Shangmian Yie

Ontogeny of Human Umbilical Cord Perivascular Cells: Molecular and Fate Potential Changes During Gestation

Human umbilical cord-derived perivascular cells (PVCs) are a recently characterized source of mesenchymal stromal cells that has gained much interest in the field of cellular therapeutics. However, very little is known about the changes in fate potential and restrictions that these cells undergo during gestational development. This study is the first to examine the phenotypic, molecular, and functional properties of first trimester (FTM)-derived PVCs, outlining properties that are unique to this population when compared to term (TERM) counterparts. FTM- and TERM-PVCs displayed analogous mesenchymal, perivascular, and immunological immunophenotypes. Both PVCs could be maintained in culture without alteration to these phenotypes or mesenchymal lineage differentiation potential. Some unique features of FTM-PVCs were uncovered in this study: (1) while the gene signatures of FTM- and TERM-PVCs were similar, key differences were observed, namely, that the Oct4A and Sox17 proteins were detected in FTM-PVCs, but not in TERM counterparts; (2) FTM-PVCs exhibited a greater proliferative potential; and (3) FTM-PVCs were more efficient in their in vitro differentiation toward selective mesenchymal cell types, including the chondrogenic and adipogenic lineages, as well as toward neuronal- and hepatocyte-like lineages, when compared to TERM-PVCs. Both PVCs were able to generate osteocytes and cardiomyocyte-like cells with similar efficiencies in vitro. Overall, FTM-PVCs show more plasticity than TERM-PVCs with regard to fate acquisition, suggesting that a restriction in multipotentiality is imposed on PVCs as gestation progresses. Taken together, our findings support the idea that PVCs from earlier in gestation may be better than later sources of multipotent stromal cells (MSCs) for some regenerative medicine applications.

Expression of survivin in human oocytes and preimplantation embryos

OBJECTIVE To determine whether [1] survivin is expressed in human oocytes and embryos; [2] embryos grown in vitro secrete survivin protein; and [3] survivin levels are correlated with embryo cleavage rates. DESIGN Experimental. SETTING University-affiliated IVF clinic. PATIENT(S) Couples undergoing IVF-ET cycles. INTERVENTION(S) Conventional reverse transcriptase-polymerase chain reaction (PCR), real-time PCR, immunohistochemistry, Western blot on oocytes, embryos and control choriocarcinoma JEG-3 cells, and ELISA analysis of conditioned culture media. MAIN OUTCOME MEASURE(S) Detection of survivin mRNA and protein in oocytes and preimplantation embryos and in JEG-3 cancer cells. Detection of survivin concentrations in embryo culture media. RESULT(S) Survivin mRNA and protein were expressed during human oocyte maturation, from germinal vesicle to metaphase II stage, and throughout embryo development, from pronuclear stage to blastocyst stage. Survivin was localized predominantly in the cytoplasm of all cells examined and in the oocytes on the chromatin of metaphase chromosomes and midbodies. Western blot analysis of human oocyte and cancer cell extracts detected a full-length (primary) survivin band of 16.5 kDa. Survivin was also detected in conditioned media samples from embryo cultures and showed a positive correlation with embryo cleavage rates. CONCLUSION(S) Our data have demonstrated for the first time that human oocytes/embryos not only express but also secret survivin, suggesting that survivin may play an important role in human oogenesis and embryogenesis.

Dietary resources shape the adaptive changes of cyanide detoxification function in giant panda ( Ailuropoda melanoleuca )

The functional adaptive changes in cyanide detoxification in giant panda appear to be response to dietary transition from typical carnivore to herbivorous bear. We tested the absorption of cyanide contained in bamboo/bamboo shoots with a feeding trial in 20 adult giant pandas. We determined total cyanide content in bamboo shoots and giant panda’s feces, levels of urinary thiocyanate and tissue rhodanese activity using color reactions with a spectrophotometer. Rhodanese expression in liver and kidney at transcription and translation levels were measured using real-time RT-PCR and immunohistochemistry, respectively. We compared differences of rhodanese activity and gene expressions among giant panda, rabbit (herbivore) and cat (carnivore), and between newborn and adult giant pandas. Bamboo shoots contained 3.2 mg/kg of cyanide and giant pandas absorbed more than 65% of cyanide. However, approximately 80% of absorbed cyanide was metabolized to less toxic thiocyanate that was discharged in urine. Rhodanese expression and activity in liver and kidney of giant panda were significantly higher than in cat, but lower than in rabbit (all P < 0.05). Levels in adult pandas were higher than that in newborn cub. Phylogenetic analysis of both nucleotide and amino acid sequences of the rhodanese gene supported a closer relationship of giant panda with carnivores than with herbivores.

Characteristics of Mesenchymal Stem Cells Isolated from Bone Marrow of Giant Panda

In present study, we report on bone marrow (BM) mesenchymal stem cells (MSCs) that are isolated from giant pandas. Cells were collected from the BM of two stillborn giant pandas. The cells were cultured and expanded in 10% fetal bovine serum medium. Cell morphology was observed under an inverted microscopy, and the proliferation potential of the cells was evaluated by counting cell numbers for eight consecutive days. Differentiation potentials of the cells were determined by using a variety of differentiation protocols for osteocytes, adipocytes, neuron cells, and cardiomyocytes. Meanwhile, the specific gene expressions for MSCs or differentiated cells were analyzed by RT-PCR. The isolated cells exhibited a fibroblast-like morphology; expressed mesenchymal specific markers such as cluster of differentiation 73 (CD73), SRY (sex determining region Y)-box 2 (SOX-2), guanine nucleotide-binding protein-like 3 (GNL3), and stem cell factor receptor (SCFR); and could be differentiated into osteocytes and adipocytes that were characterized by Alizarin Red and Oil Red O staining. Under appropriate induction conditions, these cells were also able to differentiate into neuroglial-like or myocardial-like cells that expressed specific myocardial markers such as GATA transcription factors 4 (GATA-4), cardiac troponin T (cTnT), and myosin heavy chain 7B (MYH7B), or neural specific markers such as Nestin and glial fibrillary acidic protein (GFAP). This study demonstrated stem cells recovery and growth from giant pandas. The findings suggest that cells isolated from the BM of giant pandas have a high proliferative capacity and multiple differentiation potential in vitro which might aid conservation efforts.

Urinary profiles of luteinizing hormone, estrogen and progestagen during the estrous and gestational periods in giant pandas ( Ailuropda melanoleuca )

Luteinizing hormone (LH) is one of the main pituitary hormones that regulate ovulation, however its role has not been studied in giant panda. In this study, we developed an ELISA method for the detection of panda urinary LH. We analyzed urinary hormones of 24 female pandas during 36 breeding periods, we found females could easily be impregnated if the first mating occurred within 10 hours after LH peak. We also found the patterns of the ratios of urinary LH and progestagen in pandas that bred and successfully gave birth were significantly different from those that bred but failed to give birth. These data was the first to provide the urinary LH profiles during the estrous and gestational periods in pandas, and demonstrated that the appearance of the urinary LH peak indicated the timing of ovulation. The LH detection together with estrogen analysis makes the window for successful mating narrower than previously reported. Moreover, detection of urinary LH and progestagen can be used to discriminate between pregnancies and pseudopregnancies/miscarriages in the species. Thus, our findings suggest that LH not only plays a critical role in regulating ovulation but also plays an important role in maintaining pregnancy in the giant panda.