Chunyu Bai

Combination of melatonin and Wnt-4 promotes neural cell differentiation in bovine amniotic epithelial cells and recovery from spinal cord injury.

Although melatonin has been shown to exhibit a wide variety of biological functions, its effects on promoting differentiation of neural cells remain unknown. Wnt signaling mediates major developmental processes during embryogenesis and regulates maintenance, self‐renewal, and differentiation of adult mammalian stem cells. However, the role of the noncanonical Wnt pathway during neurogenesis remains poorly understood. In this study, the amniotic epithelial cells ( AECs) were isolated from bovine amnion and incubated with various melatonin concentrations (0.01, 0.1, 1, 10, or 100 μm) and 5 × 10−5 m all‐trans retinoic acid (RA) for screening optimum culture medium of neural differentiation, compared with each groups, 1 μm melatonin and 5 × 10−5 m RA were selected to induce neural differentiation of AECs, and then siMT1, siMT2, oWnt‐4, and siWnt‐4 were expressed in AECs to research role of these genes in neural differentiation. Efficiency of neural differentiation was evaluated after expressed above genes using flow cytometry. Cell function of neural cells was demonstrated in vivo using spinal cord injury model after cell transplantation, and damage repair of spinal cord was assessed using cell tracking and Basso, Beattie, Bresnahan Locomotor Rating Scale scores. Results demonstrated that melatonin stimulated melatonin receptor 1, which subsequently increased bovine amniotic epithelial cell vitality and promoted differentiation into neural cells. This took place through cooperation with Wnt‐4. Additionally, following cotreatment with melatonin and Wnt‐4, neurogenesis gene expression was significantly altered. Furthermore, single inhibition of melatonin receptor 1 or Wnt‐4 expression decreased expression of neurogenesis‐related genes, and bovine amniotic epithelial cell‐derived neural cells were successfully colonized into injured spinal cord, which suggested participation in tissue repair.

Role of microRNA-21 in the formation of insulin-producing cells from pancreatic progenitor cells

MicroRNAs (miRNAs) regulate insulin secretion, pancreas development, and beta cell differentiation. In this study, to screen for miRNAs and their targets that function during insulin-producing cells (IPCs) formation, we examined the messenger RNA and microRNA expression profiles of pancreatic progenitor cells (PPCs) and IPCs using microarray and deep sequencing approaches, respectively. Combining our data with that from previous reports, we found that miR-21 and its targets play an important role in the formation of IPCs. However, the function of miR-21 in the formation of IPCs from PPCs is poorly understood. Therefore, we over-expressed or inhibited miR-21 and expressed small interfering RNAs of miR-21 targets in PPCs to investigate their functions in IPCs formation. We found that miR-21 acts as a bidirectional switch in the formation of IPCs by regulating the expression of target and downstream genes (SOX6, RPBJ and HES1). Small interfering RNAs were used to knock down these genes in PPCs to investigate their effects on IPCs formation. Single expression of si-RBPJ, si-SOX6 and si-HES1 in PPCs showed that si-RBPJ was an inhibitor, and that si-SOX6 and si-HES1 were promoters of IPCs formation, although si-HES1 induced formation of IPCs at higher rates than si-SOX6. These results suggest that endogenous miRNAs involved in the formation of IPCs from PPCs should be considered in the development of an effective cell transplant therapy for diabetes.

Isolation and biological characteristics of chicken adipose-derived progenitor cells.

Adipose-derived stem cells/adipose-derived progenitor cells (ADPCs) are multipotent stem cells that can differentiate in vitro into many cell types. However, the vast majority of experimental materials were obtained from human, mouse, rabbit, and other mammals but rarely from poultry. In this study, ADPCs were isolated from 1-day-old chicks. Primary ADPCs were subcultured to passage 15. The surface markers of ADPCs, CD29, CD44, CD71, and CD73, were detected by immunofluorescence and RT-polymerase chain reaction assays. The growth curves of different passages were all typically sigmoidal. In addition, ADPCs of different passages were successfully induced to differentiate into osteoblasts, adipocytes, and myocardial cells. The results suggest that the ADPCs isolated from chicken possess similar biological characteristics with those derived from other species, and their multilineage differentiation provides many potential applications.

Isolation and characterization of mesenchymal stem cells from chicken bone marrow.

The bone marrow mesenchymal stem cells (BMSCs) are multipotent stem cells, which can differentiate in vitro into many cell types. However, the vast majority of experimental materials were obtained from human, mouse, rabbit and other mammals, but rarely in poultry. So, in this study, Thirty- to sixty-day old chicken was chosen as experimental animal, to isolate and characterize BMSCs from them. To investigate the biological characteristics of chicken BMSCs, immunofluorescence and RT-PCR were used to detect the characteristic surface markers of BMSCs. Growth curves were drawn in accordance with cell numbers. To assess the differentiation capacity of the BMSCs, cells were induced to differentiate into osteoblasts, adipocytes, and endothelial cells. The surface markers of BMSCs, CD29, CD44, CD31, CD34, CD71 and CD73, were detected by immunofluorescence and RT-PCR assays. The growth curves of different passages were all typically sigmoidal. Karyotype analysis showed that these in vitro cultured cells were genetically stable. In addition, BMSCs were successfully induced to differentiate into osteoblasts, adipocytes, and endothelial cells. The results suggest that the BMSCs isolated from chicken possess similar biological characteristics with those separated from other species, and their multi-lineage differentiation potentiality herald a probable application for cellular transplant therapy in tissue engineering.

MicroRNAs can effectively induce formation of insulin-producing cells from mesenchymal stem cells: miRNA in formation of insulin-producing cells

MicroRNAs regulate insulin secretion, pancreatic development and beta cell differentiation. However, the function of microRNAs in the formation of insulin‐producing cells (IPCs) from adult stem cells is poorly understood. We examine the microRNA expression profile in nestin‐positive umbilical cord‐derived mesenchymal stem cells (N‐UCMSCs) and nestin‐positive pancreatic mesenchymal stem cells using a deep sequencing approach. We also selected specific microRNAs for overexpression in N‐UCMSCs and found that miR‐375 and miR‐26a induced IPCs differentiation from N‐UCMSCs by downregulating target genes including mtpn, sox6, bhlhe22 and ccnd1. Small interfering RNAs were also used to knock down these genes in N‐UCMSCs to induce the formation of IPCs. These results suggest that endogenous microRNAs involved in the formation of IPCs from adult stem cells show promise for advancing the development of an effective cell transplant therapy for diabetes. Copyright

Melatonin improves reprogramming efficiency and proliferation of bovine-induced pluripotent stem cells

Melatonin can modulate neural stem cell (NSC) functions such as proliferation and differentiation into NSC‐derived pluripotent stem cells (N‐iPS) in brain tissue, but the effect and mechanism underlying this are unclear. Thus, we studied how primary cultured bovine NSCs isolated from the retinal neural layer could transform into N‐iPS cell. NSCs were exposed to 0.01, 0.1, 1, 10, or 100 μm melatonin, and cell viability studies indicated that 10 μm melatonin can significantly increase cell viability and promote cell proliferation in NSCs in vitro. Thus, 10 μm melatonin was used to study miR‐302/367‐mediated cell reprogramming of NSCs. We noted that this concentration of melatonin increased reprogramming efficiency of N‐iPS cell generation from primary cultured bovine NSCs and that this was mediated by downregulation of apoptosis‐related genes p53 and p21. Then, N‐iPS cells were treated with 1, 10, 100, or 500 μm melatonin, and N‐iPS (M‐N‐iPS) cell proliferation was measured. We noted that 100 μm melatonin increased proliferation of N‐iPS cells via increased phosphorylation of intracellular ERK1/2 via activation of its pathway in M‐N‐iPS via melatonin receptors 1 (MT1). Finally, we verified that N‐iPS cells and M‐N‐iPS cells are similar to typical embryonic stem cells including the expression of pluripotency markers (Oct4 and Nanog), the ability to form teratomas in vivo, and the capacity to differentiate into all three embryonic germ layers.

Differentiation of chicken umbilical cord mesenchymal stem cells into beta-like pancreatic islet cells

Abstract In this study, we explored the possibility of using in vitro differentiation to create functional beta-like islet cells from chicken umbilical cord mesenchymal stem cells (UCMSCs). Passaged UCMSCs were induced to differentiate into pancreatic beta-like islet cells. Differentiated cells were observed through dithizone staining, and Pdx1 and insulin expressed in differentiated cells were detected with immunofluorescence. Insulin and C-peptide production from differentiated cells were analyzed using ELISA and western blotting. Differentiated cells were found to not only express Pdx1, insulin, and C-peptide, but also to display a glucose-responsive secretion of these hormones.

Biological characterization of chicken mesenchymal stem/progenitor cells from umbilical cord Wharton’s Jelly

Mesenchymal stem/progenitor cells derived from Wharton’s jelly of the umbilical cord (UCMSCs/UCMPCs) are multipotent, and can be differentiated in vitro into many cell types. Much work has been done on UCMSCs/UCMPCs from humans, mice, rabbits, and other mammals, but the relatively little literature has been published about these cells in chickens. In our work, we isolated USMSCs/USMPCs from chicken embryos. We characterized the isolated cells using immunofluorescence and reverse transcription polymerase chain reaction techniques. Primary UCMSCs/UCMPCs were subcultured to passage 30 and growth curves for each passage determined. The growth curves at different passages were all typically sigmoidal. Isolated UCMSCs/UCMPCs were induced to differentiate into adipocytes, osteoblasts, myocardial cells, and neural cells, and we were able to detect characteristic CD44, CD29, CD73, and CD71 cell surface markers. Our results suggest that UCMSCs/UCMPCs isolated from chickens possess similar biological characteristics to those from other species. Their multi-lineage differentiation capabilities herald a probable application for cellular transplant therapy in tissue engineering.Mesenchymal stem/progenitor cells derived from Wharton’s jelly of the umbilical cord (UCMSCs/UCMPCs) are multipotent, and can be differentiated in vitro into many cell types. Much work has been done on UCMSCs/UCMPCs from humans, mice, rabbits, and other mammals, but the relatively little literature has been published about these cells in chickens. In our work, we isolated USMSCs/USMPCs from chicken embryos. We characterized the isolated cells using immunofluorescence and reverse transcription polymerase chain reaction techniques. Primary UCMSCs/UCMPCs were subcultured to passage 30 and growth curves for each passage determined. The growth curves at different passages were all typically sigmoidal. Isolated UCMSCs/UCMPCs were induced to differentiate into adipocytes, osteoblasts, myocardial cells, and neural cells, and we were able to detect characteristic CD44, CD29, CD73, and CD71 cell surface markers. Our results suggest that UCMSCs/UCMPCs isolated from chickens possess similar biological characteristics to those from other species. Their multi-lineage differentiation capabilities herald a probable application for cellular transplant therapy in tissue engineering.

Isolation and Characterization of Chicken Dermis-Derived Mesenchymal Stem/Progenitor Cells

Dermis-derived mesenchymal stem/progenitor cells (DMS/PCs) were isolated from the skin tissue of 16-day-old chick embryos and then characterized by immunofluorescence and RT-PCR. We found that primary DMS/PCs could be expanded for 15 passages. Expression of β-integrin, CD44, CD71, and CD73 was observed by immunofluorescence and RT-PCR. Passage 3 DMS/PCs were successfully induced to differentiate into osteoblasts, adipocytes, and neurocytes. The results indicate the potential for multilineage differentiation of DMS/PCs that may represent an ideal candidate for cellular transplantation therapy.

Characterization of vascular endothelial progenitor cells from chicken bone marrow

BackgroundEndothelial progenitor cells (EPC) are a type of stem cell used in the treatment of atherosclerosis, vascular injury and regeneration. At present, most of the EPCs studied are from human and mouse, whereas the study of poultry-derived EPCs has rarely been reported. In the present study, chicken bone marrow-derived EPCs were isolated and studied at the cellular level using immunofluorescence and RT-PCR.ResultsWe found that the majority of chicken EPCs were spindle shaped. The growth-curves of chicken EPCs at passages (P) 1, -5 and -9 were typically “S”-shaped. The viability of chicken EPCs, before and after cryopreservation was 92.2% and 81.1%, respectively. Thus, cryopreservation had no obvious effects on the viability of chicken EPCs. Dil-ac-LDL and FITC-UAE-1 uptake assays and immunofluorescent detection of the cell surface markers CD34, CD133, VEGFR-2 confirmed that the cells obtained in vitro were EPCs. Observation of endothelial-specific Weibel-Palade bodies using transmission electron microscopy further confirmed that the cells were of endothelial lineage. In addition, chicken EPCs differentiated into endothelial cells and smooth muscle cells upon induction with VEGF and PDGF-BB, respectively, suggesting that the chicken EPCs retained multipotency in vitro.ConclusionsThese results suggest that chicken EPCs not only have strong self-renewal capacity, but also the potential to differentiate into endothelial and smooth muscle cells. This research provides theoretical basis and experimental evidence for potential therapeutic application of endothelial progenitor cells in the treatment of atherosclerosis, vascular injury and diabetic complications.