FACS selection of valuable mutant mouse round spermatids and strain rescue via round spermatid injection.

Round spermatid injection (ROSI) into mammalian oocytes can result in the development of viable embryos and offspring. One current limitation to this technique is the identification of suitable round spermatids. In the current paper, round spermatids were selected from testicular cells with phase contrast microscopy (PCM) and fluorescence-activated cell sorting (FACS), and ROSI was performed in two strains of mice. The rates of fertilization, embryonic development and offspring achieved were the same in all strains. Significantly, round spermatids selected by PCM and FACS were effectively used to rescue the infertile Pten-null mouse. The current results indicate that FACS selection of round spermatids can not only provide high-purity and viable round spermatids for use in ROSI, but also has no harmful effects on the developmental capacity of subsequently fertilized embryos. It was concluded that round spermatids selected by FACS are useful for mouse strain rederivation and rescue of infertile males; ROSI should be considered as a powerful addition to the armamentarium of assisted reproduction techniques applicable in the mouse.

Role of Nrf2 in Lipopolysaccharide-Induced Acute Kidney Injury: Protection by Human Umbilical Cord Blood Mononuclear Cells.

BACKGROUND: Acute kidney injury (AKI) is one of the common complications of sepsis. Heretofore, there is no effective treatment for septic AKI. Recent studies have revealed that besides treating hematological malignancies, human umbilical cord blood mononuclear cells (hUCBMNCs) show good therapeutic effects on other diseases. But whether hUCBMNCs can protect against septic AKI and its underlying mechanism are unknown. METHODS: The rat model of lipopolysaccharide- (LPS-) induced AKI was developed, and the injection of hUCBMNCs was executed to prevent and treat AKI. ML385, a specific nuclear factor E2-related factor 2 (Nrf2) inhibitor, was used to silence Nrf2. The cell experiments were conducted to elaborate the protective mechanism of Nrf2 pathway. RESULTS: An effective model of LPS-induced AKI was established. Compared to the rats only with LPS injection, the levels of inflammation, reactive oxygen species (ROS), and apoptosis in renal tissues after hUCBMNC injection were markedly attenuated. Pathological examination also indicated significant remission of renal tissue injury in the LPS+MNCs group, compared to rats in the LPS group. Transmission electron microscopy (TEM) showed that the damage of the mitochondria in the LPS+MNCs group was lighter than that in the LPS group. Noteworthily, the renal Nrf2/HO-1 pathway was activated and autophagy was enhanced after hUCBMNC injection. ML385 could partly reverse the renoprotective effect of hUCBMNCs, which could demonstrate that Nrf2 participated in the protection of hUCBMNCs. Cell experiments showed that increasing the expression level of Nrf2 could alleviate LPS-induced cell injury by increasing the autophagy level and decreasing the injury of the mitochondria in HK-2 cells. CONCLUSION: All results suggest that hUCBMNCs can protect against LPS-induced AKI via the Nrf2 pathway. Activating Nrf2 can upregulate autophagy to protect LPS-induced cell injury.

Human umbilical cord-derived mesenchymal stem cells and human cord blood mononuclear cells protect against cisplatin-induced acute kidney injury in rat models.

Human umbilical cord-derived mesenchymal stem cells (hUCMSCs) are a promising tool to attenuate cisplatin (CP)-induced acute kidney injury (AKI). However, whether the transplantation of human cord blood mononuclear cells (hCBMNCs) exhibits similar protective effects and their potential underlying mechanisms of action remain unclear. The present study aimed to determine the protective effects of hUCMSCs and hCBMNCs transplantation therapies on an established CP-induced rat model and explore their underlying mechanisms of action. A total of 24 Sprague-Dawley rats, selected based on body weight, were randomly assigned into 4 groups: i) normal control; ii) model (CP); iii) hCBMNCs (CP + hCBMNCs); and iv) hUCMSCs (CP + hUCMSCs). hUCMSCs (2.0x106 cells) and hCBMNCs (2.0x106 cells) were injected into the femoral vein of rats 24 h after CP (8 mg/kg) treatment. To determine the effects of hCBMNCs and hUCMSCs on CP-induced rats, renal function assessment and histological evaluations were performed. Expression levels of high mobility group box 1 (HMGB1) and the ratio of Bax/Bcl2 in renal tissues were detected to elucidate their underlying molecular mechanisms of action. The results demonstrated that transplantation of hUCMSCs and hCBMNCs significantly improved renal function in CP-induced AKI rats, as evidenced by the enhancement of renal morphology; decreased concentrations of blood urea nitrogen and serum creatinine; and a lower percentage of apoptotic renal tubular cells. The expression of HMGB1 and the ratio of Bax/Bcl-2 were significantly reduced in the hUCMSCs and hCBMNCs groups compared with CP group. In conclusion, the present study indicated that hCBMNCs exert similar protective effects to hUCMSCs on CP-induced AKI. hUCMSCs and hCBMNCs protect against CP-induced AKI by suppressing HMGB1 expression and preventing cell apoptosis.

Stanniocalcin-1 Alleviates Contrast-Induced Acute Kidney Injury by Regulating Mitochondrial Quality Control via the Nrf2 Pathway.

Contrast-induced acute kidney injury (CI-AKI) is the third common cause of acute kidney injury (AKI), which is associated with poor short- and long-term outcomes. Currently, effective therapy strategy for CI-AKI remains lacking. Stanniocalcin-1 (STC1) is a conserved glycoprotein with antiapoptosis and anti-inflammatory functions, but the role of STC1 in controlling CI-AKI is unknown. Here, we demonstrated a protective role of STC1 in contrast-induced injury in cultured renal tubular epithelial cells and CI-AKI rat models. Recombinant human STC1 (rhSTC1) regulated mitochondrial quality control, thus suppressing contrast-induced mitochondrial damage, oxidative stress, inflammatory response, and apoptotic injury. Mechanistically, activation of the Nrf2 signaling pathway contributes critically to the renoprotective effect of STC1. Together, this study demonstrates a novel role of STC1 in preventing CI-AKI and reveals Nrf2 as a molecular target of STC1. Therefore, this study provides a promising preventive target for the treatment of CI-AKI.

Novel risk models to predict acute kidney disease and its outcomes in a Chinese hospitalized population with acute kidney injury.

Acute kidney disease (AKD) is a state between acute kidney injury (AKI) and chronic kidney disease (CKD), but the prognosis of AKD is unclear and there are no risk-prediction tools to identify high-risk patients. 2,556 AKI patients were selected from 277,898 inpatients of three affiliated hospitals of Central South University from January 2015 to December 2015. The primary point was whether AKI patients developed AKD. The endpoint was death or end stage renal disease (ESRD) 90 days after AKI diagnosis. Multivariable Cox regression was used for 90-day mortality and two prediction models were established by using multivariable logistic regression. Our study found that the incidence of AKD was 53.17% (1,359/2,556), while the mortality rate and incidence of ESRD in AKD cohort was 19.13% (260/1,359) and 3.02% (41/1,359), respectively. Furthermore, adjusted hazard ratio of mortality for AKD versus no AKD was 1.980 (95% CI 1.427-2.747). In scoring model 1, age, gender, hepatorenal syndromes, organic kidney diseases, oliguria or anuria, respiratory failure, blood urea nitrogen (BUN) and acute kidney injury stage were independently associated with AKI progression into AKD. In addition, oliguria or anuria, respiratory failure, shock, central nervous system failure, malignancy, RDW-CV ≥ 13.7% were independent risk factors for death or ESRD in AKD patients in scoring model 2 (goodness-of fit, P1 = 0.930, P2 = 0.105; AUROC1 = 0.879 (95% CI 0.862-0.896), AUROC2 = 0.845 (95% CI 0.813-0.877), respectively). Thus, our study demonstrated AKD was independently associated with increased 90-day mortality in hospitalized AKI patients. A new prediction model system was able to predict AKD following AKI and 90-day prognosis of AKD patients to identify high-risk patients.

Human umbilical cord blood mononuclear cells protect against renal tubulointerstitial fibrosis in cisplatin-treated rats.

Currently, there is no effective method to prevent renal interstitial fibrosis after acute kidney injury (AKI). In this study, we established and screened a new renal interstitial fibrosis rat model after cisplatin-induced AKI. Our results indicated that rats injected with 4 mg/kg cisplatin once a week for two weeks after firstly administrated with 6.5 mg/kg loading dose of cisplatin could set up a more accurate model reflecting AKI progression to renal interstitial fibrosis. Then, we investigated the effects and possible mechanisms of human umbilical cord blood mononuclear cells (hUCBMNCs) on renal tubular interstitial fibrosis after cisplatin-induced AKI. In rats injected with hUCBMNCs for four times, level of matrix metalloproteinase 7 (MMP-7) in serum and urine, urinary albumin/creatinine ratio, tubular pathological scores, the relative collagen area of the tubulointerstitial region, endoplasmic reticulum dilation and the mitochondrial ultrastructural damage were significantly improved. The level of reactive oxygen species, α-smooth muscle actin (α-SMA), [NOD]-like pyrin domain containing protein 3 and cleaved-Caspase 3 in renal tissue decreased significantly. However, in rats injected with hUCBMNCs for two times, no significant difference was discovered in MMP-7 levels and urinary albumin/creatinine ratio. Although expression of α-SMA and the percentage areas of collagen staining in tubulointerstitial tissues were ameliorated in rats injected with hUCBMNCs for two times, the effects were significantly weaker than those in rats injected with hUCBMNCs for four times. Taken together, our study constructed a highly efficient, duplicable novel rat model of renal fibrosis after cisplatin-induced AKI. Multiple injections of hUCBMNCs may prevent renal interstitial fibrosis after cisplatin-induced AKI.

Human umbilical cord blood mononuclear cells protect against renal tubulointerstitial fibrosis in cisplatin-treated rats.

Currently, there is no effective method to prevent renal interstitial fibrosis after acute kidney injury (AKI). In this study, we established and screened a new renal interstitial fibrosis rat model after cisplatin-induced AKI. Our results indicated that rats injected with 4 mg/kg cisplatin once a week for two weeks after firstly administrated with 6.5 mg/kg loading dose of cisplatin could set up a more accurate model reflecting AKI progression to renal interstitial fibrosis. Then, we investigated the effects and possible mechanisms of human umbilical cord blood mononuclear cells (hUCBMNCs) on renal tubular interstitial fibrosis after cisplatin-induced AKI. In rats injected with hUCBMNCs for four times, level of matrix metalloproteinase 7(MMP-7)in serum and urine, urinary albumin/creatinine ratio, tubular pathological scores, the relative collagen area of the tubulointerstitial region, endoplasmic reticulum dilation and the mitochondrial ultrastructural damage were significantly improved. The level of reactive oxygen species, α-smooth muscle actin (α-SMA), [NOD]-like pyrin domain containing protein 3 and cleaved-Caspase 3 in renal tissue decreased significantly. However, in rats injected with hUCBMNCs for two times, no significant difference was discovered in MMP-7 levels and urinary albumin/creatinine ratio. Although expression of α-SMA and the percentage areas of collagen staining in tubulointerstitial tissues were ameliorated in rats injected with hUCBMNCs for two times, the effects were significantly weaker than those in rats injected with hUCBMNCs for four times. Taken together, our study constructed a highly efficient, duplicable novel rat model of renal fibrosis after cisplatin-induced AKI. Multiple injections of hUCBMNCs may prevent renal interstitial fibrosis after cisplatin-induced AKI.

[Updated dedifferentiation and pluripotency of spermatogonial stem cells].

Spermatogonial stem cells (SSCs) are the postnatal mitotic male germ cells that undergo self-renewing and differentiate into haploid sperm through spermatogenesis throughout life time. However, some recent interesting studies indicate that pluripotent cells can be derived from SSCs in culture. Thus, it seems that SSCs are a great resource of pluripotent cells for regenerative medicine, especially to meet the demand of immuno-rejection free pluripotent cells derived from patients themselves. This article aims to introduce the current understanding and advantages of the pluripotent SSCs in regenerative medicine.

17beta-estradiol stimulates proliferation of spermatogonia in experimental cryptorchid mice.

AIM: To investigate whether estrogen stimulates the proliferation of spermatogonia or induces spermatogenesis in cryptorchid mice. METHODS: Mice were surgically rendered cryptorchid, then treated with different doses of 17beta-estradiol (E2) s.c. once a day. Mice were killed at sexual maturity (45 days of age), and histological analysis and immunofluorescence were performed. Serum follicle stimulating hormone (FSH), estradiol, testosterone and luteinizing hormone (LH) were measured. RESULTS: Low doses of E2 had no notable effect on spermatogonia, but at higher doses, E2 stimulated the proliferation of spermatogonia. CONCLUSION: E2 has a dose-related mitogenic effect on spermatogonia.

Reconstruction of endometrium in vitro via rabbit uterine endometrial cells expanded by sex steroid.

OBJECTIVE: To culture rabbit endometrial cells by using sex steroids to provide adequate seeding cells for endometrium reconstruction and uterine tissue engineering. DESIGN: Prospective experimental study. SETTING: Beijing Institute of Basic Medical Sciences and Tissue Engineering Research Center, Academy of Military Medical Sciences. ANIMAL(S): New Zealand rabbit and Kunming white strain mice. INTERVENTION(S): Rabbits were primed with pregnant mare serum gonadotropin and hCG. Endometrial cells were cultured with E(2) and P(4) of different concentrations. The endometrium was reconstructed by using endometrial cells as seeding cells and collagen-basement membrane matrix as scaffolds. MAIN OUTCOME MEASURE(S): Assay with 93-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide, immunofluorescence staining, flow cytometric analysis, hematoxylin and eosin and immunohistochemical staining, and developmental rate of embryos. RESULT(S): The expression patterns of estrogen receptor and P receptor of rabbit endometrium were different before and after treatment with pregnant mare serum gonadotropin-hCG. One hundred nanomolar E(2) with 10 nmol/L P(4) facilitated the proliferation of epithelial cells whereas 100 nmol/L P(4) facilitated that of stromal cells. The epithelial cells could be stable if cultured for seven or eight passages. Cells in the epithelial layer of the reconstructed endometrium were cytokeratin positive. Some showed columnar morphology akin to the luminal epithelium in vivo. Reconstructed endometrium could improve the developmental rate and quality of one-cell mice embryos. CONCLUSION(S): Rabbit endometrial cells could be cultured with a long-standing proliferation capability by sex steroids and applied in uterine tissue engineering. Reconstructed endometrium with proliferated endometrial cells was akin to native endometrium in structure and function.

Reconstruction of engineered uterine tissues containing smooth muscle layer in collagen/matrigel scaffold in vitro.

OBJECTIVE: This study attempted to reconstruct engineered uterine tissues (EUTs) containing smooth muscle layer, akin to the normal uterine wall. METHODS: EUTs were reconstructed by seeding epithelial cells on top of the constructed stromal layer over smooth muscle layer. A self-made mold was used to keep the EUTs from contraction. At the same time, it provided static stretch to the EUTs. After 14 days of culture, the structure of the EUTs was analyzed histologically and immunohistochemically, or by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The expression of integrin beta3 subunit, heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF), and HOXA-10 was detected by reverse transcription-polymerase chain reaction (RT-PCR). The ability of the EUTs supporting the development of embryos was estimated by coculturing embryos on the EUTs. We also tried a new method to reconstruct EUTs by mixing epithelial cell and stromal cells (1:2) in collagen/Matrigel to form an endometrial layer and putting it on top of the smooth muscle layer. The self-assembling ability of the endometrial epithelial cells and stromal cells in the reconstructed EUTs was analyzed histologically and immunohistochemically. RESULTS: The results found that the constructed EUTs with the first and the second method showed three-layered structures. The epithelial layer, stromal layer, and smooth muscle layer were stained by cytokeratin 18, vimentin, and alpha-actin, respectively. TEM showed that the cells in the EUTs reconstructed by the first method were attached to each other by apical tight junctions and rivet-like desmosomes. SEM showed protruded pinopodes, microvilli, and cilium of epithelial cells. The RT-PCR analysis showed that integrin beta3 subunit, HB-EGF, and HOXA-10 were expressed in EUTs. The coculture system of EUTs improved the development rate and quality of murine embryo significantly in comparison with those of control Chatot Ziomek Bavister culture. In the EUTs reconstructed by the second method, the epithelial cells demonstrated self-assembling ability and formed epithelial cell layer on top of the stromal layer and glandular tube-like structures in the stromal layer. Columnar epithelial cells existed in some parts of the epithelial layer. CONCLUSION: We engineered EUTs containing smooth muscle layer by two methods. The reconstructed EUTs could support the development of embryos. The epithelial cells showed self-assembling ability in the EUTs.

Generation and in vitro differentiation of a spermatogonial cell line.

Spermatogenesis is the process by which spermatogonial stem cells divide and differentiate to produce sperm. In vitro sperm production has been difficult to achieve because of the lack of a culture system to maintain viable spermatogonia for long periods of time. Here we report the in vitro generation of spermatocytes and spermatids from telomerase-immortalized mouse type A spermatogonial cells in the presence of stem cell factor. This differentiation can occur in the absence of supportive cells. The immortalized spermatogonial cell line may serve as a powerful tool in elucidating the molecular mechanisms of spermatogenesis. Furthermore, through genomic modification and transplantation techniques, this male germ cell line may be used to generate transgenic mice and to develop germ cell gene therapy.