Enhancing maturity in 3D kidney micro-tissues through clonogenic cell combinations and endothelial integration.

As an advance laboratory model, three-dimensional (3D) organoid culture has recently been recruited to study development, physiology and abnormality of kidney tissue. Micro-tissues derived from primary renal cells are composed of 3D epithelial structures representing the main characteristics of original tissue. In this research, we presented a simple method to isolate mouse renal clonogenic mesenchymal (MLCs) and epithelial-like cells (ELCs). Then we have done a full characterization of MLCs using flow cytometry for surface markers which showed that more than 93% of cells expressed these markers (Cd44, Cd73 and Cd105). Epithelial and stem/progenitor cell markers characterization also performed for ELC cells and upregulating of these markers observed while mesenchymal markers expression levels were not significantly increased in ELCs. Each of these cells were cultured either alone (ME) or in combination with human umbilical vein endothelial cells (HUVECs) (MEH; with an approximate ratio of 10:5:2) to generate more mature kidney structures. Analysis of 3D MEH renal micro-tissues (MEHRMs) indicated a significant increase in renal-specific gene expression including Aqp1 (proximal tubule), Cdh1 (distal tubule), Umod (loop of Henle), Wt1, Podxl and Nphs1 (podocyte markers), compared to those groups without endothelial cells, suggesting greater maturity of the former tissue. Furthermore, ex ovo transplantation showed greater maturation in the constructed 3D kidney.

Gestational diabetes mellitus increased the number of dopaminergic neurons in the olfactory bulb of rat offspring.

Objectives: Gestational Diabetes Mellitus (GDM) is the most common metabolic complication of pregnancy that causes central nervous system and olfactory dysfunction in the offspring. It has been demonstrated that dopamine modulates several aspects of olfactory information processing in vertebrates. Materials and Methods: In this study, we investigated the effect of gestational diabetes on the expression of the Dopamine (DA) metabolism genes, tyrosine hydroxylase (TH), and dopa decarboxylase (DDC) in the olfactory bulb (OB) tissue of rats' offspring. Female Wistar rats were divided into a control group which received citrate buffer and the diabetic group which received 45 mg/kg of streptozotocin (STZ) on day 0 of gestation. Fasting blood glucose levels were measured before and 72 hr after injection. OB tissues of adult offspring were isolated, and TH-positive cells were counted by immunofluorescence staining. Also, TH and DDC expressions were analyzed by qRT- PCR and western blot. Results: The data showed that gestational diabetes could cause up-regulation of TH (P<0.01) and DDC (P<0.05) in the OB tissue of offspring. Furthermore, our results showed that GDM causes a significant increase in TH and DDC protein levels in the OB tissues of offspring. Immunohistochemistry showed a significant increase in the number of TH-positive cells in the offspring of diabetic mothers (P<0.05). Conclusion: This study showed that gestational diabetes could cause an increase in TH and DDC gene expression in the OB tissue in the offspring, which may be correlated with reduced olfactory sensitivity.

Regenerative potential of different extracellular vesicle subpopulations derived from clonal mesenchymal stem cells in a mouse model of chemotherapy-induced premature ovarian failure.

AIMS: Some studies have shown that mesenchymal stem cells (MSCs) and their derived extracellular vesicles (MSC-EVs) can restore ovarian function in premature ovarian failure (POF), however, concerns about their efficacy are attributed to the heterogeneity of the cell populations and EVs. Here, we assessed the therapeutic potential of a homogeneous population of clonal MSCs (cMSCs) and their EVs subpopulations in a mouse model of POF. MAIN METHODS: Granulosa cells were treated with cyclophosphamide (Cy) in the absence or presence of cMSCs, or cMSCs-derived EV subpopulations (EV20K and EV110K, isolated by high-speed centrifugation and differential ultracentrifugation, respectively). In addition, POF mice were treated with cMSCs, EV20K and/or EV110K. KEY FINDINGS: cMSC and both EV types protected granulosa cells from Cy-induced damage. Calcein-EVs were detected in the ovaries. Moreover, cMSC and both EV subpopulations significantly increased body weight, ovary weight, and the number of follicles, restored FSH, E2, and AMH levels, increased the granulosa cell numbers and restored the fertility of POF mice. cMSC, EV20K, and EV110K alleviated inflammatory-related genes expression (Tnf-α and IL8), and improved angiogenesis via upregulation expression of Vegf and Igf1 at the mRNA level and VEGF and αSMA at the protein level. They also inhibited apoptosis through the PI3K/AKT signaling pathway. SIGNIFICANCE: The administration of cMSCs and two cMSC-EVs subpopulations improved ovarian function and restored fertility in a POF model. EV20K is more cost-effective and feasible in terms of isolation, particularly in good manufacturing practice (GMP) facilities for treatment of POF patients in comparison with conventional EVs (EV110K).

Effect of MDMA exposure during pregnancy on cell apoptosis, astroglia, and microglia activity in rat offspring striatum.

Objectives: Ecstasy is a popular recreational psychostimulant with side effects on the central nervous system. This study examined the corpus striatum tissue of adult rats that received ecstasy during the embryonic period for histological and molecular studies. Materials and Methods: Rats were divided into control and ecstasy groups. The ecstasy group was given MDMA 15 mg/kg intraperitoneally twice daily at 8-hour intervals on days 7-15 of gestation. At the age of 15 weeks, adult offspring of both groups were examined for learning and memory study by the Morris water maze test. Then, ventral striatum tissue was harvested for TUNEL assay, Nissl staining, and real-time PCR for the expression of the GFAP and CD11b. Results: Ecstasy up-regulated the GFAP and CD11b expression in the striatum of offspring (*P˂0.05). Furthermore, the Morris water maze test showed that exposure to ecstasy significantly impaired learning and spatial memory (*P˂0.05). TUNEL assay results did not show any significant change in the number of apoptotic cells in the striatum tissue of ecstasy offspring compared with controls, while Nissl staining showed a significant decrease in the number of neurons in the ecstasy group (*P˂0.05). Conclusion: Exposure to ecstasy during pregnancy causes long-lasting changes in brain regions underlying learning and memory, including the striatum, and impaired working memory in the offspring. In addition, these data provide the first evidence that exposure to ecstasy during the embryonic period causes a persistent change in the activity of microglial cells and the number of astrocyte cells in the striatum.

Embryonic stem cells-derived mesenchymal stem cells do not differentiate into ovarian cells but improve ovarian function in POF mice.

Premature ovarian failure (POF) is characterized by amenorrhea, hypoestrogenism, elevated gonadotropin levels, and infertility. Although some of previous studies reported that Mesenchymal stem cells (MSC) transplantation could rescue the ovary function of POF animal models through the paracrine pathways, these mechanisms require further investigation. Here, we aimed to investigate the possible mechanisms of therapeutic effects of human embryonic stem cells derived MSC (ES-MSC) in a mice model of chemotherapy-induced POF. For this purpose, Cyclophosphamide (Cy) was injected intraperitoneally into female mice to induce POF. 10 days after Cy injection, we evaluated follicle count, follicle-stimulating hormone (FSH) and estradiol (E2) hormone concentrations, and TUNEL assay. Then, ES-MSC was transplanted into mice and the expression of Anti-müllerian hormone (AMH) and apoptosis was evaluated in ovary. Results indicated that ES-MSC reduced apoptosis in the follicles and increased the expression of AMH protein in the ovary of POF mice. So, ES-MSC may inhibit the apoptosis of ovarian granulosa cells. Then, to investigate the potential mechanisms of therapeutic effects of ES-MSC and their fate in the ovary, MSC were labeled with green fluorescent protein (GFP) before transplantation. Immunofluorescence staining indicated that although GFP-labeled ES-MSC was located in the ovarian stroma, they did not express granulosa cell markers: AMH and Follicle-stimulating hormone receptor (FSHR), theca cell marker: luteinizing hormone receptor (LHR), and oocyte marker: Growth/differentiation factor 9 (GDF9). Therefore, ES-MSC may not differentiate into ovarian cells directly and they might restore ovarian function in chemotherapy-induced POF mice by paracrine mechanisms.

Tracking of human embryonic stem cell-derived mesenchymal stem cells in premature ovarian failure model mice.

Premature ovarian failure (POF) is defined by amenorrhea, hypoestrogenism, elevated gonadotropin levels, and infertility. Chemotherapeutic agents are the most gonadotoxic agents that lead to POF. Although some previous studies have presented that mesenchymal stem cells (MSCs) transplantation could rescue the ovary function of POF animal models through the paracrine pathway, these mechanisms require further investigation. However, mechanisms of embryonic stem cell-derived MSCs (ES-MSCs) therapeutic effects on POF animal models have not been fully investigated yet. This study aimed to evaluate the migration and distribution of ES-MSCs in a model of chemotherapy-induced POF. Female mice received intraperitoneal injections of cyclophosphamide (Cy) to induce POF. Then, MSCs were labeled with green fluorescent protein (GFP) in vitro and injected intravenously into POF mice, and the distribution of MSCs was dynamically monitored at 1 week after transplantation. We harvested the lungs, liver, spleen, ovaries, heart, and kidneys 1 week after transplantation. The sections of these tissues were observed under the fluorescent microscope. More than 70% MSCs were successfully labeled with GFP at 72 h after labeling. MSCs were uniformly distributed in multiple organs and tissues including lungs, liver, spleen, ovaries, heart, and kidneys of POF mice. In mice, at 1week after intravenous transplantation, GFP labeled ES-MSCs were observed in the lungs, liver, spleen, ovaries, heart, and kidneys of POF mice, and the number of GFP labeled ES-MSCs in lungs, ovaries, and heart were higher than that in the spleen, kidneys, and liver. Our results revealed intravenously implanted ES-MSCs could migrate into the various tissues in chemotherapy-induced damaged POF mice.

Human embryonic stem cell-derived mesenchymal stem cells improved premature ovarian failure.

BACKGROUND: Premature ovarian failure (POF) affects many adult women less than 40 years of age and leads to infertility. According to previous reports, various tissue-specific stem cells can restore ovarian function and folliculogenesis in mice with chemotherapy-induced POF. Human embryonic stem cells (ES) provide an alternative source for mesenchymal stem cells (MSCs) because of their similarities in phenotype and immunomodulatory and anti-inflammatory characteristics. Embryonic stem cell-derived mesenchymal stem cells (ES-MSCs) are attractive candidates for regenerative medicine because of their high proliferation and lack of barriers for harvesting tissue-specific MSCs. However, possible therapeutic effects and underlying mechanisms of transplanted ES-MSCs on cyclophosphamide and busulfan-induced mouse ovarian damage have not been evaluated. AIM: To evaluate ES-MSCs vs bone marrow-derived mesenchymal stem cells (BM-MSCs) in restoring ovarian function in a mouse model of chemotherapy-induced premature ovarian failure. METHODS: Female mice received intraperitoneal injections of different doses of cyclophosphamide and busulfan to induce POF. Either human ES-MSCs or BM-MSCs were transplanted into these mice. Ten days after the mice were injected with cyclophosphamide and busulfan and 4 wk after transplantation of the ES-MSCs and/or BM-MSCs, we evaluated body weight, estrous cyclicity, follicle-stimulating hormone and estradiol hormone concentrations and follicle count were used to evaluate the POF model and cell transplantation. Moreover, terminal deoxynucleotidyl transferase mediated 2-deoxyuridine 5-triphosphate nick end labeling, real-time PCR, Western blot analysis and immunohistochemistry and mating was used to evaluate cell transplantation. Enzyme-linked immunosorbent assay was used to analyze vascular endothelial growth factor, insulin-like growth factor 2 and hepatocyte growth factor levels in ES-MSC condition medium in order to investigate the mechanisms that underlie their function. RESULTS: The human ES-MSCs significantly restored hormone secretion, survival rate and reproductive function in POF mice, which was similar to the results obtained with BM-MSCs. Gene expression analysis and the terminal deoxynucleotidyl transferase mediated 2-deoxyuridine 5-triphosphate nick end labeling assay results indicated that the ES-MSCs and/or BM-MSCs reduced apoptosis in the follicles. Notably, the transplanted mice generated new offspring. The results of different analyses showed increases in antiapoptotic and trophic proteins and genes. CONCLUSION: These results suggested that transplantation of human ES-MSCs were similar to BM-MSCs in that they could restore the structure of the injured ovarian tissue and its function in chemotherapy-induced damaged POF mice and rescue fertility. The possible mechanisms of human ES-MSC were related to promotion of follicular development, ovarian secretion, fertility via a paracrine effect and ovarian cell survival.