Generation Of Insulin-producing Cells From Human Amniotic Fluid-derived Stem Cells In Vitro

Islet cell transplantation for patients with type I diabetes holds great promise for achieving insulin independence. However, the extreme shortage of matched organ donors has made it impossible for this treatment to be used for the general diabetic population. This has evoked large scale search for alternative sources of islet cells. Stem cells have been favorites of the race because of their tremendous proliferation and differentiation potential. However, teratoma formation of embryonic stem cells or scarcity of the source and invasive procedures required to isolate and culture adult stem cells has limited their use. In this scenario, human amniotic fluid-derived stem cells (hAFSCs) are gaining much attention for cell therapy. hAFSCs are easily accessible through routine amniocentesis, which is a common, safe, reliable screening tool for the human fetus. hAFSCs have strong proliferative capacity, versatile differentiation potential and can be used for autograft. Recent researches show that hAFSCs can be directed into a wide range of cell types representing the three primary embryonic lineages of mesoderm, ectoderm and endoderm. Thus, hAFSCs may represent an important cell source for generation of surrogate islet cells. However, the capacity of hAFSCs to differentiate into functional islet cells remains unknown.Neuronal restrictive silencing factor (NRSF), also known as repressor element-1 silencing transcription factor (REST), is a transcriptional regulator that interact with gene networks involved in self-renewal of embryonic stem cell, neuron and pancreatic islet cell development. Several studies suggested that NRSF also regulated the expression of neural genes and some islet-related genes such as Pax4 in islet cells. Recently, more NRSF target genes, especially key transcription factors such as NeuroDl/Beta2, HNF4a, HNF6/Onecutl, Hes1, Neurogenin3, that regulate islet cell development were reported, which suggested that NRSF may also function as an important regulator during islet cell differentiation. In addition, The over-expression of NRSF in mouse pancreaticβTC3-cell line will impair the capacity to secrete insulin in response to mitochondrial fuels, a particularity of mature P-cells.Therefore, Using RNA interference technology to down-regulate the expression of NRSF in hAFSCs and inducing them to differentiate into insulin-producing cells is the objective of this study.The study consists of two parts:Part I:Isolation of hAFSCs and analysis of its biological characteristics:Method:Amniotic fluid samples (5-10 mL) were obtained after informed consent from pregnant women undergoing amniocentesis. The samples were centrifuged at 1600 rpm for 5 min, and the pellets were resuspended in low glucose DMEM (Invitrogen) containing 12% FBS (Hyclone),4 ng/mL bFGF (R&D), and 1% penicillin/streptomycin at 37℃in 5% CO2. We studied its basic biological characteristics, particularly, signs of pluripotency, by morphology, Giemsa-banding karyotype analysis, determination of cell proliferation, flow cytometry, immunofluorescence staining and RT-PCR experiments.Detection of surface markers:SSEA-4, CD29, CD90, CD105, CD49, CD8, CD34, CD38, CD41, CD44, CD80, CD144, HLA-ABC (MHC class I), HLA-DR (MHC class II); nuclear antigen, Nanog and Oct4, were detected by immunofluorescence staining, in this operation, cell membranes were ruptured with 100% ethanol twice, each time 20 minutes; Detection of genes, Nanog, Oct4 and Rexl was performed by RT-PCR, inner reference gene was GAPDH.Results:hAFSCs were isolated from adhering cell clones containing cells with two different morphologies:one set of cells had a fibroblastoid, spindle-shaped morphology, while the other had an epithelioid, polygonal morphology. With passaging, the epithelioid cells rapidly disappeared from culture. By passage 3, the hAFSCs showed a homogenous fibroblast-like morphology. In the culture conditions mentioned above, hAFSCs can proliferate rapidly and doubled every 36 h; karyotype remained stable during long-term subculture. Flow cytometry analysis revealed that these cells expressed signs of mesenchymal origin, such as CD29, CD90 and CD 105, also expressed some embryonic signs such as the SSES-4, Nanog, Oct4 and Rex1, and expressed the major histocompatibility complex class I antigens(HLA-ABC), but not MHC II (HLA-DR)Part II:NRSF silencing induces hAFSCs differentiation into insulin-producing cells:Method:NRSF interference vectors (siNRSF) and scrambled control vectors (siControl) were constructed, both of which containing green fluorescent protein gene, and then infected hAFSCs by lentivirus-mediated method respectively, hAFSCs infected with siNRSF were named siNRSF-hAFSCs, whereas hAFSCs infected with siControl were named siControl-hAFSCs. On day 3, we detected the efficiency of interference using quantitative PCR and western blot. Freshly infected hAFSCs were planted in 6-well plates for differentiation in a two-stage protocol. In the first stage, cells were seeded in DMEM/F12 supplemented with 2% FBS,2% B27,10 ng/mL bFGF, and 10 ng/mL Activin-A for 7 days. In the second stage, the culture medium was changed to DMEM/F12 containing 2% FBS,2% B27,10 ng/mL Activin-A,10 ng/mL BTC, and 10 mM nicotinamide for the next 7 days. On day 7 and day 14, RT-PCR and immunofluorescence staining were used to detect the gene expression in these cells. On day 14, induced cells underwent the stimulation of glucose with different density,5.5mM and 23mM, C-peptide content and secretion were analyzed by ultra-sensitive ELISA kit. Protein concentration was determined using a BCA protein assay system, then the proportion of C-peptide was calculated.Results:Using fluorescence microscopy, we found that the infection efficiency of the lentivirus in hAFSCs was above 90%.3 days after infection. qRT-PCR showed that siNRSF down-regulated NRSF mRNA levels in hAFSCs nearly by 71%. Using RT-PCR, we found that NRSF silencing caused the up-regulation of Pax4 and Insulin. When cultured in differentiation medium, siNRSF-hAFSCs gradually expressed islet cell marker genes including Pdxl, Isl-1, Nkx6.1, Glut2, and Insulin, which were not detectable in siControl-hAFSCs treated with the same differentiation medium. Immunofluorescence staining showed that differentiated siNRSF-hAFSCs expressed Pdx-1, Insulin, and C-peptide. However, the induced siControl-hAFSCs did not express these proteins. ELISA results showed that the content of intracellular C-peptide was approximately 1.24 ng/mg in the differentiated SiNRSF-hAFSCs. In contrast, there were low levels of C-peptide in the induced siControl-hAFSCs. We also found that the differentiated siNRSF-hAFSCs could release C-peptide in response to glucose stimulation. C-peptide release in the high-glucose medium was nearly three times greater than the amount released in the low-glucose medium. However, the amount of released C-peptide was almost undetectable in induced siControl-hAFSCs, and no notable changes were detected in response to different glucose concentrations.Conclusion:hAFSCs is a group of fibroblast-like stem cells, with good proliferative capacity, low immunogenicity, and multiple differentiation potential. By combination of NRSF interference and inducing medium, hAFSCs can be induced to differentiate into functional insulin-secreting cells.