Mesenchymal stem cells promote ovarian reconstruction in mice.

BACKGROUND: Studies have shown that chemotherapy and radiotherapy can cause premature ovarian failure and loss of fertility in female cancer patients. Ovarian cortex cryopreservation is a good choice to preserve female fertility before cancer treatment. Following the remission of the disease, the thawed ovarian tissue can be transplanted back and restore fertility of the patient. However, there is a risk to reintroduce cancer cells in the body and leads to the recurrence of cancer. Given the low success rate of current in vitro culture techniques for obtaining mature oocytes from primordial follicles, an artificial ovary with primordial follicles may be a good way to solve this problem. METHODS: In the study, we established an artificial ovary model based on the participation of mesenchymal stem cells (MSCs) to evaluate the effect of MSCs on follicular development and oocyte maturation. P2.5 mouse ovaries were digested into single cell suspensions and mixed with bone marrow derived mesenchymal stem cells (BM-MSCs) at a 1:1 ratio. The reconstituted ovarian model was then generated by using phytohemagglutinin. The phenotype and mechanism studies were explored by follicle counting, immunohistochemistry, immunofluorescence, in vitro maturation (IVM), in vitro fertilization (IVF), real-time quantitative polymerase chain reaction (RT-PCR), and Terminal-deoxynucleotidyl transferase mediated nick end labeling(TUNEL) assay. RESULTS: Our study found that the addition of BM-MSCs to the reconstituted ovary can enhance the survival of oocytes and promote the growth and development of follicles. After transplanting the reconstituted ovaries under kidney capsules of the recipient mice, we observed normal folliculogenesis and oocyte maturation. Interestingly, we found that BM-MSCs did not contribute to the formation of follicles in ovarian aggregation, nor did they undergo proliferation during follicle growth. Instead, the cells were found to be located around growing follicles in the reconstituted ovary. When theca cells were labeled with CYP17a1, we found some overlapped staining with green fluorescent protein(GFP)-labeled BM-MSCs. The results suggest that BM-MSCs may participate in directing the differentiation of theca layer in the reconstituted ovary. CONCLUSIONS: The presence of BM-MSCs in the artificial ovary was found to promote the survival of ovarian cells, as well as facilitate follicle formation and development. Since the cells didn't proliferate in the reconstituted ovary, this discovery suggests a potential new and safe method for the application of MSCs in clinical fertility preservation by enhancing the success rate of cryo-thawed ovarian tissues after transplantation.

Human amnion-derived mesenchymal stem cells improve subclinical hypothyroidism by immunocompetence mediating apoptosis inhibition on thyroid cells in aged mice.

Subclinical hypothyroidism (SCH) affects 10% of the global population, which is most prevalent in women and the elderly. However, it remains debatable whether the elderly with subclinical hypothyroidism needs thyroxine supplement. Human amnion-derived mesenchymal stem cells (hAMSCs) could play important roles in autoimmune diseases, suggesting that hAMSC be a candidate to regulate the thyroid function of female age-related subclinical hypothyroidism. Herein, we established the model of SCH in the aged female mice. This study was designed to investigate whether human amnion-derived mesenchymal stem cells (hAMSC) could effect on immune regulation, apoptosis inhibition of thyroid cells, thyroid function, blood lipid levels, and heart function. In addition, qualified hAMSCs were intravenously injected into aged female SCH mice via the tail vein on day 0 and day 10. The levels of thyroid hormone and blood lipids as well as cardiac function, serum immunological indexes, and apoptosis of thyroid cells were then analyzed on day 5, 10, 15, and 20; meanwhile, the quantity of Th1, Th2, Th17, and Treg immune cells in peripheral blood was evaluated before and on day 20 post-injection. Our study demonstrated that after hAMSC transplantation, the thyroid functions, blood lipid levels, and heart function indexes of age-related SCH (AR-SCH) mice were significantly improved. Consistent with this, Th1 and Treg cells increased significantly, while Th2 and Th17 cells decreased in peripheral blood. Apoptosis was also suppressed in the thyroid cells. In summary, hAMSC delivery can potentially be a safe and effective therapy for treating SCH in the elderly, improving related complications by immunomodulatory and apoptosis inhibition.

Rapid and non-invasive diagnostic techniques for embryonic developmental potential: a metabolomic analysis based on Raman spectroscopy to identify the pregnancy outcomes of IVF-ET.

The non-invasive and rapid assessment of the developmental potential of embryos is of great clinical importance in assisted reproductive technology (ART). In this retrospective study, we analyzed the metabolomics of 107 samples provided by volunteers and utilized Raman spectroscopy to detect the substance composition in the discarded culture medium of 53 embryos resulting in successful pregnancies and 54 embryos that did not result in pregnancy after implantation. The culture medium from D3 cleavage-stage embryos was collected after transplantation and a total of 535 (107 × 5) original Raman spectra were obtained. By combining several machine learning methods, we predicted the developmental potential of embryos, and the principal component analysis-convolutional neural network (PCA-CNN) model achieved an accuracy rate of 71.5%. Furthermore, the chemometric algorithm was used to analyze seven amino acid metabolites in the culture medium, and the data showed significant differences in tyrosine, tryptophan, and serine between the pregnancy and non-pregnancy groups. The results suggest that Raman spectroscopy, as a non-invasive and rapid molecular fingerprint detection technology, shows potential for clinical application in assisted reproduction.

Deacetylation of FOXP1 by HDAC7 potentiates self-renewal of mesenchymal stem cells.

BACKGROUND: Mesenchymal stem cells (MSCs) are widely used in a variety of tissue regeneration and clinical trials due to their multiple differentiation potency. However, it remains challenging to maintain their replicative capability during in vitro passaging while preventing their premature cellular senescence. Forkhead Box P1 (FOXP1), a FOX family transcription factor, has been revealed to regulate MSC cell fate commitment and self-renewal capacity in our previous study. METHODS: Mass spectra analysis was performed to identify acetylation sites in FOXP1 protein. Single and double knockout mice of FOXP1 and HDAC7 were generated and analyzed with bone marrow MSCs properties. Gene engineering in human embryonic stem cell (hESC)-derived MSCs was obtained to evaluate the impact of FOXP1 key modification on MSC self-renewal potency. RESULTS: FOXP1 is deacetylated and potentiated by histone deacetylase 7 (HDAC7) in MSCs. FOXP1 and HDAC7 cooperatively sustain bone marrow MSC self-renewal potency while attenuating their cellular senescence. A mutation within human FOXP1 at acetylation site (T176G) homologous to murine FOXP1 T172G profoundly augmented MSC expansion capacity during early passages. CONCLUSION: These findings reveal a heretofore unanticipated mechanism by which deacetylation of FOXP1 potentiates self-renewal of MSC and protects them from cellular senescence. Acetylation of FOXP1 residue T172 as a critical modification underlying MSC proliferative capacity. We suggest that in vivo gene editing of FOXP1 may provide a novel avenue for manipulating MSC capability during large-scale expansion in clinical trials.

Therapeutic effects and mechanism of human amnion-derived mesenchymal stem cells on hypercoagulability in a uremic calciphylaxis patient.

Calciphylaxis is a rare cutaneous vascular disease that manifests with intolerable pains, non-healing skin wounds, histologically characterized by calcification, fibrointimal hyperplasia, and microvessel thrombosis. Currently, there are no standardized guidelines for this disease. Recent studies have recognized a high prevalence of thrombophilias and hypercoagulable conditions in calciphylaxis patients. Here, we report a case of uremic calciphylaxis patient whom was refractory to conventional treatments and then received a salvage strategy with intravenous and local hAMSC application. In order to investigate the therapeutic mechanism of hAMSCs from the novel perspective of hypercoagulability, coagulation-related indicators, wound status, quality of life and skin biopsy were followed up. Polymerase chain reaction (PCR) was performed to determine the distribution of hAMSCs in multiple tissues including lung, kidney and muscle after infusion of hAMSCs for 24 h, 1 week and 1 month in mice aiming to investigate whether hAMSCs retain locally active roles after intravenous administration. Improvement of hypercoagulable condition involving correction of platelet, D-dimer and plasminogen levels, skin regeneration and pain alleviation were revealed after hAMSC administration over one-year period. Skin biopsy pathology suggested regenerative tissues after 1 month hAMSC application and full epidermal regeneration after 20 months hAMSC treatment. PCR analysis indicated that hAMSCs were homing in lung, kidney and muscle tissues of mice even until tail vein injection of hAMSCs for 1 month. We propose that hypercoagulability is a promising therapeutic target of calciphylaxis patients, which can be effectively improved by hAMSC treatment.

Human amnion-derived mesenchymal stem cells attenuate acute lung injury in two different acute lung injury mice models.

Acute lung injury (ALI) is one of the most common clinical emergencies with limited effective pharmaceutical treatment in the clinic, especially when it progresses to acute respiratory distress syndrome (ARDS). Currently, mesenchymal stem cells (MSCs) exhibit specific superiority for ALI/ARDS treatment. However, stem cells from different sources may result in controversial effects on similar disease conditions. This study aimed to determine the effects of human amnion-derived mesenchymal stem cells (hAMSCs) on two different ALI mice model. The administered hAMSCs effectively accumulated in the lung tissues in all hAMSC-treated groups. Compared with the model and 1% human serum albumin (HSA) groups, high-dose hAMSCs (1.0 × 106 cells) group significantly alleviated alveolar-capillary permeability, oxidative stress, inflammatory factors level and histopathological damage. In addition, the NF-κB signaling pathway is one of the key pathways activated during lipopolysaccharide (LPS) or paraquat (PQ)-induced lung injury. Our results indicated that hAMSCs (1.0 × 106 cells) obviously inhibited the expression of p-IKKα/ß, p-IκBα, and p-p65 in the lung tissue (p < 0.05). The high-dose (HD) hAMSC treatment exerted beneficial therapeutic effects on ALI mice models without detectable adverse reactions. The therapeutic effect of hAMSCs might involve NF-κB signaling pathway inhibition. hAMSC treatment is a potential candidate therapy for ALI.

Adverse effect of assisted reproductive technology-related hyperoestrogensim on the secretion and absorption of uterine fluid in superovulating mice during the peri-implantation period.

Objectives: This study aimed to investigate the potential mechanism of hyperoestrogensim elicited by ovulation induction affects endometrial receptivity and leads to embryo implantation abnormality or failure. Study design: Establishment of ovulation induction mouse model. Changes in mouse body weight, ovarian weight, serum E2 level and oestrous cycle were observed. During the peri-implantation period, morphological changes in the mouse uterus and implantation sites and the localization and protein levels of oestrogen receptors ERα and ERß, the tight junction factors CLDN3 and OCLN, the aquaporins AQP3, AQP4 and AQP8, and the sodium channel proteins SCNN1α, SCNN1ß and SCNN1γ were observed. The expression and cellular localization of ERα, CLDN3, AQP8 and SCNN1 ß in RL95-2 cell line were also detected by western blotting and immunofluorescence. Results: Ovarian and body weights were significantly higher in the 5 IU and 10 IU groups than in the CON group. The E2 level was significantly higher in the 10 IU group than in the CON group. The mice in the 10 IU group had a disordered oestrous cycle and were in oestrus for a long time. At 5.5 dpc, significantly fewer implantation sites were observed in the 10 IU group than in the CON (p<0.001) and 5 IU (p<0.05) groups. The probability of abnormal implantation and abortion was higher in the 10 IU group than in the CON and 5 IU groups. CLDN3, OCLN, AQP8 and SCNN1ß in the mouse endometrium were localized on the luminal epithelium and glandular epithelium and expression levels were lower in the 10 IU group than in the CON group. The protein expression level of ERα was increased by 50% in the 10 IU group compared to the CON group. The expressions of CLDN3, AQP8, SCNN1ß in RL95-2 cell line were significantly depressed by the superphysiological E2, ERα agonist or ERß agonist, which could be reversed by the oestrogen receptor antagonist. Conclusion: ART-induced hyperoestrogenism reduces CLDN3, AQP8 and SCNN1ß expression through ERα, thereby destroying tight junctions and water and sodium channels in the endometrial cavity epithelium, which may cause abnormal implantation due to abnormal uterine fluid secretion and absorption.

Establishing a human embryonic stem cell line (SKLRMe005-A) from a blastocyst with congenital heart disease (CHD).

GATA binding protein 6 (GATA6) is an important transcription factor of cardiovascular endothelial cells, has the potential to regulate the process of cardiac development. Consequently, its abnormal expression is related to congenital heart disease.Human GATA6 gene clones were on chromosome 18 q11.1- q 11.2, consists of 7 exons and 6 intron.Now, a human embryonic stem cell line with GATA6 c.620_647del (p.S208Afs*77) mutation was generated. Interestingly, the ESC line displayed a normal karyotype, pluripotency and morphology of stem cells.This cell line has the ability to undergo differentiation into three germ layers in vivo.

Peroxiredoxin 4 secreted by cumulus cells ameliorates the maturation of oocytes in vitro.

BACKGROUND: Peroxiredoxin 4 (Prdx4) in the endoplasmic reticulum (ER) is the only secretory member of the antioxidant Prdx family. Our previous studies demonstrated that Prdx4 in cumulus cells (CCs) ameliorated the maturation of oocytes in vitro and enhanced oocyte developmental competence by preventing CCs apoptosis caused by oxidative stress (OS) through gap junctions. In this study, we aimed to determine whether Prdx4 released by CCs can repair meiotic defects in mouse oocytes by co-culturing immature (germinal vesicle) oocytes with CCs from mature oocytes in the absence of gap junctions. RESULTS: The OS-induced meiotic defects in mouse oocytes were impeded by co-culture with CCs, as evidenced by the increased first polar body (PB1) extrusion rate and decreased ROS level. CCs increased Prdx4 expression and lowered IRE1α, Bip expression in H2O2-treated oocytes. After knockdown of Prdx4 expression in CCs, the rate of PB1 extrusion in the oocytes was significantly reduced to the level detected in H2O2 group, and ER stress was not alleviated. CO-IP and immunofluorescence co-localization experiments demonstrated that Prdx4 interacted with PDIA6 in the oocytes and the Pearson's R value was 0.69 calculated using ImageJ. CONCLUSIONS: Cumulus cells can promote the maturation of oocytes in vitro by secreting Prdx4 in a paracrine manner and serve as a promising therapeutic antioxidant for improving the quality of oocytes, especially aging oocytes, in clinical in vitro maturation (IVM).

Effects of putrescine on the quality and epigenetic modification of mouse oocytes during in vitro maturation.

CONTEXT: Low ovarian putrescine levels and decreased peak values following luteinising hormone peaks are related to poor oocyte quantity and quality in ageing women. AIMS: To investigate the effects of putrescine supplementation in in vitro maturation (IVM) medium on oocyte quality and epigenetic modification. METHODS: Germinal vesicle oocytes retrieved from the ovaries of 8-week-old and 9-month-old mice were divided into four groups (the young, young+difluoromethylornithine (DFMO), ageing and ageing+putrescine groups) and cultured in IVM medium with or without 1mM putrescine or DFMO for 16h. The first polar body extrusion (PBE), cleavage and embryonic development were evaluated. Spindles, chromosomes, mitochondria and reactive oxygen species (ROS) were measured. The expression levels of SIRT1, H3K9ac, H3K9me2, H3K9me3, and 5mC levels were evaluated. Sirt1 and imprinted genes were detected. RESULTS: The PBE was higher in the ageing+putrescine group than in the ageing group. Putrescine increased the total and inner cell mass cell numbers of blastocysts in ageing oocytes. Putrescine decreased aberrant spindles and chromosome aneuploidy, increased the mitochondrial membrane potential and decreased ROS levels. Putrescine increased SIRT1 expression and attenuated the upregulation of H3K9ac levels in ageing oocytes. Putrescine did not affect 5mC, H3K9me2 or H3K9me3 levels or imprinted gene expression. CONCLUSIONS: Putrescine supplementation during IVM improved the maturation and quality of ageing oocytes and promoted embryonic development by decreasing ROS generation, maintaining mitochondrial and spindle function and correcting aberrant epigenetic modification. IMPLICATIONS: Putrescine shows application potential for human-assisted reproduction, especially for IVM of oocytes from ageing women.

Mesenchymal Stem Cell-derived Nanovesicles as a Credible Agent for Therapy of Pulmonary Hypertension.

Extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs) have been evaluated in many studies as promising therapeutic agents for pulmonary hypertension (PH). However, low yields and heterogeneity are major barriers in the translational utility of EVs for clinical studies. To address these limitations, we fabricated MSC-derived nanovesicles (MSC-NVs) by serial extrusion through filters, resulting in MSC-NVs with characteristics similar to conventional EVs but with much higher production yields. Herein, we examined the therapeutic efficacy of MSC-NVs in preclinical models of PH in vitro and in vivo. Intervention with MSC-NVs improved the core pathologies of monocrotaline-induced PH in rats. Intravenous administration of MSC-NVs resulted in significant uptake within hypertensive lungs, pulmonary artery lesions, and especially pulmonary artery smooth muscle cells (PASMCs). In vitro, MSC-NVs inhibited PDGF-induced proliferation, migration, and phenotype switching of PASMCs. miRNA-sequencing analysis of the genetic cargo of MSC-NVs revealed that miR-125b-5p and miR-100-5p are highly abundant, suggesting that they might account for the therapeutic effects of MSC-NVs in PH. Depletion of miR-125b-5p and miR-100-5p in MSCs almost completely abolished the beneficial effects of MSC-NVs in protecting PASMCs from PDGF-stimulated changes in vitro and also diminished the protective effects of MSC-NVs in monocrotaline-induced PH in vivo. These data highlight the efficacy and advantages of MSC-NVs over MSC-EVs as a promising therapeutic strategy against PH.

Nucleoporin37 may play a role in early embryo development in human and mice.

Maternal-effect genes (MEGs) play an important role in maintaining the survival and development of mammalian embryos at the cleavage stage after fertilization. Despite long-term efforts, the MEGs that regulate preimplantation embryo development remain largely unknown. Here, using whole-exome sequencing and homozygosity mapping, we identified a potential candidate gene associated with early embryo development: nucleoporin37 (NUP37), a nucleoporin gene that encodes a member of the nuclear pore complexes and regulates nuclear pore permeability and nucleocytoplasmic transport. Moreover, we determined the temporal and spatial expression patterns of Nup37 in mouse oocytes and early embryos, and explored the role of NUP37 in oocyte maturation and preimplantation embryo development. Immunoprecipitation assays confirmed that yes-associated protein-1 (YAP1) binds to TEA domain transcription factor 4 (TEAD4) and NUP37. Furthermore, Nup37 gene knockdown reduced the nuclear import of YAP1 and down-regulated the expression of YAP1-TEAD pathway downstream genes Rrm2 and Rpl13 in early embryos. Our study provides evidence that maternal NUP37 contributes to the nuclear import of YAP1 and then activates the YAP1-TEAD pathway, a signalling pathway essential for zygotic genome activation. Nup37 may be a key gene involved in preimplantation embryo development in mammals.

A novel long-term intravenous combined with local treatment with human amnion-derived mesenchymal stem cells for a multidisciplinary rescued uremic calciphylaxis patient and the underlying mechanism.

Calciphylaxis is a rare disease characterized histologically by microvessel calcification and microthrombosis, with high mortality and no proven therapy. Here, we reported a severe uremic calciphylaxis patient with progressive skin ischemia, large areas of painful malodorous ulcers, and mummified legs. Because of the worsening symptoms and signs refractory to conventional therapies, treatment with human amnion-derived mesenchymal stem cells (hAMSCs) was approved. Preclinical release inspections of hAMSCs, efficacy, and safety assessment, including cytokine secretory ability, immunocompetence, tumorigenicity, and genetics analysis in vitro, were introduced. We further performed acute and long-term hAMSC toxicity evaluations in C57BL/6 mice and rats, abnormal immune response tests in C57BL/6 mice, and tumorigenicity tests in neonatal Balbc-nu nude mice. After the preclinical research, the patient was treated with hAMSCs by intravenous and local intramuscular injection and external supernatant application to the ulcers. When followed up to 15 months, the blood-based markers of bone and mineral metabolism improved, with skin soft tissue regeneration and a more favorable profile of peripheral blood mononuclear cells. Skin biopsy after 1-month treatment showed vascular regeneration with mature noncalcified vessels within the dermis, and 20 months later, the re-epithelialization restored the integrity of the damaged site. No infusion or local treatment-related adverse events occurred. Thus, this novel long-term intravenous combined with local treatment with hAMSCs warrants further investigation as a potential regenerative treatment for uremic calciphylaxis due to effects of inhibiting vascular calcification, stimulating angiogenesis and myogenesis, anti-inflammatory and immune modulation, multidifferentiation, re-epithelialization, and restoration of integrity.

Establishment of human embryonic stem cell (SKLRMe001-A)carrying Azoospermic factor c (AZFc) deletions.

The absence of azoospermia factor c (AZFc) is a common molecular cause of sperm failure. In men with non-obstructive azoospermia or severe oligospermia, the incidence of AZFc is around 10%. The AZFc region is located at the far end of the Yqll chromosome which has three non-overlapping sub-regions with a high frequency of deletion. Now, we generated a human embryonic stem cell line (SKLRMe001-A) that carries a deleted AZFc gene on the Y chromosome. The ESC line maintains a stem cell-like morphology, pluripotency, and has a normal karyotype. The cells can also differentiate into all three germ layers in vivo.

Sorbitol accumulation decreases oocyte quality in aged mice by altering the intracellular redox balance.

Sorbitol is a product of glucose metabolism through the polyol pathway. Many studies have demonstrated that excessive sorbitol can disrupt the intracellular redox balance. However, we still know very little about the impact of excessive intracellular sorbitol on oocyte quality, oocyte maturation, and embryo developmental potential. This study explored whether intracellular sorbitol accumulates in the oocytes of aged mice during in vitro maturation (IVM) and what roles sorbitol plays in oocyte development and maturation. Our results showed that sorbitol levels were significantly higher in in vitro-matured oocytes from aged mice than in oocytes from young mice (14.08 ± 3.78 vs. 0.23 ± 0.04 ng/oocyte). The expression of aldose reductase (AR) mRNA was significantly higher in the in vitro-cultured oocytes from 9-month-old mice than prior to culture. To decrease the excessive intracellular sorbitol in oocytes from aged mice, sorbinil, a specific inhibitor of aldose reductase, was supplemented in IVM medium, and the sorbitol level was significantly decreased (14.08 ± 3.78 vs. 0.48 ± 0.19 ng/oocyte). Our results indicated that the percentage of oocytes with first polar body extrusion (PBE) was significantly higher in the sorbinil group than in the aged group (82.4% ± 7.2% vs. 66.1% ± 6.9%), and the content of sorbitol was drastically increased in the aged group. The ROS fluorescence intensity in the sorbinil group was drastically lower than that in the aged group, while the GSH fluorescence intensity was significantly higher. Interestingly, SOD1 was upregulated in the sorbinil group. The present study suggests that excessive sorbitol accumulation is induced during IVM in aged mouse oocytes, which negatively influences oocyte quality by altering the intracellular redox balance. Inhibition of sorbitol accumulation may be a potential method to improve the nuclear maturation of aged oocytes.

Human amnion-derived mesenchymal stem cells improved the reproductive function of age-related diminished ovarian reserve in mice through Ampk/FoxO3a signaling pathway.

BACKGROUND: Age-related diminished ovarian reserve (AR-DOR) reduced the quality of oocytes, resulting in decreased female fertility. Aging is tightly related to abnormal distribution and function of mitochondria, while mitophagy is a major process to maintain normal quality and quantity of mitochondria in cells, especially in oocytes which containing a large number of mitochondria to meet the demand of energy production during oocyte maturation and subsequent embryonic development. Ampk/FoxO3a signaling is crucial in the regulation of mitophagy. It is reported mesenchymal stem cells (MSCs) can improve ovarian function. Here we aim to explore if human amnion-derived mesenchymal stem cells (hAMSCs) are effective in improving ovarian function in AR-DOR mice and whether Ampk/FoxO3a signaling is involved. METHODS: The AR-DOR model mice were established by 32-week-old mice with 3-8 litters, significantly low serum sex hormone levels and follicle counts. The old mice were divided into 5 treatment groups: normal saline (NS, control), 1% human serum albumin (HSA, resolver), low dose (LD, 5.0 × 106cells/kg), middle dose (MD, 7.5 × 106cells/kg), and high dose (HD, 10.0 × 106cells/kg). The prepared hAMSCs were injected through tail vein. Serum sex hormone level, follicle counts, fertilization rate, gestation rate, little size, apoptosis of granulosa and stromal cells, expression level of Sod2, Ampk, and ratio of phosphorylated FoxO3a to total FoxO3a in ovaries were examined. RESULTS: Our results show that after hAMSC transplantation, the ovarian function in AR-DOR mice was significantly improved, meanwhile the apoptosis of granulosa and stromal cells in the ovaries was significantly repressed, the expression level of Ampk and the ratio of phosphorylated FoxO3a to total FoxO3a both were significantly increased, meanwhile increased Sod2 expression was also observed. CONCLUSION: Our results demonstrate hAMSC transplantation via tail-injection can improve ovarian function of AR-DOR mice through Ampk/FoxO3a signaling pathway.

Single-cell analysis of nonhuman primate preimplantation development in comparison to humans and mice.

BACKGROUND: Genetic programs underlying preimplantation development and early lineage segregation are highly conserved across mammals. It has been suggested that nonhuman primates would be better model organisms for human embryogenesis, but a limited number of studies have investigated the monkey preimplantation development. In this study, we collect single cells from cynomolgus monkey preimplantation embryos for transcriptome profiling and compare with single-cell RNA-seq data derived from human and mouse embryos. RESULTS: By weighted gene-coexpression network analysis, we found that cynomolgus gene networks have greater conservation with human embryos including a greater number of conserved hub genes than that of mouse embryos. Consistently, we found that early ICM/TE lineage-segregating genes in monkeys exhibit greater similarity with human when compared to mouse, so are the genes in signaling pathways such as LRP1 and TCF7 involving in WNT pathway. Last, we tested the role of one conserved pre-EGA hub gene, SIN3A, using a morpholino knockdown of maternal RNA transcripts in monkey embryos followed by single-cell RNA-seq. We found that SIN3A knockdown disrupts the gene-silencing program during the embryonic genome activation transition and results in developmental delay of cynomolgus embryos. CONCLUSION: Taken together, our study provided new insight into evolutionarily conserved and divergent transcriptome dynamics during mammalian preimplantation development.

[High-level androgen enhances the differentiation of hESCs into male primordial germ cells].

OBJECTIVE: To observe the effects of different concentrations of testosterone on the differentiation of human embryonic stem cells (hESCs) into early male germ cells and investigate the potential impact of high-level androgen exposure in early pregnancy in women with polycystic ovary syndrome (PCOS) on the fertility and primordial germ cell reserve of the male offspring in adulthood. METHODS: We used 2 µmol/L retinoic acid to induce the differentiation of hESCs (46, XY) into male germ cells in vitro and meanwhile treated them with testosterone (T) at 0 mol/L, 3×10－7 mol/L, 5×10－7 mol/L, 15×10－7 mol/L, 45×10－7 mol/L, and 135×10－7 mol/L, respectively. We collected the cell samples at 0, 4, 7 and 14 days to determine the expressions of the specific genes and compare the differentiation process and efficiency of the male germ cells in different stages. RESULTS: There was no difference in the morphology of the hESCs treated with different concentrations of testosterone in the same differentiation stage. The expression of the marker gene DAZL in the primordial germ cells peaked on the 4th day of differentiation, significantly higher in the 15×10－7, 45×10－7 and 135×10－7 mol/L groups than in the 3×10－7 mol/L group (P < 0.05), and that of the specific gene SCP3 in the early-meiosis germ cells began to increase on the same day, more significantly in the 45×10－7mol/L than in the 3×10－7 mol/L and 5×10－7 mol/L groups (P < 0.01), and peaked on the 7th day, dramatically higher in the 15×10－7, 45×10－7 and 135×10－7 mol/L groups than in the 3×10－7 mol/L group (P < 0.01). Immunofluorescence staining and flow cytometry showed a T concentration-dependent increase in the expression of DAZL at 4 days and those of SCP3 and VASA at 7 days. Moreover, the expression of the androgen receptor (AR) in the hESCs began to rise on the 4th day and kept going up till the 14th day, higher in the high-concentration than in the low-concentration T groups in the same stage of differentiation, though with no statistically significant difference (P > 0.05). CONCLUSIONS: Exposure to high-level androgen during the differentiation of hESCs into early male germ cells can induce earlier expression of AR and earlier differentiation of hESCs into early male germ cells, which may result in insufficient reserve of male primary germ cells in the male offspring of PCOS women and affect their fertility after adulthood. hESCs can be used as an in vitro model to study the effects of intrauterine hyperandrogen on the reproductive development of male offspring in PCOS patients, which is also contributive to researches on the etiology of male infertility.

[Differentiation of embryonic stem cells from the embryos of the couples with male asthenozoospermia and Robertsonian translocation into germ cells].

OBJECTIVE: To assess the risk of male infertility in the offspring conceived through assisted reproductive technology (ART) byin vitroinductionof the differentiation of embryonic stem cells (ESCs) derived from the embryos of the couples with male asthenozoospermia and Robertsonian translocation (RT) into germ cells. METHODS: We established a CCRM16ESC line with the karyotype of 46, XY, +14, rob(13; 14) (q10; q10) from the embryo donated by a patientwithasthenozoospermiaand RT and his wife by isolation of the inner cell mass of blastula, culturing, passaging, and amplification,followed by in vitro induction and differentiationof the ESCs into germ cells with ratinoic acid(RA) at 2 mol/L. Then, we analyzed the process of differentiation and the expressions of its related genes and compared them with those in the normal CCRM23ESCs. RESULTS: CCRM16 showed the typical characteristics of ESCs, expressing the pluripotency makers of NANOG, OCT4, TRA-1-181 and SSEA4, forming embryoid bodies, and differentiating into three germlayer tissues in vitro and in vivo. Intervention with 2 mol/LRAinduced direct differentiation of the ESCs into germ cells. The expressions of the primordial germ cell marker geneDAZLand the meiosis marker geneSCP3were markedly decreased in the CCRM16 as compared with those in the normal CCRM23 ESCs. CONCLUSIONS: The CCRM16ESC linewith the karyotype of46, XY, +14, rob(13; 14) (q10; q10) has thetypical characteristics of ESCs but an abnormal process of differentiation into germ cells in the early stage. In vitroinductionof the differentiation of ESCs into germ cells can be used for assessing the risk of male infertility in the offspring conceived through ART for asthenozoospermia patients.

Nano-loaded human umbilical cord mesenchymal stem cells as targeted carriers of doxorubicin for breast cancer therapy.

The main challenge of anticancer drugs is poor tumor targeting. Now cellular carriers are widely investigated to deliver anticancer agents. As an ideal cellular candidate, human umbilical cord derived mesenchymal stem cells (hUC-MSCs) possess inherent tropism potential to tumor. Here, we constructed hUC-MSCs carrying transferrin-inspired-nanoparticles that contain doxorubicin(hUC-MSCs-Tf-inspired-NPs) to achieve enhanced anti-tumor efficacy and made an evaluation. Results represented that hUC-MSCs-Tf-inspired-NPs not only exhibit the controlled-release property of Tf-inspired-NPs, but also integrate tumor tropism and penetrative abilities of MSCs. The tumor volume of nude mice bearing breast cancer MCF-7 treated with hUC-MSCs-Tf-inspired-NPs, was remarkably reduced compared to those treated with free drug or Tf-inspired-NPs. Thus, Tf-inspired-NPs loaded hUC-MSCs have a potential to deliver anticancer drugs.