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A synthetic peptide-acrylate surface for production of insulin-producing cells from human embryonic stem cells.

ABSTRACT: Human embryonic stem cells (hESCs), due to their self-renewal capacity and pluripotency, have become a potential source of transplantable ?-cells for the treatment of diabetes. However, it is imperative that the derived cells fulfill the criteria for clinical treatment. In this study, we replaced common Matrigel with a synthetic peptide-acrylate surface (Synthemax) to expand undifferentiated hESCs and direct their differentiation in a defined and serum-free medium. We confirmed that the cells still expressed pluripotent markers, had the ability to differentiate into three germ layers, and maintained a normal karyotype after 10 passages of subculture. Next, we reported an efficient protocol for deriving nearly 86% definitive endoderm cells from hESCs under serum-free conditions. Moreover, we were able to obtain insulin-producing cells within 21 days following a simple three-step protocol. The results of immunocytochemical and quantitative gene expression analysis showed that the efficiency of induction was not significantly different between the Synthemax surface and the Matrigel-coated surface. Thus, we provided a totally defined condition from hESC culture to insulin-producing cell differentiation, and the derived cells could be a therapeutic resource for diabetic patients in the future.

SUBMITTER: Lin PY

PROVIDER: S-EPMC3920848 | biostudies-other | 2014 Feb

REPOSITORIES: biostudies-other

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A synthetic peptide-acrylate surface for production of insulin-producing cells from human embryonic stem cells.

Lin Pei-Yi PY Hung Shih-Han SH Yang Yao-Chen YC Liao Li-Chuan LC Hsieh Yi-Cheng YC Yen Hsan-Jan HJ Lu Huai-En HE Lee Maw-Sheng MS Chu I-Ming IM Hwang Shiaw-Min SM

Stem cells and development 20131116 4

Human embryonic stem cells (hESCs), due to their self-renewal capacity and pluripotency, have become a potential source of transplantable β-cells for the treatment of diabetes. However, it is imperative that the derived cells fulfill the criteria for clinical treatment. In this study, we replaced common Matrigel with a synthetic peptide-acrylate surface (Synthemax) to expand undifferentiated hESCs and direct their differentiation in a defined and serum-free medium. We confirmed that the cells st ...[more]

PMID: 24083371

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Project description:The PEC-01 cell population, differentiated from human embryonic stem cells (hESCs), contains pancreatic progenitors (PPs) that, when loaded into macroencapsulation devices (to produce the VC-01 candidate product) and transplanted into mice, can mature into glucose-responsive insulin-secreting cells and other pancreatic endocrine cells involved in glucose metabolism. We modified the protocol for making PEC-01 cells such that 73%-80% of the cell population consisted of PDX1-positive (PDX1+) and NKX6.1+ PPs. The PPs were further differentiated to islet-like cells (ICs) that reproducibly contained 73%-89% endocrine cells, of which approximately 40%-50% expressed insulin. A large fraction of these insulin-positive cells were single hormone-positive and expressed the transcription factors PDX1 and NKX6.1. To preclude a significant contribution of progenitors to the in vivo function of ICs, we used a simple enrichment process to remove remaining PPs, yielding aggregates that contained 93%-98% endocrine cells and 1%-3% progenitors. Enriched ICs, when encapsulated and implanted into mice, functioned similarly to the VC-01 candidate product, demonstrating conclusively that in vitro-produced hESC-derived insulin-producing cells can mature and function in vivo in devices. A scaled version of our suspension culture was used, and the endocrine aggregates could be cryopreserved and retain functionality. Although ICs expressed multiple important ? cell genes, the cells contained relatively low levels of several maturity-associated markers. Correlating with this, the time to function of ICs was similar to PEC-01 cells, indicating that ICs required cell-autonomous maturation after delivery in vivo, which would occur concurrently with graft integration into the host.Type 1 diabetes (T1D) affects approximately 1.25 million people in the U.S. alone and is deadly if not managed with insulin injections. This paper describes the production of insulin-producing cells in vitro and a new protocol for producing the cells, representing another potential cell source for a diabetes cell therapy. These cells can be loaded into a protective device that is implanted under the skin. The device is designed to protect the cells from immune rejection by the implant recipient. The implant can engraft and respond to glucose by secreting insulin, thus potentially replacing the ? cells lost in patients with T1D.

Project description:ObjectiveGlucagon-like peptide-1 (GLP-1) has attracted tremendous attention for treatment of diabetes. Likewise, it seems that active ingredients of chamomile oil might have anti-diabetic effects. This work was conducted to investigate the effects of the combination of GLP-1 and chamomile oil on differentiation of mesenchymal stem cells (MSCs) into functional insulin-producing cells (IPCs).Materials and methodsIn this experimental study, adipose MSCs derived from the adult male New Zealand white rabbits were assigned into four groups: control (without any treatment); GLP-1 (in which cells were treated with 10 nM GLP-1 every other day for 5 days); chamomile oil (in which cells were treated with 100 ug/ml Matricaria chamomilla L. flower oil every other day for 5 days); and GLP-1+ chamomile oil (in which cells were treated with 10 nM GLP-1 and 100 μg/ml M. chamomilla flower oil every other day for 5 days). Characterization of isolated MSCs was performed using flow cytometry, Alizarin red S staining and Oil red O staining. The expressions of genes specific for IPCs were measured using reverse transcriptase-polymerase chain reaction (RT-PCR) assay. Measurement of insulin and the cleaved connecting peptide (C-peptide) in response to different concentrations of glucose, were performed using ELISA kits.ResultsOur results demonstrated that isolated cells highly expressed MSC markers and were able to differentiate into osteocytes and adipocytes. Additionally, using GLP-1 in combination with chamomile oil exhibited higher levels of IPCs gene markers including NK homeobox gene 2.2 (NKX-2.2), paired box gene 4 (PAX4), insulin (INS) and pancreatic duodenal homeobox-1 (PDX1) as well as insulin and C-peptide secretion in response to different glucose concentrations compared to GLP-1 or chamomile oil alone (P<0.05).ConclusionCollectively, these findings establish a substantial foundation for using peptides in combination with natural products to obtain higher efficiency in regenerative medicine and peptide therapy.

Project description:Mesenchymal stromal cells (MSCs) are an attractive option for cell therapy for type 1 diabetes mellitus (DM). These cells can be obtained from many sources, but bone marrow and adipose tissue are the most studied. MSCs have distinct advantages since they are nonteratogenic, nonimmunogenic and have immunomodulatory functions. Insulin-producing cells (IPCs) can be generated from MSCs by gene transfection, gene editing or directed differentiation. For directed differentiation, MSCs are usually cultured in a glucose-rich medium with various growth and activation factors. The resulting IPCs can control chemically-induced diabetes in immune-deficient mice. These findings are comparable to those obtained from pluripotent cells. PD-L1 and PD-L2 expression by MSCs is upregulated under inflammatory conditions. Immunomodulation occurs due to the interaction between these ligands and PD-1 receptors on T lymphocytes. If this function is maintained after differentiation, life-long immunosuppression or encapsulation could be avoided. In the clinical setting, two sites can be used for transplantation of IPCs: the subcutaneous tissue and the omentum. A 2-stage procedure is required for the former and a laparoscopic procedure for the latter. For either site, cells should be transplanted within a scaffold, preferably one from fibrin. Several questions remain unanswered. Will the transplanted cells be affected by the antibodies involved in the pathogenesis of type 1 DM? What is the functional longevity of these cells following their transplantation? These issues have to be addressed before clinical translation is attempted. Graphical Abstract Bone marrow MSCs are isolated from the long bone of SD rats. Then they are expanded and through directed differentiation insulin-producing cells are formed. The differentiated cells are loaded onto a collagen scaffold. If one-stage transplantation is planned, a drug delivery system must be incorporated to ensure immediate oxygenation, promote vascularization and provide some growth factors. Some mechanisms involved in the immunomodulatory function of MSCs. These are implemented either by cell to cell contact or by the release of soluble factors. Collectively, these pathways results in an increase in T-regulatory cells.

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A synthetic peptide-acrylate surface for production of insulin-producing cells from human embryonic stem cells.

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A synthetic peptide-acrylate surface for production of insulin-producing cells from human embryonic stem cells.

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