Zhongying Dou

Derivation and characterization of human embryonic germ cells: serum-free culture and differentiation potential

This study examined the effects of a chemically defined culture medium supplement, knock-out serum replacement (KSR), on the growth and differentiation of human embryonic germ cells (hEgc) and found that the efficiency of the initial establishment of hEGC lines in KSR medium was significantly higher than in fetal calf serum (FCS) medium. The percentage of undifferentiated hEGC colonies growing in KSR medium was significantly higher than in FCS-based medium (P < 0.05). The hEGC colonies showed typical mouse embryonic germ cell-like morphology. They showed normal and stable diploid karyotype and expressed alkaline phosphatase (AP), stage-specific embryonic antigens (SSEA) and other specific markers of pluripotent cells. In addition, hEGC could form simple and cystic embryoid bodies (EB) that consisted of various cell types including neural, epithelial and rhythmically beating cardiac cells, even sperm-like and oocyte-like cells. Tumour-like outgrowths were formed in nude mice and found to contain a variety of cell types, including uterine epithelium, adipocytes, squamous tissue and skin structures. In conclusion, an appropriate serum-free culture system has been developed for the establishment of hEGC lines. This may provide an in-vitro model to study differentiation and can be used as a potential source of therapy for infertility and regenerative medicine.

Pancreatic Islet-Like Clusters from Bone Marrow Mesenchymal Stem Cells of Human First-Trimester Abortus Can Cure Streptozocin-Induced Mouse Diabetes

Bone marrow mesenchymal stem cells (BMSCs) have been reported to possess low immunogenicity and cause immunosuppression of recipients when allografted. They can differentiate into insulin-producing cells and may be a valuable source for islet formation. However, the extremely low differentiating rate of adult BMSCs toward insulin-producing cells and the insufficient insulin secretion of the differentiated BMSCs in vitro prevent their clinical use in diabetes treatment. Little is known about the potential of cell replacement therapy with human BMSCs. Previously, we isolated and identified human first-trimester fetal BMSCs (hfBMSCs). Under a novel four-step induction procedure established in this study, the hfBMSCs effectively differentiated into functional pancreatic islet-like cell clusters that contained 62 ± 14% insulin-producing cells, expressed a broad gene profile related to pancreatic islet β-cell development, and released high levels of insulin (2.245 ± 0.222 pmol/100 clusters per 30 min) and C-peptide (2.200 ± 0.468 pmol/100 clusters per 30 min) in response to 25 mmol/L glucose stimulus in vitro. The pancreatic islet-like cell clusters normalized the blood glucose level of diabetic model mice for at least 9 weeks when xenografted; blood glucose levels in these mice rose abnormally again when the grafts were removed. Examination of the grafts indicated that the transplanted cells survived in recipients and produced human insulin and C-peptide in situ. These results demonstrate that hfBMSCs derived from a human first-trimester abortus can differentiate into pancreatic islet-like cell clusters following an established four-step induction. The insulin-producing clusters present advantages in cell replacement therapy of type 1 diabetic model mice.

The Four Reprogramming Factors and Embryonic Development in Mice

The transcription factors (Oct4, Sox2, c-Myc, and Klf4) play an important role in the generation of induced pluripotent stem cells. These factors are expressed in metaphase II oocytes and embryonic stem cells (ESCs). The mechanisms responsible for the reprogramming of ooplasm during nuclear transfer are expected to be associated with the four factors. Here, we show that different paternal genetic backgrounds are able to influence the in vitro development of parthenogenetic and cloned embryos. Using real- time polymerase chain reaction (PCR) we found that the expression level of Oct4 in oocytes was less than that of ESCs, whereas oocytes from KM x C3H females showed the highest expression level of Sox2 than the other strains tested or in G1 ESCs. c-Myc mRNA levels in oocytes from KM mice were greater than those found in ESCs or oocytes of KM x C3H mice. These data demonstrate that the expression of the four transcription factors was different among the oocytes, which may be a contributing factor for the different efficiencies of parthenogenesis and the development of cloned embryos in vitro.

Reconstruction of damaged corneal epithelium using Venus-labeled limbal epithelial stem cells and tracking of surviving donor cells

Limbal epithelial stem cells are responsible for the self-renewal and replenishment of the corneal epithelium. Although it is possible to repair the ocular surface using limbal stem cell transplantation, the mechanisms behind this therapy are unclear. To investigate the distribution of surviving donor cells in a reconstructed corneal epithelium, we screened a Venus-labeled limbal stem cell strain in goats. Cells were cultivated on denuded human amniotic membrane for 21 days to produce Venus-labeled corneal epithelial sheets. The Venus-labeled corneal epithelial sheets were transplanted to goat models of limbal stem cell deficiency. At 3 months post-surgery, the damaged corneal epithelia were obviously improved in the transplanted group compared with the non-transplanted control, with the donor cells still residing in the reconstructed ocular surface epithelium. Using Venus as a marker, our results indicated that the location and survival of donor cells varied, depending on the corneal epithelial region. Additionally, immunofluorescent staining of the reconstructed corneal epithelium demonstrated that many P63(+) cells were unevenly distributed among basal and suprabasal epithelial layers. Our study provides a new model, and reveals some of the mechanisms involved in corneal epithelial cell regeneration research.

Plasticity of marrow mesenchymal stem cells from human first-trimester fetus: from single-cell clone to neuronal differentiation.

Recent results have shown that bone marrow mesenchymal stem cells (BMSCs) from human first-trimester abortus (hfBMSCs) are closer to embryonic stem cells and perform greater telomerase activity and faster propagation than mid- and late-prophase fetal and adult BMSCs. However, no research has been done on the plasticity of hfBMSCs into neuronal cells using single-cell cloned strains without cell contamination. In this study, we isolated five single cells from hfBMSCs and obtained five single-cell cloned strains, and investigated their biological property and neuronal differentiation potential. We found that four of the five strains showed similar expression profile of surface antigen markers to hfBMSCs, and most of them differentiated into neuron-like cells expressing Nestin, Pax6, Sox1, β-III Tubulin, NF-L, and NSE under induction. One strain showed different expression profile of surface antigen markers from the four strains and hfBMSCs, and did not differentiate toward neuronal cells. We demonstrated for the first time that some of single-cell cloned strains from hfBMSCs can differentiate into nerve tissue-like cell clusters under induction in vitro, and that the plasticity of each single-cell cloned strain into neuronal cells is different.

Transplantation of ex vivo expanded stem cell amniotic sheet for ocular surface reconstruction

Transplantation of ex vivo expanded stem cell amniotic sheet for ocular surface reconstruction

Reconstruction of corneal epithelium with cryopreserved corneal limbal stem cells in a goat model

The integrity and transparency of the cornea plays a key role in preserving vision. This paper reports a procedure to create an artificial sheet of corneal epithelium from cryopreserved limbal stem cells (LSCs) and to use this for corneal transplantation. Corneal LSCs were isolated from biopsy specimens of rabbit limbal lamellar and cryopreserved in liquid nitrogen at 2-4 passages. The cells were grown in culture medium for 12-14 days on top of a cell-free human amniotic membrane framed on a nitrocellulose sheet. The corneal epithelium generated was transplanted into the right eyes of 14 LSC deficient (LSCD) rabbits (seven experimental animals, seven controls) with corneal damage. The seven LSCD rabbits in the experimental group were transplanted with a corneal epithelial sheet generated from the cryopreserved corneal LSCs. Four LSCD rabbits were used as the vehicle control and were transplanted with a cell-free amniotic membrane, and the remaining three LSCD rabbits were negative controls without transplantation. Over a 2-month recovery period, 2/7 animals in the experimental group recovered completely, four recovered partially and one did not respond. In the control groups, three negative controls and three vehicle controls lost their vision completely, and one of the vehicle controls partially recovered transparency of the cornea Following treatment, corneal transparency of the experimental rabbits was significantly improved compared to controls (P<0.05). The results indicated that cryopreserved corneal LSCs can repair damaged rabbit cornea, suggesting a possible new clinical approach to reconstruction of corneal epithelium.