Original Data

Rev Diabet Stud, 2010, 7(2):158-167

DOI 10.1900/RDS.2010.7.158

Turning Human Epidermis Into Pancreatic Endoderm

Pere Santamaria¹^,²^,³, Ignacio Rodríguez-Pizá¹, Xavier Clemente-Casares¹^,², Jun Yamanouchi², Lola Mulero-Perez¹, Trond Aasen¹^,⁴, Angel Raya¹^,⁴^,⁵, Juan Carlos Izpisúa Belmonte¹^,⁶

¹Center of Regenerative Medicine in Barcelona, Dr. Aiguader 88, 08003 Barcelona, Spain
²Julia McFarlane Diabetes Research Centre, Department of Microbiology and Infectious Diseases, and Institute of Inflammation, Infection and Immunity, Faculty of Medicine, The University of Calgary, 3330 Hospital Dr. N.W., Calgary, Alberta T2N 4N1, Canada
³On sabbatical leave
⁴Networking Center of Biomedical Research in Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN)
⁵Institució Catalana de Recerca i Estudis Avançats (ICREA)
⁶Gene Expression Laboratory, Salk Institute for Biological Studies, 10010 North Torrey Pines Rd., La Jolla, California 92037, USA
Address correspondence to: Pere Santamaria, e-mail: psantama@ucalgary.ca

Abstract

OBJECTIVE: Human embryonic stem (hES) cells can be differentiated into pancreatic endoderm structures in vitro. The study was performed to determine whether induced pluripotent stem (iPS) cells can be differentiated into similar structures with comparable efficiency. METHODS: We compared the ability of hES cells and iPS cells derived from human epidermal keratinocytes to progressively differentiate into pancreatic endoderm. Human foreskin keratinocytes were reprogrammed to pluripotency by transduction with retroviruses encoding Oct4, Sox2, and Klf4. The resulting keratinocyte-derived iPS (KiPS) cell lines and a hES cell line were subjected to a modified pancreatic endoderm differentiation protocol. Cells and embryoid-body structures derived from both hES and KiPS cells were compared at different stages of development for expression of stem cell and differentiation markers, including Sox2, Oct4, Mixl1, Brachyury, Gsc, FoxA2, Sox17, Hnf4α, Hnf1β, Nkx2.2, Nkx6.1, Hex, Isl1, Pdx1, and Slc2A, via Taqman real-time PCR, flow-cytometry, and/or immunocytochemistry. RESULTS: hES cells and KiPS cells expressed similar levels of the stem cell factors Sox2 and Oct4. Upon differentiation, both cell types underwent remarkably similar changes in gene expression. They acquired the definitive endoderm markers Sox17 and FoxA2. Most Sox17⁺ and FoxA2⁺ cells co-expressed Hnf4α and Hnf1β, found in the primitive gut tube, a pancreas precursor. Most FoxA2⁺ cells were also Pdx1⁺, and many expressed Nkx2.2, Nkx6.1, and Isl1. CONCLUSIONS: Keratinocyte-derived iPS cells can be differentiated into pancreatic endoderm, and the efficiency of this process is comparable to that seen for hES cells. Thus keratinocytes have the potential to serve as a source of patient-specific pancreatic endoderm for transplantation.

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Rev Diabet Stud, 2010, 7(2):168-182

DOI 10.1900/RDS.2010.7.168

Human Placenta-Derived Mesenchymal Stem Cells and Islet-Like Cell Clusters Generated From These Cells as a Novel Source for Stem Cell Therapy in Diabetes

Sachin Kadam¹, Sudhakar Muthyala², Prabha Nair², Ramesh Bhonde³

¹National Center for Cell Science, Ganeshkhind, Pune 411007, MS, India
²Division of Tissue Engineering and Regeneration Technologies, BMT Wing, Sree Chitra Tirunal Institute for Medical Sciences and Technology, Poojappura, Thiruvananthapuram 695012, Kerala, India
³Stempeutics Research Malaysia SDN BHD ,Technology Park Malaysia, Bukit Jalil, 57000 Kuala Lumpur, Malaysia
Address correspondence to: Ramesh Bhonde, e-mail: rrbhonde@gmail.com

Abstract

Placental tissue holds great promise as a source of cells for regenerative medicine due to its plasticity, and easy availability. Human placenta-derived mesenchymal stem cells (hPDMSCs) have the potential to differentiate into insulin-producing cells. Upon transplantation, they can reverse experimental diabetes in mice. However, it is not known whether culture-expanded undifferentiated hPDMSCs are capable of restoring normoglycemia upon transplantation in streptozotocin (STZ)-induced diabetic mice. Hence we prepared long-term cultures of hPDMSCs from the chorionic villi of full-term human placenta. Flow cytometry analyses and immunocytochemistry study revealed bonafide mesenchymal nature of the isolated hPDMSCs. These cultures could differentiate into adipogenic, oesteogenic, chondrogenic, and neuronal lineages on exposure to lineage-specific cocktails. Furthermore, we showed that hPDMSCs can form islet-like cell clusters (ILCs) on stepwise exposure to serum-free defined media containing specific growth factors and differentiating agents. qRT-PCR showed the expression of insulin, glucagon, and somatostatin in undifferentiated hPDMSCs and in ILCs. Differentiated ILCs were found to express human insulin, glucagon, and somatostatin by immunocytochemistry. Additionally, ILCs also showed abundance of pancreatic transcription factors ngn3 and isl1. Both undifferentiated hPDMSCs and ILCs exihibited insulin secretion in response to glucose. Transplantation of hPDMSCs or ILCs derived from hPDMSCs in STZ-induced diabetic mice led to restoration of normoglycemia. Our results demonstrate, for the first time, reversal of hyperglycemia by undifferentiated hPDMSCs and ILCs derived from hPDMSCs. These results suggest human placenta-derived MSCs as an alternative source for cell replacement therapy in diabetes.

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