Induced in vitro differentiation of neural-like cells from human amnion-derived fibroblast-like cells

There is growing evidence that the human amnion contains various types of stem cell. As amniotic tissue is readily available, it has the potential to be an important source of material for regenerative medicine. In this study, we evaluated the potential of human amnion-derived fibroblast-like (HADFIL) cells to differentiate into neural cells. Two HADFIL cell populations, derived from two different neonates, were analyzed. The expression of neural cell-specific genes was examined before and after in vitro induction of cellular differentiation. We found that neuron specific enolase, neurofilament-medium, beta-tubulin isotype III, and glial fibrillary acidic protein (GFAP) showed significantly increased expression following the induction of differentiation. In addition, immunostaining demonstrated that neuron specific enolase, GFAP and myelin basic protein (MBP) were present in HADFIL cells following the induction of differentiation, although one of the HADFIL cell populations showed a lower expression of GFAP and MBP. These results indicate that HADFIL cell populations have the potential to differentiate into neural cells. Although further studies are necessary to determine whether such in vitro-differentiated cells can function in vivo as neural cells, these amniotic cell populations might be of value in therapeutic applications that require human neural cells.     

Induction of pancreatic β-cell-like cells from CD44+/CD105+ human amniotic fluids via epigenetic regulation of the pancreatic and duodenal homeobox factor 1 promoter

Pancreatic and duodenal homeobox factor 1 (PDX-1) maintains β-cell function and differentiation via direct regulation of multiple islet cell genes. However, the molecular mechanisms involved in this process remain unknown. Here, we show that PDX-1 plays an important role in the induction of CD44+/CD105+ human amniotic fluid cells (HuAFCs) into functional pancreatic β-cell-like cells in vitro. CD44+/CD105+ HuAFCs were transfected with either siRNA targeting PDX-1 (siRNA-PDX-1) or mock plasmid (siRNA-MOCK). Following induction, siRNA-MOCK-transfected cells differentiated into β-cell-like cells that expressed multiple islet cell markers and produced insulin and C-peptide in a glucose-regulated manner. However, siRNA-PDX-1-transfected cells did not fully differentiate into β-cell-like cells. Further, we observed epigenetic changes at the PDX-1 gene locus in induced CD44(+)/CD105(+) HuAFCs. Therefore, CD44+/CD105+ HuAFCs could be a source of human pancreatic β-cell-like cells with potential uses in cell replacement therapy for diabetes.     

再生胰腺提取物诱导人羊膜间充质干细胞定向分化为胰岛素分泌细胞

利用天然生物诱导剂大鼠再生胰腺提取物(Rgenerating pancreatic extract,RPE)定向诱导人羊膜间充质干细胞(Human amniotic mesenchymal stem cells,hAMSCs)向胰岛素分泌细胞分化。切除大鼠60%胰腺刺激胰腺再生,而后制备RPE,以终浓度为20 mg/L的RPE诱导hAMSCs。实验通过形态学鉴定、双硫腙染色、免疫荧光分析、RT-PCR基因检测和高糖刺激胰岛素分泌等实验鉴定细胞诱导结果。实验结果显示P3代hAMSCs经RPE诱导后形态变化明显,诱导15 d后细胞呈簇状生长,经双硫腙染色可见棕红色细胞团;免疫荧光染色结果显示诱导细胞呈胰岛素阳性表达;RT-PCR实验证明诱导细胞阳性表达人胰岛相关基因Pdx1和insulin;高糖刺激实验证明培养液中有胰岛素成分产生,且分泌量随刺激时间的延长先增加而后趋于稳定。实验结果表明hAMSCs在体外经RPE诱导可以分化为胰岛素分泌细胞。     

Transient transcriptional activation of gastrin during sodium butyrate-induced differentiation of islet cells

Transient expression of pancreatic gastrin corresponds to a period of rapid islet cell development. After birth gastrin expression silencing is coincidental with islet cell terminal differentiation, while persistent expression is accompanied with nesidioblastosis and reexpression observed in islet cell tumors. Experiments with transgenic animals suggested that gastrin might act synergistically with growth factors to stimulate islet cell development. The present study intended to establish an in vitro cell culture model to analyse the molecular events controlling gastrin gene activation and repression dependent on islet cell differentiation. Sodium butyrate, a proliferation-arresting compound has previously been shown to differentiate insulinoma cells while increasing insulin production. The present paper demonstrates concomitant transient increase in gastrin mRNA, intracellular and secreted gastrin during sodium butyrate treatment. Increased gastrin expression was due to activation or derepression of gastrin promoter activity as revealed by promoter analyses. This in vitro model mimics the expression pattern of gastrin and insulin observed during fetal islet cell development and provides an excellent tool to analyse the molecular mechanisms controlling gastrin gene activation and selective repression during islet cell differentiation.     

A feasibility study of an in vitro differentiation potential toward insulin-producing cells by dental tissue-derived mesenchymal stem cells

Dental tissue-derived mesenchymal stem cells have been proposed as an alternative source for mesenchymal stem cells. Here, we investigated the differentiation ability toward insulin producing cells (IPCs) of human dental pulp stem cells (hDPSCs) and human periodontal ligament stem cells (hPDLSCs). These cells expressed mesenchymal stem cell surface markers and were able to differentiate toward osteogenic and adipogenic lineages. Upon 3 step-IPCs induction, hDPSCs exhibited more colony number than hPDLSCs. The mRNA upregulation of pancreatic endoderm/islet markers was noted. However, the significant increase was noted only for PDX-1, NGN-3, and INSULIN mRNA expression of hDPSCs. The hDPSCs-derived IPCs expressed PRO-INSULIN and released C-PEPTIDE upon glucose stimulation in dose-dependent manner. After IPCs induction, the Notch target, HES-1 and HEY-1, mRNA expression was markedly noted. Notch inhibition during the last induction step or throughout the protocol disturbed the ability of C-PEPTIDE release upon glucose stimulation. The results suggested that hDPSCs had better differentiation potential toward IPCs than hPDLSCs. In addition, the Notch signalling might involve in the differentiation regulation of hDPSCs into IPCs.     

Neuronal cell differentiation of mesenchymal stem cells originating from canine amniotic fluid

The amniotic fluid contains mesenchymal stem cells (MSCs) and can be readily available for tissue engineering. Regenerative treatments such as tissue engineering, cell therapy, and transplantation show potential in clinical trials of degenerative diseases. Disease presentation and clinical responses in the Canis familiaris not only are physiologically similar to human compared with other traditional mammalian models but is also a suitable model for human diseases. The aim of this study was to investigate whether canine amniotic-fluid-derived mesenchymal stem cells (cAF-MSCs) can differentiate into neural precursor cells in vitro when exposed to neural induction reagent. During neural differentiation, cAF-MSCs progressively acquire neuron-like morphology. Messenger RNA (mRNA) expression levels of neural-specific genes, such as NEFL, NSE, and TUBB3 (βIII-tubulin) dramatically increased in the differentiated cAF-MSCs after induction. In addition, protein expression levels of nestin, βIII-tubulin, and tyrosine hydroxylase remarkably increased in differentiated cAF-MSCs. This study demonstrates that cAF-MSCs have great potential for neural precursor differentiation in vitro. Therefore, amniotic fluid may be a suitable alternative source of stem cells and can be applied to cell therapy in neurodegenerative diseases.     

Differentiation of mesenchymal cells derived from human amniotic membranes into hepatocyte-like cells in vitro

Mesenchymal stem cells are believed to be involved in the formation of mesenchymal tissues, including bone, cartilage, muscle, tendon and adipose tissue. Interestingly, it has previously been reported that mesenchymal stem cells could also differentiate into endoderm-derived cells, such as hepatocytes. The amniotic membrane contains mesenchymal cells and is a readily available human tissue. Therefore, we investigated the potential of mesenchymal cells derived from human amniotic membrane (MC-HAM) to differentiate into hepatocytes. We analyzed the expression of hepatocyte-specific genes in MC-HAM before and after induction of differentiation into hepatocytes. We observed the expression of mRNAs encoding albumin, a-fetoprotein, cytokeratin 18 and alpha1-antitrypsin, but not those encoding glucose-6-phosphatase or ornithine transcarbamylase, prior to the induction of differentiation. However, immunocytochemistry revealed that albumin and alpha-fetoprotein were abundantly produced only after the induction of differentiation into hepatocytes. In addition, we observed the storage of glycogen, a characteristic feature of hepatocytes, using periodic acid-Schiff staining of MC-HAM induced to differentiate into hepatocytes. Overall, MC-HAM appear to be able to differentiate into cells possessing some characteristics of hepatocytes. Although further studies should be carried out to determine whether such in vitro-differentiated cells can function in vivo as hepatocytes. These cells may be useful in various applications that require human hepatocytes.     

Mesenchymal stem cells: biology and clinical potential in type 1 diabetes therapy

Mesenchymal stem cells (MSCs) can be derived from adult bone marrow, fat and several foetal tissues. In vitro , MSCs have the capacity to differentiate into multiple mesodermal and non-mesodermal cell lineages. Besides, MSCs possess immunosuppressive effects by modulating the immune function of the major cell populations involved in alloantigen recognition and elimination. The intriguing biology of MSCs makes them strong candidates for cell-based therapy against various human diseases. Type 1 diabetes is caused by a cell-mediated autoimmune destruction of pancreatic β-cells. While insulin replacement remains the cornerstone treatment for type 1 diabetes, the transplantation of pancreatic islets of Langerhans provides a cure for this disorder. And yet, islet transplantation is limited by the lack of donor pancreas. Generation of insulin-producing cells (IPCs) from MSCs represents an attractive alternative. On the one hand, MSCs from pancreas, bone marrow, adipose tissue, umbilical cord blood and cord tissue have the potential to differentiate into IPCs by genetic modification and/or defined culture conditions In vitro . On the other hand, MSCs are able to serve as a cellular vehicle for the expression of human insulin gene. Moreover, protein transduction technology could offer a novel approach for generating IPCs from stem cells including MSCs. In this review, we first summarize the current knowledge on the biological characterization of MSCs. Next, we consider MSCs as surrogate β-cell source for islet transplantation, and present some basic requirements for these replacement cells. Finally, MSCs-mediated therapeutic neovascularization in type 1 diabetes is discussed.     

Epigenetic memory and preferential lineage-specific differentiation in induced pluripotent stem cells derived from human pancreatic islet beta cells

Human induced pluripotent stem cells (HiPSCs) appear to be highly similar to human embryonic stem cells (HESCs). Using two genetic lineage-tracing systems, we demonstrate the generation of iPSC lines from human pancreatic islet beta cells. These reprogrammed cells acquired markers of pluripotent cells and differentiated into the three embryonic germ layers. However, the beta cell-derived iPSCs (BiPSCs) maintained open chromatin structure at key beta-cell genes, together with?a unique DNA methylation signature that distinguishes them from other PSCs. BiPSCs also demonstrated an increased ability to differentiate into insulin-producing cells both in?vitro and in?vivo, compared with ESCs and isogenic non-beta iPSCs. Our results suggest that the epigenetic memory may predispose?BiPSCs to differentiate more readily into insulin producing cells. These findings demonstrate that HiPSC phenotype may be influenced by their cells of origin, and suggest that their skewed differentiation potential may be advantageous for cell replacement therapy.     

In vitro transdifferentiation of adult pancreatic acinar cells into insulin-expressing cells

Despite a recent breakthrough in human islet transplantation for treating diabetes mellitus, the limited availability of insulin-producing tissue is still a major obstacle. Here, we studied whether adult pancreatic acinar cells have the potential to transdifferentiate into islet or beta cells. Pancreatic acini were isolated from 7- to 8-weeks-old male Sprague-Dawley rats and cultured in suspension. Within 1 week, most of the acinar cells lost amylase expression and converted to cells with a duct cell phenotype. Insulin-positive cells were also observed, mainly at the periphery of the acini-derived spheroids. Insulin gene and protein expression was increased. Presence of a few insulin-positive cells coexpressing cytokeratins suggests that a spontaneous acinar to ductal cell transdifferentiation process was further going on towards beta cells. This study provides the first evidence that adult pancreatic acinar cells could be differentiated into insulin-expressing cells in vitro.     

miRNA-375 promotes beta pancreatic differentiation in human induced pluripotent stem (hiPS) cells

Islet transplantation is considered as an ultimate option for the treatment of type I diabetes. Human induced pluripotent stem cells (hiPSCs) have raised the possibility that patient-specific insulin-secreting cells might be derived from somatic cells through cell fate reprogramming. However, current protocols mostly rely on the use of several cytokines and inhibitors for directing differentiation towards pancreatic fate. Given the high manufacturing cost of these recombinant proteins, this approach is prohibitive for clinical applications. Knowing that microRNAs (miRNAs) are key players in various stages of pancreatic development, we present a novel and cost-effective strategy in which over-expression of miR-375 promotes pancreatic differentiation in hiPSCs in the absence of any other stimulator. We used a polycistronic viral vector expressing Sox2, Klf4, c-Myc, and Oct4 to drive hiPSCs from human foreskin fibroblasts. The established hiPSCs are similar to human embryonic stem cells in many aspects including morphology, passaging, surface and pluripotency markers, and gene expression. For differentiation induction, miR-375 was lentivirally overexpressed in these hiPSCs. Morphological assessment, immunocytochemistry, and expression analysis of islet marker genes confirmed that islet like cells were obtained in miR-375 transduced cells compared to controls. Our differentiated clusters secreted insulin in a glucose-dependant manner, showing in vitro functionality. We demonstrated for the first time that miRNAs might be ideal substitutes to induce pancreatic differentiation in hiPSCs. This work provides a new approach to study the role of miRNAs in pancreatic specification and increase the feasibility of using patient-specific iPSCs for beta cell replacement therapy for type I diabetes.