Yingjian Zhu

Human Wharton’s jelly-derived mesenchymal stromal cells reduce renal fibrosis through induction of native and foreign hepatocyte growth factor synthesis in injured tubular epithelial cells

IntroductionBased on some well-documented reports, we attempted to clarify the antifibrotic mechanisms of human Wharton’s-jelly-derived mesenchymal stromal cells (WJ-MSCs) from the perspective of induction of hepatocyte growth factor (HGF) expression in tubular epithelial cells (TECs).MethodsA rat model of acute kidney injury (AKI) was established through unilateral renal ischemia for 1 hour. Two days later, a single intravenous cell or vehicle injection, or contralateral nephrectomy, was performed. Rats were sacrificed at 1 day, 1 week, 4 weeks, or 6 weeks after the intervention. Renal fibrosis was evaluated by Masson trichrome staining and Sircol collagen assay. The upregulation of α-smooth muscle actin (α-SMA) versus E-cadherin expression was adopted as an indicator of tubular epithelial-mesenchymal transition (EMT). Gene and protein expression of HGF or transforming growth factor-beta1 (TGF-β1) was determined by real-time polymerase chain reaction (RT-PCR) and Western blot, respectively. HGF expression in TECs was detected with immunostaining. In vitro, rat TECs subjected to hypoxia injury were incubated with or without conditioned medium (CM) from WJ-MSCs for 1, 3, 24, or 48 hours. Rat or human HGF synthesis in TECs was assessed with immunostaining, RT-PCR, or ELISA.ResultsCell delivery or nephrectomy led to abrogation of renal scarring. At the incipient period of AKI, through induction of HGF expression, either of them remarkably promoted the upregulation of HGF versus TGF-β1 expression in damaged kidney. Rat TECs were not only the principal cells expressing HGF but also exhibited human HGF expression after cell infusion. During fibrogenesis, the downregulation of HGF versus TGF-β1 expression was greatly prevented by WJ-MSCs or kidney removal, thereby resulting in tubular EMT delay. In vitro, after 24 or 48 hours of incubation, CM not only robustly induced the upregulation of rat HGF gene expression in TECs but substantially amplified the release of rat HGF. Under the induction of CM, human HGF mRNA and protein were detected in rat TECs.ConclusionsWJ-MSCs contribute to tubular EMT delay and the alleviation of renal fibrosis. Induction of native and foreign HGF synthesis in damaged TECs at the initial stage of AKI leads to recovery of the disturbed balance of HGF/TGF-β1 during scar formation, being one of the vital mechanisms.

Molecular and toxicologic research in newborn hypospadiac male rats following in utero exposure to di-n-butyl phthalate (DBP)

The objective of this study was to first evaluate the developmental abnormalities and carry out the molecular analysis of external genitalia in newborn hypospadiac male rats induced by maternal exposure to di-n-butyl phthalate (DBP). Timed-pregnant rats were given DBP by gastric intubation at dose of 750 mg/kg body weight (bw)/day from gestation day (GD) 14 to GD18 to establish a hypospadiac rat model. The incidence of hypospadias was 46.67% in male offsprings. On postnatal day (PND) 7, at the newborn stage, decreased body weight and anogenital distance (AGD)/body weight ratio were observed in newborn hypospadiac male rats. The general image and transverse serial histological analysis of genitalia of newborn hypospadiac male rats confirmed the malformation. Autopsy analysis revealed development of reproductive organs (testes, genital tubercle (GT)), hollow organs (stomach, bladder), and solid organs (brain, heart, liver, spleen, lung, kidney, pancreas) in newborn hypospadiac male rats affected by DBP. Moreover, significantly decreased gene expression of important signaling molecules necessary for GT formation including sonic hedgehog signaling molecules (Shh and Ptched 1), bone morphogenetic proteins signaling molecules (Bmp4 and Bmp7), fibroblast growth factor signaling molecules (Fgf8, Fgf10 and Fgfr2), and the transforming growth factor-beta superfamily signaling molecules (TGF-beta1 and TGF-beta receptor III) were observed, for the first time, in the GT of newborn hypospadias induced by DBP. These results showed that the reproductive system and development conditions of newborn hypospadiac rats were damaged by DBP. These disturbed signaling pathways which orchestrating genital development might play an important role in the toxic process of DBP induced hypospadias.

Microvesicles derived from human umbilical cord mesenchymal stem cells facilitate tubular epithelial cell dedifferentiation and growth via hepatocyte growth factor induction.

During acute kidney injury (AKI), tubular cell dedifferentiation initiates cell regeneration; hepatocyte growth factor (HGF) is involved in modulating cell dedifferentiation. Mesenchymal stem cell (MSC)-derived microvesicles (MVs) deliver RNA into injured tubular cells and alter their gene expression, thus regenerating these cells. We boldly speculated that MVs might induce HGF synthesis via RNA transfer, thereby facilitating tubular cell dedifferentiation and regeneration. In a rat model of unilateral AKI, the administration of MVs promoted kidney recovery. One of the mechanisms of action is the acceleration of tubular cell dedifferentiation and growth. Both in vivo and in vitro, rat HGF expression in damaged rat tubular cells was greatly enhanced by MV treatment. In addition, human HGF mRNA present in MVs was delivered into rat tubular cells and translated into the HGF protein as another mechanism of HGF induction. RNase treatment abrogated all MV effects. In the in vitro experimental setting, the conditioned medium of MV-treated injured tubular cells, which contains a higher concentration of HGF, strongly stimulated cell dedifferentiation and growth, as well as Erk1/2 signaling activation. Intriguingly, these effects were completely abrogated by either c-Met inhibitor or MEK inhibitor, suggesting that HGF induction is a crucial contributor to the acceleration of cell dedifferentiation and growth. All these findings indicate that MV-induced HGF synthesis in damaged tubular cells via RNA transfer facilitates cell dedifferentiation and growth, which are important regenerative mechanisms.

The Anti-Oxidative Role of Micro-Vesicles Derived from Human Wharton-Jelly Mesenchymal Stromal Cells through NOX2/gp91(phox) Suppression in Alleviating Renal Ischemia-Reperfusion Injury in Rats

Oxidative stress is known as one of the main contributors in renal ischemia/reperfusion injury (IRI). Here we hypothesized that Micro-vesicles (MVs) derived from human Wharton Jelly mesenchymal stromal cells (hWJMSCs) could protect kidney against IRI through mitigating oxidative stress. MVs isolated from hWJMSCs conditioned medium were injected intravenously in rats immediately after unilateral kidney ischemia for 60 min. The animals were sacrificed at 24h, 48h and 2 weeks respectively after reperfusion. Our results show that the expression of NOX2 and reactive oxygen species (ROS) in injured kidney tissues was declined and the oxidative stress was alleviated in MVs group at 24h and 48h in parallel with the reduced apoptosis and enhanced proliferation of cells. IRI-initiated fibrosis was abrogated by MVs coincident with renal function amelioration at 2 weeks. NOX2 was also found down-regulated by MVs both in human umbilical vein endothelial cells (HUVEC) and NRK-52E cell line under hypoxia injury model in vitro. In conclusion, a single administration of hWJMSC-MVs might protect the kidney by alleviation of the oxidative stress in the early stage of kidney IRI through suppressing NOX2 expression. Moreover, it could reduce the fibrosis and improved renal function.

Ureteral Reconstruction Using Autologous Tubular Grafts for the Management of Ureteral Strictures and Defects: An Experimental Study

Objective: To investigate whether the peritoneal cavity could function as a bioreactor to produce autologous tubular grafts for ureteral reconstruction in beagles. Materials and Methods: 8-Fr Silastic tubes were implanted into the peritoneal cavities of 6 female beagles. At 3 weeks, the tubes were harvested and the tubular tissue covering the tubes was gently everted. A segment 3 cm in length of the right mid-ureter, involving two thirds of its diameter, was removed parallel to the ureteral axis, leaving a third of the ureteral wall. A 5-Fr double-J stent was inserted into the ureter through the created defect, and two thirds of the graft were anastomosed to both edges of the ureteral defect. One third of the graft was overlapped with the retained normal ureter and anastomosed to the external surface of the lumens. Thus, the graft was partly encapsulated by the remainder of ureteral wall. The stent was maintained for 6 weeks and removed. Excretory urography was performed at 8 (n = 3) and 12 weeks (n = 3), postoperatively. Meanwhile, the neoureter was harvested and analyzed. The left ureter served as the control and a simple intubated ureterotomy was performed. Results: Histological analysis of the tubular tissue demonstrated transversely arranged myofibroblasts and an outer layer of mesothelium. The tissue was easily everted and transplanted as a ureteral graft. Eight weeks postoperatively, the neoureter demonstrated normal ureteral architecture, composed of multilayers of urothelium surrounded by smooth muscle bundles, which became increasingly organized with time. Excretory urography indicated no stenosis or hydronephrosis. Conclusions: These results show that autologous tubular tissue grown within the recipients’ peritoneal cavity can be used for ureteral reconstruction in the beagle model.

Mesenchymal Stromal Cell-Derived Extracellular Vesicles Protect Against Acute Kidney Injury Through Anti-Oxidation by Enhancing Nrf2/ARE Activation in Rats.

Background/Aims: Anti-oxidation is an effective strategy for curing acute kidney injury (AKI). Herein, we suggest that extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs) might play an anti-oxidative role by enhancing Nrf2/ARE activation in AKI. Methods: EVs isolated from the conditioned medium of human Whartons Jelly mesenchymal stromal cells and human foreskin fibroblast were intravenously injected in rats immediately after 45 min of unilateral kidney ischemia. Animals were sacrificed 24 h after injury. Results: Results showed that renal tubular injury was alleviated and renal function was improved by MSC-EVs. Cell apoptosis and sNGAL levels, which reflect kidney cell injury, were reduced. Moreover, MSC-EVs decreased oxidative stress in injured kidney tissues and NRK-52E cells under hypoxia injury. Nrf2/antioxidant response element (ARE) enhancement and HO-1 up-regulation were further observed after MSC-EV treatment both in vivo and in vitro. Conclusions: MSC-EVs may protect against AKI possibly through anti-oxidation by enhancing Nrf2/ARE activation.

Microvesicles Derived from Human Wharton's Jelly Mesenchymal Stem Cells Promote Human Renal Cancer Cell Growth and Aggressiveness through Induction of Hepatocyte Growth Factor

In our previous study, microvesicles (MVs) released from human Whartons jelly mesenchymal stem cells (hWJ-MSCs) retard the growth of bladder cancer cells. We would like to know if MVs have a similar effect on human renal cell carcinoma (RCC). By use of cell culture and the BALB/c nu/nu mice xeno-graft model, the influence of MVs upon the growth and aggressiveness of RCC (786-0) was assessed. Cell counting kit-8 (CCK-8) assay, incidence of tumor, tumor size, Ki-67 or TUNEL staining was used to evaluate tumor cell growth in vitro or in vivo. Flow cytometry assay (in vitro) or examination of cyclin D1 expression (in vivo) was carried out to determine the alteration of cell cycle. The aggressiveness was analyzed by Wound Healing Assay (in vitro) or MMP-2 and MMP-9 expression (in vivo). AKT/p-AKT, ERK1/2/p-ERK1/2 or HGF/c-MET expression was detected by real-time PCR or western blot. Our data demonstrated that MVs promote the growth and aggressiveness of RCC both in vitro and in vivo. In addition, MVs facilitated the progression of cell cycle from G0/1 to S. HGF expression in RCC was greatly induced by MVs, associated with activation of AKT and ERK1/2 signaling pathways. RNase pre-treatment abrogated all effects of MVs. In summary, induction of HGF synthesis via RNA transferred by MVs activating AKT and ERK1/2 signaling is one of crucial contributors to the pro-tumor effect.

Tubularized Urethral Replacement Using Tissue-Engineered Peritoneum-Like Tissue in a Rabbit Model

Introduction: This study aims to investigate whether mesothelial cells could function as seed cells to construct tissue-engineered peritoneum-like tissue for urethral reconstruction in a rabbit model. Materials and Methods: Bladder acellular matrices were prepared and trimmed to 1.5 × 1 cm. Nine male rabbits underwent omentum biopsy and autologous mesothelial cells were isolated. After in vitro expansion, the cells were seeded onto the matrices and incubated for 7 days. In 18 rabbits, a pendulous urethral segment 1.5 cm long was totally excised and urethroplasty was performed with tubularized matrices seeded with cells in 9 animals and without cells in 9 as controls. Urethrography was performed at 1, 2 and 6 months postoperatively. Meanwhile, the neourethras were harvested and analyzed grossly and histologically. Results: Histological analysis of the cell-seeded grafts revealed a loose collagen matrix covered with a single layer of mesothelim. Serial urethrography indicated a wide urethral caliber without stricture formation in animals implanted with cell-seeded matrices, while all animals of the control group developed stricture. Histological analysis of the implanted cell-seeded matrices demonstrated a normal urethral architecture by 1 month, composed of multilayers of urothelium surrounded by smooth muscle bundles, which became increasingly organized with time. By 6 months, the neourethra could be hardly distinguished from native urethra both grossly and histologically. Conclusions: Autologous mesothelial cells could be successfully used as seed cells for tubularized urethral reconstruction in male rabbits.

Mesenchymal Stromal Cells Derived Extracellular Vesicles Ameliorate Acute Renal Ischemia Reperfusion Injury by Inhibition of Mitochondrial Fission through miR-30

Background. The immoderation of mitochondrial fission is one of the main contributors in ischemia reperfusion injury (IRI) and mesenchymal stromal cells (MSCs) derived extracellular vesicles have been regarded as a potential therapy method. Here, we hypothesized that extracellular vesicles (EVs) derived from human Wharton Jelly mesenchymal stromal cells (hWJMSCs) ameliorate acute renal IRI by inhibiting mitochondrial fission through miR-30b/c/d. Methods. EVs isolated from the condition medium of MCS were injected intravenously in rats immediately after monolateral nephrectomy and renal pedicle occlusion for 45 minutes. Animals were sacrificed at 24 h after reperfusion and samples were collected. MitoTracker Red staining was used to see the morphology of the mitochondria. The expression of DRP1 was measured by western blot. miR-30 in EVs and rat tubular epithelial cells was assessed by qRT-PCR. Apoptosis pathway was identified by immunostaining. Results. We found that the expression of miR-30 in injured kidney tissues was declined and mitochondrial dynamics turned to fission. But they were both restored in EVs group in parallel with reduced cell apoptosis. What is more, when the miR-30 antagomirs were used to reduce the miRNA levels, all the related effects of EVs reduced remarkably. Conclusion. A single administration of hWJMSC-EVs could protect the kidney from IRI by inhibition of mitochondrial fission via miR-30.

Differentiation of human umbilical cord mesenchymal stem cells into steroidogenic cells in vitro

Although previous studies have shown that stem cells can be differentiated into Leydig cells by gene transfection, a simple, safe and effective induction method has not yet been reported. Therefore, the present study investigated novel methods for the induction of human umbilical cord mesenchymal stem cell (HUMSC) differentiation into Leydig-like, steroidogenic cells. HUMSCs were acquired using the tissue block culture attachment method, and the expression of MSC surface markers was evaluated by flow cytometry. Leydig cells were obtained by enzymatic digestion and identified by lineage-specific markers via immunofluorescence. Third-passage HUMSCs were cultured with differentiation-inducing medium (DIM) or Leydig cell-conditioned medium (LC-CM), and HUMSCs before induction were used as the control group. Following the induction of HUMSCs, Leydig cell lineage-specific markers (CYP11A1, CYP17A1 and 3β-HSD) were positively identified using immunofluorescence analysis. Additionally, reverse transcription-quantitative polymerase chain reaction and western blot analysis were performed to evaluate the expression levels of these genes and enzymes. In contrast, the control group cells did not show the characteristics of Leydig cells. Collectively, these results indicate that, under in vitro conditions, LC-CM can achieve a comparable effect to that of DIM on inducing HUMSCs differentiation into steroidogenic cells.