a transcription factor, has a key role in regulating the maturation of pancreatic β-cells (pβCs). In the present study, it was investigated whether Pax4 is able to synergistically act with Pdx1, Ngn3 and MafA to induce human umbilical cord mesenchymal stem cells (HuMSCs) to differentiate into functional pβCs in vitro. HuMSCs were isolated, cultured and separately transfected with adenovirus (Ad) expressing enhanced green fluorescence protein, Pax4 (Ad-Pax4), Pdx1+MafA+Ngn3 (Ad-3F) or Ad-Pxa4 + Ad-3F. The expression of C-peptide, insulin and glucagon was detected by immunofluorescence. The transcription of a panel of genes was determined by reverse transcription-quantitative PCR, including glucagon (GCG), insulin (INS), NK6 homeobox 1 (NKX6-1), solute carrier family 2 member 2 (SLC2A2), glucokinase (GCK), proprotein convertase subtilisin/kexin type 1 (PCSK1), neuronal differentiation 1 (NEUROD1), ISL LIM homeobox 1 (ISL 1), Pax6 and PCSK type 2 (PCSK2). Insulin secretion stimulated by glucose was determined using ELISA. The results suggested that, compared with Ad-3F alone, cells co-transfected with Ad-Pax4 and Ad-3F expressed higher levels of INS and C-peptide, as well as genes expressed in pancreatic β precursor cells, and secreted more insulin in response to high glucose. Furthermore, the expression of GCG in cells transfected with Ad-3F was depressed by Ad-Pax4. The present study demonstrated that Pax4 was able to synergistically act with the transcription factors Pdx1, Ngn3 and MafA to convert HuMSCs to functional pβLCs. HuMSCs may be potential seed cells for generating functional pβLCs in the therapy of diabetes.
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Affiliation(s)
- Ting Zhang
- Department of Hematology and Oncology, Shanghai Children's Hospital, Shanghai Jiao Tong University, Shanghai 200062, P.R. China
| | - Hongwu Wang
- Department of Pediatrics, The Second Affiliated Hospital of Shantou University Medical College, Shantou, Guangdong 515041, P.R. China
| | - Tianyou Wang
- Hematological Tumor Center, Beijing Children's Hospital Affiliated to Capital Medical University, Beijing 100045, P.R. China
| | - Chiju Wei
- Multidisciplinary Research Center, Shantou University, Shantou, Guangdong 515063, P.R. China
| | - Hui Jiang
- Department of Hematology and Oncology, Shanghai Children's Hospital, Shanghai Jiao Tong University, Shanghai 200062, P.R. China
| | - Shayi Jiang
- Department of Hematology and Oncology, Shanghai Children's Hospital, Shanghai Jiao Tong University, Shanghai 200062, P.R. China
| | - Jingwei Yang
- Department of Hematology and Oncology, Shanghai Children's Hospital, Shanghai Jiao Tong University, Shanghai 200062, P.R. China
| | - Jingbo Shao
- Department of Hematology and Oncology, Shanghai Children's Hospital, Shanghai Jiao Tong University, Shanghai 200062, P.R. China
- Correspondence to: Dr Jingbo Shao, Department of Hematology and Oncology, Shanghai Children's Hospital, Shanghai Jiao Tong University, 355 Luding Road, Shanghai 200062, P.R. China, E-mail:
| | - Lian Ma
- Department of Pediatrics, The Second Affiliated Hospital of Shantou University Medical College, Shantou, Guangdong 515041, P.R. China
- Department of Hematology and Oncology, Shenzhen Children's Hospital, Shenzhen, Guangdong 518038, P.R. China
- Shenzhen Public Service Platform of Molecular Medicine in Pediatric Hematology and Oncology, Shenzhen, Guangdong 518038, P.R. China
- Dr Lian Ma, Department of Hematology and Oncology, Shenzhen Children's Hospital, 7019 Yitian Road, Shenzhen, Guangdong 518038, P.R. China, E-mail:
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Kaya-Sezginer E, Yilmaz-Oral D, Gur S. Administration of human umbilical cord blood mononuclear cells restores bladder dysfunction in streptozotocin-induced diabetic rats. Low Urin Tract Symptoms 2019; 11:232-240. [PMID: 31207098 DOI: 10.1111/luts.12268] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/21/2018] [Revised: 02/01/2019] [Accepted: 04/24/2019] [Indexed: 12/17/2022]
Abstract
OBJECTIVE This study evaluated the effect of human umbilical cord blood mononuclear cells (HUCB-MNCs) on bladder dysfunction in streptozotocin (STZ; 35 mg/kg, i.v.)-induced diabetic rats. METHODS Adult male Sprague-Dawley rats (n = 30) were equally divided into three groups: control group, STZ-diabetic group, and HUCB-MNC-treated group (1 × 106 cells). HUCB-MNCs were isolated by density gradient centrifugation from eight healthy donors and injected into the corpus cavenosum in STZ-diabetic rats 4 weeks after the induction of diabetes. Studies were performed 4 weeks after HUCB-MNC or vehicle injection. In vitro organ bath studies were performed on bladder strips, whereas protein expression of hypoxia-inducible factor (HIF)-1α, vascular endothelial growth factor (VEGF), and α-smooth muscle actin (SMA) in the bladder and the ratio of smooth muscle cells (SMCs) to collagen were determined using western blotting and Masson trichrome staining. RESULTS Neurogenic contractions of detrusor smooth muscle strips were 55% smaller in the diabetic group than control group (P < 0.05); these contractions were normalized by HUCB-MNC treatment. In addition, HUCB-MNC treatment restored the impaired maximal carbachol-induced contractile response in detrusor strips in the diabetic group (29%; P < 0.05). HUCB-MNC treatment improved the KCl-induced contractile response in the diabetic bladder (68%; P < 0.05), but had no effect on ATP-induced contractile responses. Increased expression of HIF-1α and VEGF protein and decreased expression of α-SMA protein and the SMC/collagen ratio in diabetic rats were reversed by HUCB-MNC. CONCLUSION Administration of HUCB-MNCs facilitates bladder function recovery, which is likely related to downregulation of HIF-1α expression and attenuation of fibrosis in STZ-diabetic rats.
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Affiliation(s)
- Ecem Kaya-Sezginer
- Department of Biochemistry, Faculty of Pharmacy, Ankara University, Ankara, Turkey
| | - Didem Yilmaz-Oral
- Department of Pharmacology, Faculty of Pharmacy, Ankara University, Ankara, Turkey.,Department of Pharmacology, Faculty of Pharmacy, Cukurova University, Adana, Turkey
| | - Serap Gur
- Department of Pharmacology, Faculty of Pharmacy, Ankara University, Ankara, Turkey
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Luo Y, Xu Y, Wang ZY, Li X, Xing WB, Zhang TC. The Synergy of Two Factors on Insulin Expression. Cell Reprogram 2018; 20:49-54. [PMID: 29303357 DOI: 10.1089/cell.2017.0026] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022] Open
Abstract
As a potential cure for diabetes, more and more attentions have been paid to organ transplants to replace insulin therapy. As a result, many researchers have explored out many programs to get insulin-producing cells (IPCs) to replace the defective β cells. Currently, more and more new induction methods are being proposed, and at the same time, more and more possible induction molecular mechanisms are being revealed. The purpose of this study was to explore whether and how the two factors pdx-1 and myocardin affected the differentiation of rat mesenchymal stem cells (rMSCs) into IPCs. In this study, we investigated the process of transfecting myocardin and/or pdx-1 in rMSCs in vitro. The results showed that rMSCs were able to secrete insulin after cotransfected with myocardin and pdx-1. At the same time, we explored the possible mechanism that myocardin and pdx-1 coinduced rMSCs into IPCs by forming a complex to promote the transcriptional activity of insulin. Our results may provide a theoretical basis to the study of islet transplantation in the future.
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Affiliation(s)
- Ying Luo
- 1 Institute of Biology and Medicine, Wuhan University of Science and Technology , Wuhan, China
| | - Yao Xu
- 1 Institute of Biology and Medicine, Wuhan University of Science and Technology , Wuhan, China
| | - Zhen-Yu Wang
- 1 Institute of Biology and Medicine, Wuhan University of Science and Technology , Wuhan, China
| | - Xi Li
- 1 Institute of Biology and Medicine, Wuhan University of Science and Technology , Wuhan, China
| | - Wei-Bing Xing
- 1 Institute of Biology and Medicine, Wuhan University of Science and Technology , Wuhan, China
| | - Tong-Cun Zhang
- 1 Institute of Biology and Medicine, Wuhan University of Science and Technology , Wuhan, China .,2 Key Laboratory of Industrial Microbiology, Ministry of Education and Tianjin City, College of Biotechnology, Tianjin University of Science and Technology , Tianjin, China
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Vieira A, Druelle N, Avolio F, Napolitano T, Navarro-Sanz S, Silvano S, Collombat P. β-Cell Replacement Strategies: The Increasing Need for a "β-Cell Dogma". Front Genet 2017. [PMID: 28634486 PMCID: PMC5459879 DOI: 10.3389/fgene.2017.00075] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
Type 1 diabetes is an auto-immune disease resulting in the loss of pancreatic β-cells and, consequently, in chronic hyperglycemia. Insulin supplementation allows diabetic patients to control their glycaemia quite efficiently, but treated patients still display an overall shortened life expectancy and an altered quality of life as compared to their healthy counterparts. In this context and due to the ever increasing number of diabetics, establishing alternative therapies has become a crucial research goal. Most current efforts therefore aim at generating fully functional insulin-secreting β-like cells using multiple approaches. In this review, we screened the literature published since 2011 and inventoried the selected markers used to characterize insulin-secreting cells generated by in vitro differentiation of stem/precursor cells or by means of in vivo transdifferentiation. By listing these features, we noted important discrepancies when comparing the different approaches for the initial characterization of insulin-producing cells as true β-cells. Considering the recent advances achieved in this field of research, the necessity to establish strict guidelines has become a subject of crucial importance, especially should one contemplate the next step, which is the transplantation of in vitro or ex vivo generated insulin-secreting cells in type 1 diabetic patients.
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Affiliation(s)
- Andhira Vieira
- Centre National de la Recherche Scientifique, Institut National de la Santé et de la Recherche Médicale, iBV, Université Côte d'AzurNice, France
| | - Noémie Druelle
- Centre National de la Recherche Scientifique, Institut National de la Santé et de la Recherche Médicale, iBV, Université Côte d'AzurNice, France
| | - Fabio Avolio
- Centre National de la Recherche Scientifique, Institut National de la Santé et de la Recherche Médicale, iBV, Université Côte d'AzurNice, France
| | - Tiziana Napolitano
- Centre National de la Recherche Scientifique, Institut National de la Santé et de la Recherche Médicale, iBV, Université Côte d'AzurNice, France
| | - Sergi Navarro-Sanz
- Centre National de la Recherche Scientifique, Institut National de la Santé et de la Recherche Médicale, iBV, Université Côte d'AzurNice, France
| | - Serena Silvano
- Centre National de la Recherche Scientifique, Institut National de la Santé et de la Recherche Médicale, iBV, Université Côte d'AzurNice, France
| | - Patrick Collombat
- Centre National de la Recherche Scientifique, Institut National de la Santé et de la Recherche Médicale, iBV, Université Côte d'AzurNice, France
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Manzar GS, Kim EM, Zavazava N. Demethylation of induced pluripotent stem cells from type 1 diabetic patients enhances differentiation into functional pancreatic β cells. J Biol Chem 2017; 292:14066-14079. [PMID: 28360105 DOI: 10.1074/jbc.m117.784280] [Citation(s) in RCA: 29] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/03/2017] [Revised: 03/29/2017] [Indexed: 12/31/2022] Open
Abstract
Type 1 diabetes (T1D) can be managed by transplanting either the whole pancreas or isolated pancreatic islets. However, cadaveric pancreas is scarcely available for clinical use, limiting this approach. As such, there is a great need to identify alternative sources of clinically usable pancreatic tissues. Here, we used induced pluripotent stem (iPS) cells derived from patients with T1D to generate glucose-responsive, insulin-producing cells (IPCs) via 3D culture. Initially, T1D iPS cells were resistant to differentiation, but transient demethylation treatment significantly enhanced IPC yield. The cells responded to high-glucose stimulation by secreting insulin in vitro The shape, size, and number of their granules, as observed by transmission electron microscopy, were identical to those found in cadaveric β cells. When the IPCs were transplanted into immunodeficient mice that had developed streptozotocin-induced diabetes, they promoted a dramatic decrease in hyperglycemia, causing the mice to become normoglycemic within 28 days. None of the mice died or developed teratomas. Because the cells are derived from "self," immunosuppression is not required, providing a much safer and reliable treatment option for T1D patients. Moreover, these cells can be used for drug screening, thereby accelerating drug discovery. In conclusion, our approach eliminates the need for cadaveric pancreatic tissue.
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Affiliation(s)
- Gohar S Manzar
- From the Department of Internal Medicine and University of Iowa, Iowa City, Iowa; Department of Biomedical Engineering, University of Iowa, Iowa City, Iowa; Veterans Affairs Medical Center, Iowa City, Iowa,; Mayo Clinic College of Medicine, Rochester, Minnesota, and Daejeon 34114, Republic of Korea
| | - Eun-Mi Kim
- From the Department of Internal Medicine and University of Iowa, Iowa City, Iowa; Veterans Affairs Medical Center, Iowa City, Iowa,; Predictive Model Research Center, Korea Institute of Toxicology, Daejeon 34114, Republic of Korea
| | - Nicholas Zavazava
- From the Department of Internal Medicine and University of Iowa, Iowa City, Iowa; Department of Biomedical Engineering, University of Iowa, Iowa City, Iowa; Veterans Affairs Medical Center, Iowa City, Iowa,.
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14
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Wallet MA, Santostefano KE, Terada N, Brusko TM. Isogenic Cellular Systems Model the Impact of Genetic Risk Variants in the Pathogenesis of Type 1 Diabetes. Front Endocrinol (Lausanne) 2017; 8:276. [PMID: 29093700 PMCID: PMC5651267 DOI: 10.3389/fendo.2017.00276] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/28/2017] [Accepted: 10/02/2017] [Indexed: 12/31/2022] Open
Abstract
At least 57 independent loci within the human genome confer varying degrees of risk for the development of type 1 diabetes (T1D). The majority of these variants are thought to contribute to overall genetic risk by modulating host innate and adaptive immune responses, ultimately resulting in a loss of immunological tolerance to β cell antigens. Early efforts to link specific risk variants with functional alterations in host immune responses have employed animal models or genotype-selected individuals from clinical bioresource banks. While some notable genotype:phenotype associations have been described, there remains an urgent need to accelerate the discovery of causal variants and elucidate the molecular mechanisms by which susceptible alleles alter immune functions. One significant limitation has been the inability to study human T1D risk loci on an isogenic background. The advent of induced pluripotent stem cells (iPSCs) and genome-editing technologies have made it possible to address a number of these outstanding questions. Specifically, the ability to drive multiple cell fates from iPSC under isogenic conditions now facilitates the analysis of causal variants in multiple cellular lineages. Bioinformatic analyses have revealed that T1D risk genes cluster within a limited number of immune signaling pathways, yet the relevant immune cell subsets and cellular activation states in which candidate risk genes impact cellular activities remain largely unknown. In this review, we summarize the functional impact of several candidate risk variants on host immunity in T1D and present an isogenic disease-in-a-dish model system for interrogating risk variants, with the goal of expediting precision therapeutics in T1D.
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Affiliation(s)
- Mark A. Wallet
- Department of Pathology, Immunology, and Laboratory Medicine, University of Florida Diabetes Institute, College of Medicine, Gainesville, FL, United States
| | - Katherine E. Santostefano
- Department of Pathology, Immunology, and Laboratory Medicine, University of Florida Diabetes Institute, College of Medicine, Gainesville, FL, United States
| | - Naohiro Terada
- Department of Pathology, Immunology, and Laboratory Medicine, University of Florida Diabetes Institute, College of Medicine, Gainesville, FL, United States
| | - Todd M. Brusko
- Department of Pathology, Immunology, and Laboratory Medicine, University of Florida Diabetes Institute, College of Medicine, Gainesville, FL, United States
- *Correspondence: Todd M. Brusko,
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15
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Shao Y, Chen QZ, Zeng YH, Li Y, Ren WY, Zhou LY, Liu RX, Wu K, Yang JQ, Deng ZL, Yu Y, Sun WJ, He BC. All-trans retinoic acid shifts rosiglitazone-induced adipogenic differentiation to osteogenic differentiation in mouse embryonic fibroblasts. Int J Mol Med 2016; 38:1693-1702. [PMID: 27779644 PMCID: PMC5117762 DOI: 10.3892/ijmm.2016.2782] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/05/2016] [Accepted: 10/14/2016] [Indexed: 12/19/2022] Open
Abstract
Rosiglitazone (RSG) is a potent drug used in the treatment of insulin resistance; however, it is associated with marked skeletal toxicity. RSG-induced osteoporosis may contribute to the promotion of adipogenic differentiation at the expense of osteogenic differentiation in bone marrow stromal cells. The aim of this study was to investigate whether RSG-induced bone toxicity can be reversed by combined treatment with all-trans retinoic acid (ATRA). We examined different osteogenic markers in mouse embryonic fibroblasts (MEFs) following treatment with RSG, ATRA, or RSG and ATRA in combination. We examined the effects of RSG and/or ATRA on ectopic bone formation, and dissected the possible molecular mechanisms underlying this process. We found that ATRA or RSG both induced alkaline phosphatase (ALP) activity in the MEFs, and that the ATRA-induced ALP activity was enhanced by RSG and vice versa. However, only the combination of RSG and ATRA increased the expression of osteopontin and osteocalcin, promoted matrix mineralization, and induced ectopic ossification in MEFs. Mechanistically, we found that the osteogenic differentiation induced by the combination of RSG and ATRA may be mediated partly by suppressing RSG-induced adipogenic differentiation and activating bone morphogenetic protein (BMP)/Smad signaling. On the whole, our findings demonstrate that RSG in combination with ATRA promotes the commitment of MEFs to the osteoblast lineage. Thus, the combination of these two agents may prove to be a promising and novel therapeutic regimen for insulin resistance without skeletal toxicity. It may also be a better strategy with which to prevent RSG-induced osteoporosis.
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Affiliation(s)
- Ying Shao
- Department of Pharmacology, School of Pharmacy, Chongqing Medical University, Chongqing, Sichuan 400016, P.R. China
| | - Qian-Zhao Chen
- Department of Pharmacology, School of Pharmacy, Chongqing Medical University, Chongqing, Sichuan 400016, P.R. China
| | - Yu-Hua Zeng
- Department of Pharmacology, School of Pharmacy, Chongqing Medical University, Chongqing, Sichuan 400016, P.R. China
| | - Yang Li
- Department of Pharmacology, School of Pharmacy, Chongqing Medical University, Chongqing, Sichuan 400016, P.R. China
| | - Wen-Yan Ren
- Department of Pharmacology, School of Pharmacy, Chongqing Medical University, Chongqing, Sichuan 400016, P.R. China
| | - Lin-Yun Zhou
- Department of Pharmacology, School of Pharmacy, Chongqing Medical University, Chongqing, Sichuan 400016, P.R. China
| | - Rong-Xin Liu
- Department of Pharmacology, School of Pharmacy, Chongqing Medical University, Chongqing, Sichuan 400016, P.R. China
| | - Ke Wu
- Department of Pharmacology, School of Pharmacy, Chongqing Medical University, Chongqing, Sichuan 400016, P.R. China
| | - Jun-Qing Yang
- Department of Pharmacology, School of Pharmacy, Chongqing Medical University, Chongqing, Sichuan 400016, P.R. China
| | - Zhong-Liang Deng
- Key Laboratory for Biochemistry and Molecular Pharmacology of Chongqing, Chongqing Medical University, Chongqing, Sichuan 400016, P.R. China
| | - Yu Yu
- Department of Pharmacology, School of Pharmacy, Chongqing Medical University, Chongqing, Sichuan 400016, P.R. China
| | - Wen-Juan Sun
- Department of Pharmacology, School of Pharmacy, Chongqing Medical University, Chongqing, Sichuan 400016, P.R. China
| | - Bai-Cheng He
- Department of Pharmacology, School of Pharmacy, Chongqing Medical University, Chongqing, Sichuan 400016, P.R. China
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16
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Wong CH, Li YJ, Chen YC. Therapeutic potential of targeting acinar cell reprogramming in pancreatic cancer. World J Gastroenterol 2016; 22:7046-57. [PMID: 27610015 PMCID: PMC4988312 DOI: 10.3748/wjg.v22.i31.7046] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/09/2016] [Revised: 06/10/2016] [Accepted: 06/28/2016] [Indexed: 02/06/2023] Open
Abstract
Pancreatic ductal adenocarcinoma (PDAC) is a common pancreatic cancer and the fourth leading cause of cancer death in the United States. Treating this life-threatening disease remains challenging due to the lack of effective prognosis, diagnosis and therapy. Apart from pancreatic duct cells, acinar cells may also be the origin of PDAC. During pancreatitis or combined with activating KRas(G12D) mutation, acinar cells lose their cellular identity and undergo a transdifferentiation process called acinar-to-ductal-metaplasia (ADM), forming duct cells which may then transform into pancreatic intraepithelial neoplasia (PanIN) and eventually PDAC. During ADM, the activation of mitogen-activated protein kinases, Wnt, Notch and phosphatidylinositide 3-kinases/Akt signaling inhibits the transcription of acinar-specific genes, including Mist and amylase, but promotes the expression of ductal genes, such as cytokeratin-19. Inhibition of this transdifferentiation process hinders the development of PanIN and PDAC. In addition, the transdifferentiated cells regain acinar identity, indicating ADM may be a reversible process. This provides a new therapeutic direction in treating PDAC through cancer reprogramming. Many studies have already demonstrated the success of switching PanIN/PDAC back to normal cells through the use of PD325901, the expression of E47, and the knockdown of Dickkopf-3. In this review, we discuss the signaling pathways involved in ADM and the therapeutic potential of targeting reprogramming in order to treat PDAC.
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17
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Sunitha MM, Srikanth L, Santhosh Kumar P, Chandrasekhar C, Sarma PVGK. In vitro differentiation potential of human haematopoietic CD34(+) cells towards pancreatic β-cells. Cell Biol Int 2016; 40:1084-93. [PMID: 27514733 DOI: 10.1002/cbin.10654] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/22/2016] [Accepted: 07/17/2016] [Indexed: 11/06/2022]
Abstract
Haematopoietic stem cells (HSCs) possess multipotent ability to differentiate into various types of cells on providing appropriate niche. In the present study, the differentiating potential of human HSCs into β-cells of islets of langerhans was explored. Human HSCs were apheretically isolated from a donor and cultured. Phenotypic characterization of CD34 glycoprotein in the growing monolayer HSCs was confirmed by immunocytochemistry and flow cytometry techniques. HSCs were induced by selection with beta cell differentiating medium (BDM), which consists of epidermal growth factor (EGF), fibroblast growth factor (FGF), transferrin, Triiodo-l-Tyronine, nicotinamide and activin A. Distinct morphological changes of differentiated cells were observed on staining with dithizone (DTZ) and expression of PDX1, insulin and synaptophysin was confirmed by immunocytochemistry. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis revealed distinct expression of specific β-cell markers, pancreatic and duodenal homeobox-1 (PDX1), glucose transporter-2 (GLUT-2), synaptophysin (SYP) and insulin (INS) in these differentiated cells compared to HSCs. Further, these cells exhibited elevated expression of INS gene at 10 mM glucose upon inducing with different glucose concentrations. The prominent feature of the obtained β-cells was the presence of glucose sensors, which was determined by glucokinase activity and high glucokinase activity compared with CD34(+) stem cells. These findings illustrate the differentiation of CD34(+) HSCs into β-cells of islets of langerhans.
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Affiliation(s)
- Manne Mudhu Sunitha
- Stem Cell laboratory, Department of Biotechnology, Sri Venkateswara Institute of Medical Sciences, Tirupati, 517 507, Andhra Pradesh, India
| | - Lokanathan Srikanth
- Stem Cell laboratory, Department of Biotechnology, Sri Venkateswara Institute of Medical Sciences, Tirupati, 517 507, Andhra Pradesh, India
| | - Pasupuleti Santhosh Kumar
- Stem Cell laboratory, Department of Biotechnology, Sri Venkateswara Institute of Medical Sciences, Tirupati, 517 507, Andhra Pradesh, India
| | - Chodimella Chandrasekhar
- Department of Haematology, Sri Venkateswara Institute of Medical Sciences, Tirupati, Andhra Pradesh, India
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18
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Dong X, Zhang T, Liu Q, Zhu J, Zhao J, Li J, Sun B, Ding G, Hu X, Yang Z, Zhang Y, Li L. Beneficial effects of urine-derived stem cells on fibrosis and apoptosis of myocardial, glomerular and bladder cells. Mol Cell Endocrinol 2016; 427:21-32. [PMID: 26952874 DOI: 10.1016/j.mce.2016.03.001] [Citation(s) in RCA: 39] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/09/2015] [Revised: 02/17/2016] [Accepted: 03/02/2016] [Indexed: 12/19/2022]
Abstract
Urine-derived stem cells (USCs) are isolated from voided urine and display high proliferative activity and multiple differentiation potentials. The applicability of USCs in the treatment of bladder dysfunction and in cell-based urological tissue engineering has been demonstrated. Whether they could serve as a potential stem cell source for the treatment of diabetes mellitus (DM) and its complications has not been investigated. Here, we report the repairing and protective effects of USCs on pancreatic islets, the myocardium, the renal glomerulus and the bladder detrusor in diabetic rat models. Type 2 diabetic rat models were induced by means of a high fat diet and intraperitoneal injection with streptozotocin. USCs isolated from voided urine were administered via tail veins. The functional changes of pancreatic islets, left ventricle, glomerulus and bladder micturition were assessed by means of insulin tolerance tests, echocardiography, urine biochemical indexes and cystometry. The histologic changes were evaluated by hematoxylin and eosin staining, Masson's trichrome staining and TUNEL staining. Treatment with USCs significantly alleviated the histological destruction and functional decline. Although the USC treatment did not decrease fasting blood glucose to a significantly different level, the fibrosis and apoptosis of the myocardium, glomerulus and detrusor were significantly inhibited. This study indicates that administration of USCs may be useful for the treatment of the complications of DM.
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Affiliation(s)
- Xingyou Dong
- Department of Urology, Second Affiliated Hospital, Third Military Medical University, Chongqing, China
| | - Teng Zhang
- Department of Urology, Second Affiliated Hospital, Third Military Medical University, Chongqing, China
| | - Qian Liu
- Department of Urology, Second Affiliated Hospital, Third Military Medical University, Chongqing, China
| | - Jingzhen Zhu
- Department of Urology, Second Affiliated Hospital, Third Military Medical University, Chongqing, China
| | - Jiang Zhao
- Department of Urology, Second Affiliated Hospital, Third Military Medical University, Chongqing, China
| | - Jia Li
- Department of Urology, Second Affiliated Hospital, Third Military Medical University, Chongqing, China
| | - Bishao Sun
- Department of Urology, Second Affiliated Hospital, Third Military Medical University, Chongqing, China
| | - Guolin Ding
- Department of Urology, Second Affiliated Hospital, Third Military Medical University, Chongqing, China
| | - Xiaoyan Hu
- Department of Urology, Second Affiliated Hospital, Third Military Medical University, Chongqing, China
| | - Zhenxing Yang
- Department of Urology, Second Affiliated Hospital, Third Military Medical University, Chongqing, China
| | - Yuanyuan Zhang
- Wake Forest Institute of Regenerative Medicine, Wake Forest University, Winston Salem, NC, USA
| | - Longkun Li
- Department of Urology, Second Affiliated Hospital, Third Military Medical University, Chongqing, China.
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19
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Iacovacci V, Ricotti L, Menciassi A, Dario P. The bioartificial pancreas (BAP): Biological, chemical and engineering challenges. Biochem Pharmacol 2016; 100:12-27. [DOI: 10.1016/j.bcp.2015.08.107] [Citation(s) in RCA: 24] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/25/2015] [Accepted: 08/26/2015] [Indexed: 01/05/2023]
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20
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Nekoei SM, Azarpira N, Sadeghi L, Kamalifar S. In vitro differentiation of human umbilical cord Wharton’s jelly mesenchymal stromal cells to insulin producing clusters. World J Clin Cases 2015; 3:640-649. [PMID: 26244156 PMCID: PMC4517339 DOI: 10.12998/wjcc.v3.i7.640] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/10/2014] [Revised: 04/23/2015] [Accepted: 05/18/2015] [Indexed: 02/05/2023] Open
Abstract
AIM: To investigate the differentiation of human Wharton’s jelly derived mesenchymal stromal cells (WJ-MSCs) to insulin producing clusters (IPC) this study was conducted.
METHODS: The umbilical cords samples were collected from full term caesarian section mothers and the WJ-MSCS were cultured from tissue explants in High glucose-Dulbecco’s Modified Eagle Medium (H-DMEM); H-DMEM supplemented with 10% fetal bovine serum (FBS) and antibiotics. The expression of CD90, CD44, CD105, CD34 and CD133 as well as osteogenic and adipogenic differentiation of cells in appropriate medium were also evaluated. The cells were differentiated toward IPC with changing the culture medium and adding the small molecules such as nicotinic acid, epidermal growth factor, and exendin-4 during 3 wk period. The gene expression of PDX1, NGN3, Glut2, insulin was monitored by reveres transcription polymerase chain reaction method. The differentiated clusters were stained with Dithizone (DTZ) which confirms the presence of insulin granules. The insulin challenge test (low and high glucose concentration in Krebs-Ringer HEPES buffer) was also used to evaluate the functional properties of differentiated clusters.
RESULTS: WJ-MSCS were positive for mesenchymal surface markers (CD90, CD44, CD105), and negative for CD34 and CD133. The accumulation of lipid vacuoles and deposition of calcium mineral in cells were considered as adipogenic and osteogenic potential of WJ-MSCS. The cells also expressed the transcriptional factors such as Nanog and OCT4. During this three step differentiation, the WJ-MSCS morphology was gradually changed from spindle shaped cells in to epithelioid cells and eventually to three dimensional clusters. The clusters expressed PDX1, NGN3, Glut2, and insulin. The cells became bright red color when stained with DTZ and the insulin secretion was also confirmed. In glucose challenge test a significant increase in insulin secretion from 0.91 ± 0.04 μIu/mL (2.8 mmol/L glucose) to to 8.34 ± 0.45 μIu/mL (16.7 mmol/L glucose) was recorded (P < 0.05). The insulin secretion of undifferentiated WJ-MSCS was not changed in this challenge test.
CONCLUSION: WJ-MSCs have the ability to differentiate in to islet-like cells in vitro. However, this process needs further optimization in order to generate efficient and functional IPCs.
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21
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Jian RL, Mao LB, Xu Y, Li XF, Wang FP, Luo XG, Zhou H, He HP, Wang N, Zhang TC. Generation of insulin-producing cells from C3H10T1/2 mesenchymal progenitor cells. Gene 2015; 562:107-116. [PMID: 25724395 DOI: 10.1016/j.gene.2015.02.061] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/17/2014] [Revised: 02/10/2015] [Accepted: 02/18/2015] [Indexed: 12/29/2022]
Abstract
Mesenchymal stem cells (MSCs) have been reported to be an attractive source for the generation of transplantable surrogate β cells. A murine embryonic mesenchymal progenitor cell line C3H10T1/2 has been recognized as a model for MSCs, because of its multi-lineage differentiation potential. The purpose of this study was to explore whether C3H/10T1/2 cells have the potential to differentiate into insulin-producing cells (IPCs). Here, we investigated and compared the in vitro differentiation of rat MSCs and C3H10T1/2 cells into IPCs. After the cells underwent IPC differentiation, the expression of differentiation markers were detected by immunocytochemistry, reverse transcription-polymerase chain reaction (RT-PCR), quantitative real-time RT-PCR (qRT-PCR) and Western blotting. The insulin secretion was evaluated by enzyme-linked immunosorbent assay (ELISA). Furthermore, these differentiated cells were transplanted into streptozotocin-induced diabetic mice and their biological functions were tested in vivo. This study reports a 2-stage method to generate IPCs from C3H10T1/2 cells. Under specific induction conditions for 7-8 days, C3H10T1/2 cells formed three-dimensional spheroid bodies (SBs) and secreted insulin, while generation of IPCs derived from rat MSCs required a long time (more than 2 weeks). Furthermore, these IPCs derived from C3H10T1/2 cells were injected into diabetic mice and improves basal glucose, body weight and exhibited normal glucose tolerance test. The present study provided a simple and faithful in vitro model for further investigating the mechanism underlying IPC differentiation of MSCs and cell replacement therapy for diabetes.
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Affiliation(s)
- Ruo-Lei Jian
- Key Laboratory of Industrial Microbiology, Ministry of Education and Tianjin City, College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, China
| | - Li-Bin Mao
- Key Laboratory of Industrial Microbiology, Ministry of Education and Tianjin City, College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, China
| | - Yao Xu
- Key Laboratory of Industrial Microbiology, Ministry of Education and Tianjin City, College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, China
| | - Xiao-Fan Li
- Key Laboratory of Industrial Microbiology, Ministry of Education and Tianjin City, College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, China
| | - Feng-Po Wang
- Key Laboratory of Industrial Microbiology, Ministry of Education and Tianjin City, College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, China
| | - Xue-Gang Luo
- Key Laboratory of Industrial Microbiology, Ministry of Education and Tianjin City, College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, China
| | - Hao Zhou
- Key Laboratory of Industrial Microbiology, Ministry of Education and Tianjin City, College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, China
| | - Hong-Peng He
- Key Laboratory of Industrial Microbiology, Ministry of Education and Tianjin City, College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, China
| | - Nan Wang
- Key Laboratory of Industrial Microbiology, Ministry of Education and Tianjin City, College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, China.
| | - Tong-Cun Zhang
- Key Laboratory of Industrial Microbiology, Ministry of Education and Tianjin City, College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, China; Department of Biochemistry, Medical College, Wuhan University of Science and Technology, Wuhan 430081, China.
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22
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Khorsandi L, Khodadadi A, Nejad-Dehbashi F, Saremy S. Three-dimensional differentiation of adipose-derived mesenchymal stem cells into insulin-producing cells. Cell Tissue Res 2015; 361:745-53. [PMID: 25795142 DOI: 10.1007/s00441-015-2140-9] [Citation(s) in RCA: 39] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/04/2014] [Accepted: 01/28/2015] [Indexed: 01/17/2023]
Abstract
The aim of this study is to evaluate the collagen/hyaluronic acid (Col/HA) scaffold effect on the differentiation of insulin-producing cells (IPCs) from adipose-derived mesenchymal stem cells (ASCs). In this experimental study, ASCs were cultured and seeded in a Col/HA scaffold (3D culture) and then treated with induction media. After induction, the presence of IPCs was evaluated using gene expression (PDX-1, GLUT-2 and insulin) analysis and immunocytochemistry, while functional maturity was determined by measuring insulin release in response to low- and high-glucose media. The induced IPCs were morphologically similar to pancreatic islet-like cells. Expression of the islet-associated genes PDX-1, GLUT-2 and insulin genes in 3D-cultured cells was markedly higher than the 2D-cultured cells exposure differentiation media. Compared to the 2D culture of ASCs-derived IPCs, the insulin release from 3D ASCs-derived IPCs showed a nearly 4-fold (p < 0.05) increase when exposed to a high glucose (25 mmol) medium. The percentage of insulin-positive cells in the 3D experimental group showed an approximately 4-fold increase compared to the 2D experimental culture cells. The results of this study demonstrated that the COL/HA scaffold can enhance the differentiation of IPCs from rat ASCs.
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Affiliation(s)
- Layasadat Khorsandi
- Cell & Molecular Research Center, Faculty of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran, P.O. Box: 61335,
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23
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Bose B, Sudheer PS. In Vitro Differentiation of Pluripotent Stem Cells into Functional β Islets Under 2D and 3D Culture Conditions and In Vivo Preclinical Validation of 3D Islets. Methods Mol Biol 2015; 1341:257-84. [PMID: 25783769 DOI: 10.1007/7651_2015_230] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022]
Abstract
Since the advent of pluripotent stem cells, (embryonic and induced pluripotent stem cells), applications of such pluripotent stem cells are of prime importance. Indeed, scientists are involved in studying the basic biology of pluripotent stem cells, but equal impetus is there to direct the pluripotent stem cells into multiple lineages for cell therapy applications. Scientists across the globe have been successful, to a certain extent, in obtaining cells of definitive endoderm and also pancreatic β islets by differentiating human pluripotent stem cells. Pluripotent stem cell differentiation protocols aim at mimicking in vivo embryonic development. As in vivo embryonic development is a complex process and involves interplay of multiple cytokines, the differentiation protocols also involve a stepwise use of multiple cytokines. Indeed the novel markers for pancreas organogenesis serve as the roadmaps to develop new protocols for pancreatic differentiation from pluripotent stem cells. Earliest developed protocols for pancreas differentiation involved "Nestin selection pathway," a pathway common for both neuronal and pancreatic differentiation lead to the generation of cells that were a combination of cells from neuronal lineage. Eventually with the discovery of hierarchy of β cell transcription factors like Pdx1, Pax4, and Nkx2.2, forced expression of such transcription factors proved successful in converting a pluripotent stem cell into a β cell. Protocols developed almost half a decade ago to the recent ones rather involve stepwise differentiations involving various cytokines and could generate as high as 25 % functional insulin-positive cells in vitro. Most advanced protocols for β islet differentiations from human pluripotent stem cells focused on 3D culture conditions, which reportedly produced 60-65 % functional β islet cells. Here, we describe the protocol for differentiation of human pluripotent stem cells into functional β cells under both 2D and 3D culture conditions.
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Affiliation(s)
- Bipasha Bose
- Level 03, Stem Cell Biology and Tissue Engineering Division, Yenepoya Research Centre, Yenepoya University, University Road, Derlakatte, Mangalore, 575018, Karnataka, India.
| | - P Shenoy Sudheer
- Molecular Genetics and Cell Biology, School of Biological Sciences, Nanyang Technological University, NTU/SBS Lab location @ Level 2, Singapore Institute for Clinical Sciences Brenner Centre for Molecular Medicine 30 Medical Drive, Singapore, 117609, Singapore
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24
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Ren M, Shang C, Zhong X, Guo R, Lao G, Wang X, Cheng H, Min J, Yan L, Shen J. Insulin-producing cells from embryonic stem cells rescues hyperglycemia via intra-spleen migration. Sci Rep 2014; 4:7586. [PMID: 25533571 PMCID: PMC4274503 DOI: 10.1038/srep07586] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/10/2014] [Accepted: 11/24/2014] [Indexed: 02/07/2023] Open
Abstract
Implantation of embryonic stem cells (ESC)-derived insulin-producing cells has been extensively investigated for treatment of diabetes in animal models. However, the in vivo behavior and migration of transplanted cells in diabetic models remains unclear. Here we investigated the location and migration of insulin-producing cells labeled with superparamagnetic iron oxide (SPIO) using a dynamic MRI tracking method. SPIO labeled cells showed hypointense signal under the kidney subcapsules of diabetic mice on MRI, and faded gradually over the visiting time. However, new hypointense signal appeared in the spleen 1 week after transplantation, and became obvious with the time prolongation. Further histological examination proved the immigrated cells were insulin and C-peptide positive cells which were evenly distributed throughout the spleen. These intra-spleen insulin-producing cells maintained their protective effects against hyperglycemia in vivo, and these effects were reversed upon spleen removal. Transplantation of insulin-producing cells through spleen acquired an earlier blood glucose control as compared with that through kidney subcapsules. In summary, our data demonstrate that insulin-producing cells transplanted through kidney subcapsules were not located in situ but migrated into spleen, and rescues hyperglycemia in diabetic models. MRI may provide a novel tracking method for preclinical cell transplantation therapy of diabetes continuously and non-invasively.
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Affiliation(s)
- Meng Ren
- Department of Endocrinology, Sun Yat-sen Memorial Hospital of Sun Yat-sen University, Guangzhou. 510120, China
| | - Changzhen Shang
- Department of Hepatology, Sun Yat-sen Memorial Hospital of Sun Yat-sen University, Guangzhou. 510120, China
| | - Xiaomei Zhong
- Department of Radiology, Sun Yat-sen Memorial Hospital of Sun Yat-sen University, Guangzhou. 510120, China
| | - Ruomi Guo
- Department of Radiology, Sun Yat-sen Memorial Hospital of Sun Yat-sen University, Guangzhou. 510120, China
| | - Guojuan Lao
- Department of Endocrinology, Sun Yat-sen Memorial Hospital of Sun Yat-sen University, Guangzhou. 510120, China
| | - Xiaoyi Wang
- Department of Endocrinology, Sun Yat-sen Memorial Hospital of Sun Yat-sen University, Guangzhou. 510120, China
| | - Hua Cheng
- Department of Endocrinology, Sun Yat-sen Memorial Hospital of Sun Yat-sen University, Guangzhou. 510120, China
| | - Jun Min
- Department of Hepatology, Sun Yat-sen Memorial Hospital of Sun Yat-sen University, Guangzhou. 510120, China
| | - Li Yan
- Department of Endocrinology, Sun Yat-sen Memorial Hospital of Sun Yat-sen University, Guangzhou. 510120, China
| | - Jun Shen
- Department of Radiology, Sun Yat-sen Memorial Hospital of Sun Yat-sen University, Guangzhou. 510120, China
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Khorsandi L, Nejad-Dehbashi F, Ahangarpour A, Hashemitabar M. Three-dimensional differentiation of bone marrow-derived mesenchymal stem cells into insulin-producing cells. Tissue Cell 2014; 47:66-72. [PMID: 25554603 DOI: 10.1016/j.tice.2014.11.005] [Citation(s) in RCA: 31] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/24/2014] [Revised: 11/22/2014] [Accepted: 11/23/2014] [Indexed: 12/20/2022]
Abstract
Fibrin glue (FG) is used in a variety of clinical applications and in the laboratory for localized and sustained release of factors potentially important for tissue engineering. The aim of this study was to evaluate FG scaffold effect on differentiation of insulin-producing cells (IPCs) from bone marrow-derived mesenchymal stem cells (BM-MSCs). In this experimental study BM-MSCs were cultured and the cells characterized by analysis of cell surface markers using flow cytometry. BM-MSCs were seeded in FG scaffold (3D culture) and then treated with induction media. After induction, the presence of IPCs was demonstrated using gene expression profiles for pancreatic cell differentiation markers (PDX-1, GLUT-2 and insulin) and insulin detection in cytoplasm. Release of insulin by these cells was confirmed by radioimmunoassay. Expression of the islet-associated genes PDX-1, GLUT-2 and Insulin genes in 3D cultured cells was markedly higher than the 2D cultured cells exposure differentiation media. Compared to 2D culture of BM-MSCs-derived IPCs, the insulin release from 3D BM-MSCs-derived IPCs showed a nearly 3 fold (p<0.05) increase when exposed to a high glucose (25 mM) medium. Percentage of insulin positive cells in 3D experimental group showed an approximately 3.5-fold increase in compared to 2D experimental culture cells. The results of this study demonstrated that FG scaffold can enhance the differentiation of IPCs from rats BM-MSCs.
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Affiliation(s)
- Layasadat Khorsandi
- Cell & Molecular Research Center, Faculty of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran; Department of Anatomical Sciences, Faculty of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran.
| | - Fereshteh Nejad-Dehbashi
- Cell & Molecular Research Center, Faculty of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
| | - Akram Ahangarpour
- Diabetes Research Center, Health research institute and Department of Physiology, School of Medicine, Jundishapur University of Medical Sciences, Ahvaz 61335-189, Iran
| | - Mahmoud Hashemitabar
- Cell & Molecular Research Center, Faculty of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
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Tan G, Elefanty AG, Stanley EG. β-cell regeneration and differentiation: how close are we to the 'holy grail'? J Mol Endocrinol 2014; 53:R119-29. [PMID: 25385843 DOI: 10.1530/jme-14-0188] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
Diabetes can be managed by careful monitoring of blood glucose and timely delivery of exogenous insulin. However, even with fastidious compliance, people with diabetes can suffer from numerous complications including atherosclerosis, retinopathy, neuropathy, and kidney disease. This is because delivery of exogenous insulin coupled with glucose monitoring cannot provide the fine level of glucose control normally provided by endogenous β-cells in the context of intact islets. Moreover, a subset of people with diabetes lack awareness of hypoglycemic events; a status that can have grave consequences. Therefore, much effort has been focused on replacing lost or dysfunctional β-cells with cells derived from other sources. The advent of stem cell biology and cellular reprogramming strategies have provided impetus to this work and raised hopes that a β-cell replacement therapy is on the horizon. In this review, we look at two components that will be required for successful β-cell replacement therapy: a reliable and safe source of β-cells and a mechanism by which such cells can be delivered and protected from host immune destruction. Particular attention is paid to insulin-producing cells derived from pluripotent stem cells because this platform addresses the issue of scale, one of the more significant hurdles associated with potential cell-based therapies. We also review methods for encapsulating transplanted cells, a technique that allows grafts to evade immune attack and survive for a long term in the absence of ongoing immunosuppression. In surveying the literature, we conclude that there are still several substantial hurdles that need to be cleared before a stem cell-based β-cell replacement therapy for diabetes becomes a reality.
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Affiliation(s)
- Gemma Tan
- Department of Anatomy and Developmental BiologyMonash University, Building 73, Clayton, Victoria 3800, AustraliaMurdoch Childrens Research InstituteThe Royal Children's Hospital, Flemington Road, Parkville, Victoria 3052, AustraliaDepartment of PaediatricsThe Royal Children's Hospital, University of Melbourne, Flemington Road, Parkville, Victoria 3052, Australia Department of Anatomy and Developmental BiologyMonash University, Building 73, Clayton, Victoria 3800, AustraliaMurdoch Childrens Research InstituteThe Royal Children's Hospital, Flemington Road, Parkville, Victoria 3052, AustraliaDepartment of PaediatricsThe Royal Children's Hospital, University of Melbourne, Flemington Road, Parkville, Victoria 3052, Australia
| | - Andrew G Elefanty
- Department of Anatomy and Developmental BiologyMonash University, Building 73, Clayton, Victoria 3800, AustraliaMurdoch Childrens Research InstituteThe Royal Children's Hospital, Flemington Road, Parkville, Victoria 3052, AustraliaDepartment of PaediatricsThe Royal Children's Hospital, University of Melbourne, Flemington Road, Parkville, Victoria 3052, Australia Department of Anatomy and Developmental BiologyMonash University, Building 73, Clayton, Victoria 3800, AustraliaMurdoch Childrens Research InstituteThe Royal Children's Hospital, Flemington Road, Parkville, Victoria 3052, AustraliaDepartment of PaediatricsThe Royal Children's Hospital, University of Melbourne, Flemington Road, Parkville, Victoria 3052, Australia Department of Anatomy and Developmental BiologyMonash University, Building 73, Clayton, Victoria 3800, AustraliaMurdoch Childrens Research InstituteThe Royal Children's Hospital, Flemington Road, Parkville, Victoria 3052, AustraliaDepartment of PaediatricsThe Royal Children's Hospital, University of Melbourne, Flemington Road, Parkville, Victoria 3052, Australia
| | - Edouard G Stanley
- Department of Anatomy and Developmental BiologyMonash University, Building 73, Clayton, Victoria 3800, AustraliaMurdoch Childrens Research InstituteThe Royal Children's Hospital, Flemington Road, Parkville, Victoria 3052, AustraliaDepartment of PaediatricsThe Royal Children's Hospital, University of Melbourne, Flemington Road, Parkville, Victoria 3052, Australia Department of Anatomy and Developmental BiologyMonash University, Building 73, Clayton, Victoria 3800, AustraliaMurdoch Childrens Research InstituteThe Royal Children's Hospital, Flemington Road, Parkville, Victoria 3052, AustraliaDepartment of PaediatricsThe Royal Children's Hospital, University of Melbourne, Flemington Road, Parkville, Victoria 3052, Australia Department of Anatomy and Developmental BiologyMonash University, Building 73, Clayton, Victoria 3800, AustraliaMurdoch Childrens Research InstituteThe Royal Children's Hospital, Flemington Road, Parkville, Victoria 3052, AustraliaDepartment of PaediatricsThe Royal Children's Hospital, University of Melbourne, Flemington Road, Parkville, Victoria 3052, Australia
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Pettinato G, Vanden Berg-Foels WS, Zhang N, Wen X. ROCK inhibitor is not required for embryoid body formation from singularized human embryonic stem cells. PLoS One 2014; 9:e100742. [PMID: 25365581 PMCID: PMC4217711 DOI: 10.1371/journal.pone.0100742] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/14/2014] [Accepted: 05/30/2014] [Indexed: 12/18/2022] Open
Abstract
We report a technology to form human embryoid bodies (hEBs) from singularized human embryonic stem cells (hESCs) without the use of the p160 rho-associated coiled-coil kinase inhibitor (ROCKi) or centrifugation (spin). hEB formation was tested under four conditions: +ROCKi/+spin, +ROCKi/-spin, -ROCKi/+spin, and -ROCKi/-spin. Cell suspensions of BG01V/hOG and H9 hESC lines were pipetted into non-adherent hydrogel substrates containing defined microwell arrays. hEBs of consistent size and spherical geometry can be formed in each of the four conditions, including the -ROCKi/-spin condition. The hEBs formed under the -ROCKi/-spin condition differentiated to develop the three embryonic germ layers and tissues derived from each of the germ layers. This simplified hEB production technique offers homogeneity in hEB size and shape to support synchronous differentiation, elimination of the ROCKi xeno-factor and rate-limiting centrifugation treatment, and low-cost scalability, which will directly support automated, large-scale production of hEBs and hESC-derived cells needed for clinical, research, or therapeutic applications.
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Affiliation(s)
- Giuseppe Pettinato
- Department of Chemical and Life Science Engineering, Virginia Commonwealth University, Richmond, Virginia, United States of America
- Department of Biomedical Engineering, Virginia Commonwealth University, Richmond, Virginia, United States of America
- Department of Bioengineering, Clemson University, Clemson, South Carolina, United States of America
| | - Wendy S. Vanden Berg-Foels
- Department of Chemical and Life Science Engineering, Virginia Commonwealth University, Richmond, Virginia, United States of America
- Department of Bioengineering, Clemson University, Clemson, South Carolina, United States of America
- Department of Craniofacial Biology, Medical University of South Carolina, Charleston, South Carolina, United States of America
| | - Ning Zhang
- Department of Biomedical Engineering, Virginia Commonwealth University, Richmond, Virginia, United States of America
- Department of Bioengineering, Clemson University, Clemson, South Carolina, United States of America
- * E-mail: (NZ); (XW)
| | - Xuejun Wen
- Department of Chemical and Life Science Engineering, Virginia Commonwealth University, Richmond, Virginia, United States of America
- Department of Bioengineering, Clemson University, Clemson, South Carolina, United States of America
- Department of Craniofacial Biology, Medical University of South Carolina, Charleston, South Carolina, United States of America
- Institute for Biomedical Engineering and Nano Science (iNANO), Shanghai East Hospital, Tongji Medical School, Tongji University, Shanghai, People's Republic of China
- * E-mail: (NZ); (XW)
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Li JT, Sun FX. Myocardin and pdx-1 synergistically induce hMSCs to differentiate into insulin secreting cells. Biochem Biophys Res Commun 2014:S0006-291X(14)01747-1. [PMID: 25301554 DOI: 10.1016/j.bbrc.2014.09.110] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/19/2014] [Accepted: 09/26/2014] [Indexed: 01/09/2023]
Abstract
Mesenchymal stem cells (MSCs) have been reported as an attractive source for the generation of transplantable surrogate β cells. The objective of this study was to investigate a new method to induce the differentiation of hMSCs into insulin secretion cells and to explore its molecular mechanisms. In this study, we investigated in vitro differentiation of hMSCs by overexpression of myocardin and pdx-1. Differentiated cells were evaluated by immunocytochemistry, reverse transcription-polymerase chain reaction (RT-PCR), quantificational real-time RT-PCR (qRT-PCR) and Western blotting. Furthermore, the molecular mechanisms were evaluated by chip assay, CO-IP and Luciferase assay. This study reported a new method to induce the differentiation of hMSCs into insulin secretion cells. The method is cotransduction of myocardin and pdx-1 for 7days. At the same time, we find myocardin and pdx-1 can form a complex to promote the transactivities of insulin by affecting the formation of the pdx-1/myocardin/SRF/CArG complex both in vitro and in vitro. The present study provided a simple and faithful in vitro model for further investigating the cell replacement therapy for diabetes.
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Affiliation(s)
- Jing-Ting Li
- College of Resource and Environment Science, Pingdingshan University, Pingdingshan 467000, China.
| | - Fang-Xing Sun
- College of Architecture & Urban Planning, Henan University of Urban Construction, Pingdingshan 467036, China
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Ikehara S, Li M. Stem cell transplantation improves aging-related diseases. Front Cell Dev Biol 2014; 2:16. [PMID: 25364723 PMCID: PMC4206983 DOI: 10.3389/fcell.2014.00016] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/11/2014] [Accepted: 04/14/2014] [Indexed: 01/20/2023] Open
Abstract
Aging is a complex process of damage accumulation, and has been viewed as experimentally and medically intractable. The number of patients with age-associated diseases such as type 2 diabetes mellitus (T2DM), osteoporosis, Alzheimer's disease (AD), Parkinson's disease, atherosclerosis, and cancer has increased recently. Aging-related diseases are related to a deficiency of the immune system, which results from an aged thymus and bone marrow cells. Intra bone marrow-bone marrow transplantation (IBM-BMT) is a useful method to treat intractable diseases. This review summarizes findings that IBM-BMT can improve and treat aging-related diseases, including T2DM, osteoporosis and AD, in animal models.
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Affiliation(s)
- Susumu Ikehara
- Department of Stem Cell Disorders, Kansai Medical University Hirakata, Osaka, Japan
| | - Ming Li
- Department of Stem Cell Disorders, Kansai Medical University Hirakata, Osaka, Japan
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Chhabra P, Brayman KL. Overcoming barriers in clinical islet transplantation: current limitations and future prospects. Curr Probl Surg 2014; 51:49-86. [PMID: 24411187 DOI: 10.1067/j.cpsurg.2013.10.002] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/05/2023]
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Transplantation of Encapsulated Pancreatic Islets as a Treatment for Patients with Type 1 Diabetes Mellitus. Adv Med 2014; 2014:429710. [PMID: 26556410 PMCID: PMC4590955 DOI: 10.1155/2014/429710] [Citation(s) in RCA: 41] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/21/2013] [Accepted: 11/30/2013] [Indexed: 12/19/2022] Open
Abstract
Encapsulation of pancreatic islets has been proposed and investigated for over three decades to improve islet transplantation outcomes and to eliminate the side effects of immunosuppressive medications. Of the numerous encapsulation systems developed in the past, microencapsulation have been studied most extensively so far. A wide variety of materials has been tested for microencapsulation in various animal models (including nonhuman primates or NHPs) and some materials were shown to induce immunoprotection to islet grafts without the need for chronic immunosuppression. Despite the initial success of microcapsules in NHP models, the combined use of islet transplantation (allograft) and microencapsulation has not yet been successful in clinical trials. This review consists of three sections: introduction to islet transplantation, transplantation of encapsulated pancreatic islets as a treatment for patients with type 1 diabetes mellitus (T1DM), and present challenges and future perspectives.
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Bose B, Katikireddy KR, Shenoy PS. Regenerative medicine for diabetes: differentiation of human pluripotent stem cells into functional β-cells in vitro and their proposed journey to clinical translation. VITAMINS AND HORMONES 2014; 95:223-48. [PMID: 24559920 DOI: 10.1016/b978-0-12-800174-5.00009-0] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/04/2022]
Abstract
Diabetes is a group of metabolic diseases, rising globally at an alarming rate. Type 1 (juvenile diabetes) is the autoimmune version of diabetes where the pancreas is unable to produce insulin, whereas type 2 (adult onset diabetes) is caused due to insulin resistance of the cells. In either of the cases, elevated blood glucose levels are observed which leads to progressive comorbidity like renal failure, cardiovascular disease, retinopathy, etc. Metformin, sulphonyl urea group of drugs, as well as insulin injections are the available therapies. In advanced cases of diabetes, the drug alone or drug in combination with insulin injections are not able to maintain a steady level of blood glucose. Moreover, frequent insulin injections are rather cumbersome for the patient. So, regenerative medicine could be a permanent solution for fighting diabetes. Islet transplantation has been tried with a limited amount of success on a large population of diabetics because of the shortage of cadaveric pancreas. Therefore, the best proposed alternative is regenerative medicine involving human pluripotent stem cell (hPSC)-derived beta islet transplantation which can be obtained in large quantities. Efficient protocols for in vitro differentiation of hPSC into a large number of sustained insulin-producing beta cells for transplantation will be considered to be a giant leap to address global rise in diabetic cases. Although most of the protocols mimic in vivo pancreatic development in humans, considerable amount of lacuna persists for near-perfect differentiation strategies. Moreover, beta islets differentiated from hPSC have not yet been successfully translated under clinical scenario.
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Affiliation(s)
- Bipasha Bose
- Nanyang Technological University, School of Biological Sciences, NTU Lab Location @ Level 2 Singapore Institute for Clinical Sciences, Brenner Centre for Molecular Medicine, Singapore, Singapore.
| | | | - P Sudheer Shenoy
- Nanyang Technological University, School of Biological Sciences, NTU Lab Location @ Level 2 Singapore Institute for Clinical Sciences, Brenner Centre for Molecular Medicine, Singapore, Singapore
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Lee YS, Jun HS. Anti-diabetic actions of glucagon-like peptide-1 on pancreatic beta-cells. Metabolism 2014; 63:9-19. [PMID: 24140094 DOI: 10.1016/j.metabol.2013.09.010] [Citation(s) in RCA: 212] [Impact Index Per Article: 19.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/07/2013] [Revised: 09/05/2013] [Accepted: 09/14/2013] [Indexed: 12/11/2022]
Abstract
Glucagon-like peptide-1 (GLP-1), an incretin hormone, is released from intestinal L-cells in response to nutrients. GLP-1 lowers blood glucose levels by stimulating insulin secretion from pancreatic beta-cells in a glucose-dependent manner. In addition, GLP-1 slows gastric emptying, suppresses appetite, reduces plasma glucagon, and stimulates glucose disposal, which are beneficial for glucose homeostasis. Therefore, incretin-based therapies such as GLP-1 receptor agonists and inhibitors of dipeptidyl peptidase IV, an enzyme which inactivates GLP-1, have been developed for treatment of diabetes. This review outlines our knowledge of the actions of GLP-1 on insulin secretion and biosynthesis, beta-cell proliferation and regeneration, and protection against beta-cell damage, as well as the involvement of recently discovered signaling pathways of GLP-1 action, mainly focusing on pancreatic beta-cells.
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Affiliation(s)
- Young-Sun Lee
- Lee Gil Ya Cancer and Diabetes Institute, Gachon University, 7-45 Songdo-dong, Yeonsu-ku, Incheon 406-840, South Korea
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Chhabra P, Brayman KL. Stem cell therapy to cure type 1 diabetes: from hype to hope. Stem Cells Transl Med 2013; 2:328-336. [PMID: 23572052 PMCID: PMC3667565 DOI: 10.5966/sctm.2012-0116] [Citation(s) in RCA: 112] [Impact Index Per Article: 9.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/07/2012] [Accepted: 02/01/2013] [Indexed: 02/05/2023] Open
Abstract
Type 1 diabetes mellitus (T1D) is a chronic, multifactorial autoimmune disease that involves the progressive destruction of pancreatic β-cells, ultimately resulting in the loss of insulin production and secretion. The goal of clinical intervention is to prevent or arrest the onset and progression of autoimmunity, reverse β-cell destruction, and restore glycometabolic and immune homeostasis. Despite promising outcomes observed with islet transplantation and advancements in immunomodulatory therapies, the need for an effective cell replacement strategy for curing T1D still persists. Stem cell therapy offers a solution to the cited challenges of islet transplantation. While the regenerative potential of stem cells can be harnessed to make available a self-replenishing supply of glucose-responsive insulin-producing cells, their immunomodulatory properties may potentially be used to prevent, arrest, or reverse autoimmunity, ameliorate innate/alloimmune graft rejection, and prevent recurrence of the disease. Herein, we discuss the therapeutic potential of stem cells derived from a variety of sources for the cure of T1D, for example, embryonic stem cells, induced pluripotent stem cells, bone marrow-derived hematopoietic stem cells, and multipotent mesenchymal stromal cells derived from bone marrow, umbilical cord blood, and adipose tissue. The benefits of combinatorial approaches designed to ensure the successful clinical translation of stem cell therapeutic strategies, such as approaches combining effective stem cell strategies with islet transplantation, immunomodulatory drug regimens, and/or novel bioengineering techniques, are also discussed. To conclude, the application of stem cell therapy in the cure for T1D appears extremely promising.
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Affiliation(s)
- Preeti Chhabra
- Department of Surgery, University of Virginia School of Medicine, Charlottesville, Virginia, USA
| | - Kenneth L. Brayman
- Department of Surgery, University of Virginia School of Medicine, Charlottesville, Virginia, USA
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Bose B, Shenoy P S. Non insulin producing cell line, MIA PaCa-2 is rendered insulin producing in vitro via mesenchymal epithelial transition. J Cell Biochem 2013; 114:1642-52. [PMID: 23386380 DOI: 10.1002/jcb.24506] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/20/2012] [Accepted: 01/16/2013] [Indexed: 11/12/2022]
Abstract
We used non-insulin producing pancreatic carcinoma cell line, MIA PaCa-2 and have modulated its culture conditions by using 1% matrigel as extracellular matrix, N2, B27 growth supplements and serum free conditions. Expression of markers was analyzed using qRT-PCR, immunofluorescence and in vitro functional assay for insulin and C-peptide release was assessed using insulin and C-peptide ELISA, respectively. The cells grown under this altered culture conditions have exhibited a transition in the morphology from mesenchymal to epithelial with extensive piling up of cells. A reduction in doubling time from 40 to 18 h, upregulation of beta islet specific markers like pancreatic duodenal homeobox-1 (Pdx-1), C-peptide, insulin, and disappearance of markers like vimentin were observed. On the functional level, the altered morphology bearing cells released high levels of insulin in response to 10 µM tolbutamide (an activator of insulin pathway) and reduced insulin secretion in response to 50 µM nifedipine (inhibitor of the pathway). On the contrary, the original cells (mesenchymal morphology) had failed to release any insulin in response to varying concentrations of glucose and also the activators and inhibitors of the insulin pathway. This investigation thus provides a basis for using this basic developmental biology phenomenon mesenchymal to epithelial transition as a strategy to generate a large number of functional islets from stem cells of mesenchymal origin.
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Affiliation(s)
- Bipasha Bose
- Embryonic Stem Cell Group Reliance Life Sciences Pvt. Ltd. Dhirubhai Ambani Life Sciences Centre, Navi Mumbai, India.
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36
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Animal models of diabetes mellitus for islet transplantation. EXPERIMENTAL DIABETES RESEARCH 2012; 2012:256707. [PMID: 23346100 PMCID: PMC3546491 DOI: 10.1155/2012/256707] [Citation(s) in RCA: 49] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 11/19/2012] [Accepted: 12/12/2012] [Indexed: 01/09/2023]
Abstract
Due to current improvements in techniques for islet isolation and transplantation and protocols for immunosuppressants, islet transplantation has become an effective treatment for severe diabetes patients. Many diabetic animal models have contributed to such improvements. In this paper, we focus on 3 types of models with different mechanisms for inducing diabetes mellitus (DM): models induced by drugs including streptozotocin (STZ), pancreatomized models, and spontaneous models due to autoimmunity. STZ-induced diabetes is one of the most commonly used experimental diabetic models and is employed using many specimens including rodents, pigs or monkeys. The management of STZ models is well established for islet studies. Pancreatomized models reveal different aspects compared to STZ-induced models in terms of loss of function in the increase and decrease of blood glucose and therefore are useful for evaluating the condition in total pancreatomized patients. Spontaneous models are useful for preclinical studies including the assessment of immunosuppressants because such models involve the same mechanisms as type 1 DM in the clinical setting. In conclusion, islet researchers should select suitable diabetic animal models according to the aim of the study.
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