Small extracellular vesicles from hypoxia-preconditioned bone marrow mesenchymal stem cells attenuate spinal cord injury via miR-146a-5p-mediated regulation of macrophage polarization

Spinal cord injury is a disabling condition with limited treatment options.Multiple studies have provided evidence suggesting that small extracellular vesicles(SEVs)secreted by bone marrow mesenchymal stem cells(MSCs)help mediate the beneficial effects conferred by MSC transplantation following spinal cord injury.Strikingly,hypoxia-preconditioned bone marrow mesenchymal stem cell-derived SEVs(HSEVs)exhibit increased therapeutic potency.We thus explored the role of HSEVs in macrophage immune regulation after spinal cord injury in rats and their significance in spinal cord repair.SEVs or HSEVs were isolated from bone marrow MSC supernatants by density gradient ultracentrifugation.HSEV administration to rats via tail vein injection after spinal cord injury reduced the lesion area and attenuated spinal cord inflammation.HSEVs regulate macrophage polarization towards the M2 phenotype in vivo and in vitro.Micro RNA sequencing and bioinformatics analyses of SEVs and HSEVs revealed that mi R-146a-5p is a potent mediator of macrophage polarization that targets interleukin-1 receptor-associated kinase 1.Reducing mi R-146a-5p expression in HSEVs partially attenuated macrophage polarization.Our data suggest that HSEVs attenuate spinal cord inflammation and injury in rats by transporting mi R-146a-5p,which alters macrophage polarization.This study provides new insights into the application of HSEVs as a therapeutic tool for spinal cord injury.

Transplantation of human placental chorionic plate-derived mesenchymal stem cells for repair of neurological damage in neonatal hypoxic-ischemic encephalopathy

Neonatal hypoxic-ischemic encephalopathy is often associated with permanent cerebral palsy,neurosensory impairments,and cognitive deficits,and there is no effective treatment for complications related to hypoxic-ischemic encephalopathy.The therapeutic potential of human placental chorionic plate-derived mesenchymal stem cells for various diseases has been explored.However,the potential use of human placental chorionic plate-derived mesenchymal stem cells for the treatment of neonatal hypoxic-ischemic encephalopathy has not yet been investigated.In this study,we injected human placental chorionic plate-derived mesenchymal stem cells into the lateral ventricle of a neonatal hypoxic-ischemic encephalopathy rat model and observed significant improvements in both cognitive and motor function.Protein chip analysis showed that interleukin-3 expression was significantly elevated in neonatal hypoxic-ischemic encephalopathy model rats.Following transplantation of human placental chorionic plate-derived mesenchymal stem cells,interleukin-3 expression was downregulated.To further investigate the role of interleukin-3 in neonatal hypoxic-ischemic encephalopathy,we established an in vitro SH-SY5Y cell model of hypoxic-ischemic injury through oxygen-glucose deprivation and silenced interleukin-3 expression using small interfering RNA.We found that the activity and proliferation of SH-SY5Y cells subjected to oxygen-glucose deprivation were further suppressed by interleukin-3 knockdown.Furthermore,interleukin-3 knockout exacerbated neuronal damage and cognitive and motor function impairment in rat models of hypoxic-ischemic encephalopathy.The findings suggest that transplantation of hpcMSCs ameliorated behavioral impairments in a rat model of hypoxic-ischemic encephalopathy,and this effect was mediated by interleukin-3-dependent neurological function.

Small extracellular vesicles secreted by induced pluripotent stem cell-derived mesenchymal stem cells improve postoperative cognitive dysfunction in mice with diabetes

Postoperative cognitive dysfunction(POCD)is a common surgical complication.Diabetes mellitus(DM)increases risk of developing POCD after surgery.DM patients with POCD seriously threaten the quality of patients’life,however,the intrinsic mechanism is unclear,and the effective treatment is deficiency.Previous studies have demonstrated neuronal loss and reduced neurogenesis in the hippocampus in mouse models of POCD.In this study,we constructed a mouse model of DM by intraperitoneal injection of streptozotocin,and then induced postoperative cognitive dysfunction by transient bilateral common carotid artery occlusion.We found that mouse models of DM-POCD exhibited the most serious cognitive impairment,as well as the most hippocampal neural stem cells(H-NSCs)loss and neurogenesis decline.Subsequently,we hypothesized that small extracellular vesicles secreted by induced pluripotent stem cell-derived mesenchymal stem cells(iMSC-sEVs)might promote neurogenesis and restore cognitive function in patients with DM-POCD.iMSC-sEVs were administered via the tail vein beginning on day 2 after surgery,and then once every 3 days for 1 month thereafter.Our results showed that iMSC-sEVs treatment significantly recovered compromised proliferation and neuronal-differentiation capacity in H-NSCs,and reversed cognitive impairment in mouse models of DM-POCD.Furthermore,miRNA sequencing and qPCR showed miR-21-5p and miR-486-5p were the highest expression in iMSC-sEVs.We found iMSC-sEVs mainly transferred miR-21-5p and miR-486-5p to promote H-NSCs proliferation and neurogenesis.As miR-21-5p was demonstrated to directly targete Epha4 and CDKN2C,while miR-486-5p can inhibit FoxO1 in NSCs.We then demonstrated iMSC-sEVs can transfer miR-21-5p and miR-486-5p to inhibit EphA4,CDKN2C,and FoxO1 expression in H-NSCs.Collectively,these results indicate significant H-NSC loss and neurogenesis reduction lead to DM-POCD,the application of iMSC-sEVs may represent a novel cell-free therapeutic tool for diabetic patients with postoperative cognitive dysfunction.

Embryonic-like stem cells derived from postpartum placenta delivered after spotaneous labor emerging as universal prophylactic cancer vaccine

In the eighth decade of the last century extensive clinical delayed-type hypersensitivity (DTH) skin tests to an intradermal injection of a pharmaceutical allogeneic human Placenta Suspension (phPS) performed in obstetrical, gynecological and control group patients have shown positive reaction in 239 patients with clinical conditions having been as histopatrhological substratum, a hypoxia-induced adaptive/reactive epithelial cell proliferation, e.g. syncytiotrophoblastic cell hyperplasia, endometrial cell hyperplasia, or different gynecological cancers. Because the immune response against phPS has shown antigenc similarities between normal placental and endometrial hyperplastic cells and different kinds of cancer cells and because many cancers adopt an embryonic stem-like gene expression pattern, it is suggested that the profile of hypoxia-promoting placental and endometrial stem cell proliferation is more embryonic-like, and that the immune respose against phPS is expected to cross-react with tumor cells in vivo. In the process of persistent growth and accelerated oxygen consumption by hyperplastic cytotrophoblastic cells and neoplastic cells in a hypoxic microenvironment, a basic shift in energy metabolism is accompanied by appearance of heat shock proteins (HSPs), of fetal isoenzymes and of membrane glycoproteins (reappearance of oncofetal antigens, OFAs), which, as result of their overexpression/amplification may induce a host immunological response. Thus, it is assumed that phPS prepared from full-term human placentas delivered after a spontaneous labor comprises stem/progenitor cells reverted to a proliferative embryonic stem cell-like-state upon exposure to labor-inducing intrmittent placental hypoxia and that by expressing HSP/OFAs could immunize to generate immune response againjst a variety of antigens that are shared by different kinds of epithelial cancers. This immunological feature of phPS qualifies is as a vaccine-related product that may be used for a preventive cancer vaccine when mixed with a potent adjuvant (BCG-Vaccine) and given normal healthy individuals.

Small regulators making big impacts:regulation of neural stem cells by small non-coding RNAs

Neurodegeneration and traumatic brain injuries are leading causes of disability and present an enormous disease burden both in terms of patient suffering and healthcare cost.Treatment of brain lesions remains as a major challenge in medicine largely because of the limited regenerative capacity of the adult brain.

Effect of CXCR3/HO-1 genes modified bone marrow mesenchymal stem cells on small bowel transplant rejection

AIM To investigate whether bone marrow mesenchymal stem cells(BMMSCs) modified with the HO-1 and CXCR3 genes can augment the inhibitory effect of BMMSCs on small bowel transplant rejection.METHODS Lewis rat BMMSCs were cultured in vitro. Thirdpassage BMMSCs were transduced with the CXCR3/HO-1 genes or the HO-1 gene alone. The rats were divided into six groups and rats in the experimental group were pretreated with BMMSCs 7 d prior to smallbowel transplant. Six time points(instant, 1 d, 3 d, 7 d, 10 d, and 14 d)(n = 6) were chosen for each group. Hematoxylin-eosin staining was used to observe pathologic rejection, while immunohistochemistry and Western blot were used to detect protein expression. Flow cytometry was used to detect T lymphocytes and enzyme linked immunosorbent assay was used to detect cytokines.RESULTS The median survival time of BMMSCs from the CXCR3/HO-1 modified group(53 d) was significantly longer than that of the HO-1 modified BMMSCs group(39 d), the BMMSCs group(26 d), and the NS group(control group)(16 d)(P < 0.05). Compared with BMMSCs from the HO-1 modified BMMSCs, BMMSCs, and NS groups, rejection of the small bowel in the CXCR3/HO-1 modified group was significantly reduced, while the weight of transplant recipients was also significantly decreased(P < 0.05). Furthermore, IL-2, IL-6, IL-17, IFN-γ, and TNF-α levels were significantly decreased and the levels of IL-10 and TGF-β were significantly increased(P < 0.05). CONCLUSION BMMSCs modified with the CXCR3 and HO-1 genes can abrogate the rejection of transplanted small bowel more effectively and significantly increase the survival time of rats that receive a small bowel transplant.

Transient Folate Deprivation in Combination with Small-molecule Compounds Facilitates the Generation of Somatic Cell-derived Pluripotent Stem Cells in Mice

Induced pluripotent stem cells （iPSCs） can be propagated indefinitely, while maintaining the capacity to differentiate into all cell types in the body except for the extra-embryonic tissues. This iPSC technology not only represents a new way to use individual-specific stem cells for regenerative medicine but also constitutes a novel method to obtain large numbers of disease-specific cells for biomedical re- search. However, the low efficiency of reprogramming and genomic integration of oncogenes and viral vectors limit the potential application of iPSCs. Chemical-induced reprogramming offers a novel ap- proach to generating iPSCs. In this study, a new combination of small-molecule compounds （SMs） （so- dium butyrate, A-83-01, CHIR99021, Y-27632） under conditions of transient folate deprivation was used to generate iPSC. It was found that transient folate deprivation combined with SMs was sufficient to permit reprogramming from mouse embryonic fibroblasts （MEFs） in the presence of transcription factors, Oct4 and Klf4, within 25 days, replacing Sox2 and c-Myc, and accelerated the generation of mouse iPSCs The resulting cell lines resembled mouse embryonic stem （ES） cells with respect to proliferation rate, morphology, pluripotency-associatedmarkers and gene expressions. Deprivation of folic acid, combined with treating MEFs with SMs, can improve the inducing efficiency of iPSCs and reduce their carcino- genicity and the use of exogenous reprogramming factors.

A review from mesenchymal stem-cells and their small extracellular vesicles in tissue engineering

This review aims to offer a vision of the clinical reality of cell therapy today in intensive medicine.For this,it has been carried out a description of the properties,functions,and Mesenchymal Stem Cells(MSCS)sources to subsequently address the evidence in preclinical models and studies clinical trials with whole cells and models attributed to small extracellular vesicles(sEVs),nanoparticles made up of microvesicles secreted by cells with an effect on the extracellular matrix,and their impact as an alternative towards cell-free regenerative medicine.MSCs are cells that enhance the regenerative capacity which can be differentiated typically in different lineages committed as bone,cartilage,and adipose tissue.On the other hand,small extracellular vesicles are structures that participate notoriously and crucially in intercellular communication,which has led to a change in the concept of the functions and the role that these vesicles play in living organisms,in the restoration of damaged tissues and the inflammatory response and immunological.We present the mechanisms that are involved in the applications of MSCS as whole cells and their sEVs in cell therapy and cell-free therapy as an alternative in regenerative medicine.Considering the structural loss that occurs after surgical procedures for cystic and tumoral pathology in periodontitis,as well as the maxillary atrophy that determines the rehabilitation with dental implants,it is imperative to find satisfactory solutions.The opportunity provided by the findings in stem cells is a recent introduction in the field of oral surgery,based on the regenerative potential that these cells possess to restore defects at different levels of the oral cavity.This review aims to discover the real applications that stem cells may have in our treatments shortly.

Very small embryonic like(VSEL) stem cells

It is generally accepted that adult bone marrow（BM） contains both hematopoietic stem cells（HSCs） and mesenchymal stem cells （MSCs）. Recently, a rare population of stem cells different from HSCs and MSCs were identified in murine BM and human cord blood （CB）, named as very small embryonic like（VSEL） stem cells. These cells are tiny round and CXCR4^＋ Sca-1^＋ Lin^- CD45^-, expressing SSEA-1/4, Oct-4 and Nanog, which have potent of differentiation into all three germ-layer lineages, such as cardiornyocytes, neural and pancreatic ceils.

An Insight on Small Molecule Induced Foot-Print Free Naive Pluripotent Stem Cells in Livestock

Bona fide embryonic stem cell (ESC) lines from livestock species have been challenging to derive and maintain, contrasting mouse and human ESCs. However, induced pluripotent stem cells (iPSC) generated by reprogramming somatic cells tender an option, as they display characteristic features of ESC. The comprehension that induced pluripotent stem cells (iPSC) could be created with in no time also holds the potential of allowing pluripotent cells to be derived from animal models vital in biomedical research. Endeavors to produce bona fide pluripotent stem cells (PSC) from livestock have been going on for more than two decades. But, attempts to derive bona fide livestock iPS cells have met with limited success. Recently it’s been reported that small molecules can augment reprogramming efficiency and may be used to substitute few or all transcription factors used for reprogramming. It is assumed that the reprogramming factors are conserved among species, and this small molecule reprogramming approach will probably apply to livestock species as well. So this review will focus mainly on the accomplishments of small molecules on accelerating cell reprogramming and obtaining naive pluripotency, and raise a new insight on, exogenous genes free, livestock naive iPSC generation with a new bullet, small molecule.

Protective effect of bone marrow mesenchymal stem cells in intestinal barrier permeability after heterotopic intestinal transplantation

AIM:To explore the protective effect of bone marrow mesenchymal stem cells(BM MSCs)in the small intestinal mucosal barrier following heterotopic intestinal transplantation(HIT)in a rat model.METHODS:BM MSCs were isolated from male Lewis rats by density gradient centrifugation,cultured,and analyzed by flow cytometry.The HIT models were divided into a non-rejection group,saline-treated rejection group(via penile vein),and BM MSC–treated group(via penile vein).Intestinal mucosal barrier injury was estimated by diamine oxidase(DAO)and D-lactic acid(D-LA)expression levels.Tumor necrosis factor-α(TNF-α),interferon-γ(INF-γ),interleukin-10(IL-10),and transforming growth factor-β(TGF-β)were detected by enzyme-linked immunosorbent assay.Ultrastructural change of tight junctions(TJs)was observed under transmission electron microscope.Expression levels of the TJ proteins occludin and zona occludens(ZO)-1,affected by the inflammatory factors,were measured using real-time polymerase chain reaction and Western blotting.RESULTS:The pathological score at each time point after surgery indicated significantly less serious injury in the BM MSCs-treated group than in the rejection group(P<0.05).In the former,graft levels of DAO and D-LA were reduced,and TNF-αand INF-γproduction was inhibited(at day 7:10.6473±0.0710vs 17.2128±0.4991,P<0.05;545.1506±31.9416vs 810.2637±25.1175,P<0.05).IL-10 and TGF-βproduction was increased greatly(at day 7:125.7773±4.7719 vs 80.3756±2.5866,P<0.05;234.5273±9.3980 vs 545.1506±31.9416,P<0.05).There was increased expression of occludin and ZO-1 protein(at day 7:0.2674±0.0128 vs 0.1352±0.0142,P<0.05;at day 5:0.7189±0.0289 vs 0.4556±0.0242,P<0.05)and mRNA(at day 7:0.3860±0.0254 vs 0.1673±0.0369,P<0.05;at day 5:0.5727±0.0419 vs0.3598±0.0242,P<0.05).CONCLUSION:BM MSCs can improve intestinal barrier permeability,repair TJs,and increase occludin and ZO-1 protein expression.With altered cytokine levels,they can protect the intestinal mucosa after transplantation.

Small molecules for mesenchymal stem cell fate determination

Mesenchymal stem cells(MSCs)are adult stem cells harboring self-renewal and multilineage differentiation potential that are capable of differentiating into osteoblasts,adipocytes,or chondrocytes in vitro,and regulating the bone marrow microenvironment and adipose tissue remodeling in vivo.The process of fate determination is initiated by signaling molecules that drive MSCs into a specific lineage.Impairment of MSC fate determination leads to different bone and adipose tissue-related diseases,including aging,osteoporosis,and insulin resistance.Much progress has been made in recent years in discovering small molecules and their underlying mechanisms control the cell fate of MSCs both in vitro and in vivo.In this review,we summarize recent findings in applying small molecules to the trilineage commitment of MSCs,for instance,genistein,medicarpin,and icariin for the osteogenic cell fate commitment;isorhamnetin,risedronate,and arctigenin for pro-adipogenesis;and atractylenolides and dihydroartemisinin for chondrogenic fate determination.We highlight the underlying mechanisms,including direct regulation,epigenetic modification,and post-translational modification of signaling molecules in the AMPK,MAPK,Notch,PI3K/AKT,Hedgehog signaling pathways etc.and discuss the small molecules that are currently being studied in clinical trials.The target-based manipulation of lineage-specific commitment by small molecules offers substantial insights into bone marrow microenvironment regulation,adipose tissue homeostasis,and therapeutic strategies for MSC-related diseases.

Mesenchymal stem cell-derived small extracellular vesicles in the treatment of human diseases:Progress and prospect

Mesenchymal stem cells(MSCs)are self-renewing,multipotent cells that could differentiate into multiple tissues.MSC-based therapy has become an attractive and promising strategy for treating human diseases through immune regulation and tissue repair.However,accumulating data have indicated that MSC-based therapeutic effects are mainly attributed to the properties of the MSC-sourced secretome,especially small extracellular vesicles(sEVs).sEVs are signaling vehicles in intercellular communication in normal or pathological conditions.sEVs contain natural contents,such as proteins,mRNA,and microRNAs,and transfer these functional contents to adjacent cells or distant cells through the circulatory system.MSC-sEVs have drawn much attention as attractive agents for treating multiple diseases.The properties of MSC-sEVs include stability in circulation,good biocompatibility,and low toxicity and immunogenicity.Moreover,emerging evidence has shown that MSC-sEVs have equal or even better treatment efficacies than MSCs in many kinds of disease.This review summarizes the current research efforts on the use of MSC-sEVs in the treatment of human diseases and the existing challenges in their application from lab to clinical practice that need to be considered.

Presence and role of stem cells in ovarian cancer

Ovarian cancer is the deadliest gynecological malignancy.It is typically diagnosed at advanced stages of the disease,with metastatic sites disseminated widely within the abdominal cavity.Ovarian cancer treatment is challenging due to high disease recurrence and further complicated pursuant to acquired chemoresistance.Cancer stem cell(CSC)theory proposes that both tumor development and progression are driven by undifferentiated stem cells capable of self-renewal and tumor-initiation.The most recent evidence revealed that CSCs in terms of ovarian cancer are not only responsible for primary tumor growth,metastasis and relapse of disease,but also for the development of chemoresistance.As the elimination of this cell population is critical for increasing treatment success,a deeper understanding of ovarian CSCs pathobiology,including epithelial-mesenchymal transition,signaling pathways and tumor microenvironment,is needed.Finally,before introducing new therapeutic agents for ovarian cancer,targeting CSCs,accurate identification of different ovarian stem cell subpopulations,including the very small embryoniclike stem cells suggested as progenitors,is necessary.To these ends,reliable markers of ovarian CSCs should be identified.In this review,we present the current knowledge and a critical discussion concerning ovarian CSCs and their clinical role.

Differentiation of human embryonic stem cells derived mesenchymal stem cells into corneal epithelial cells after being seeded on decellularized SMILE-derived lenticules

AIM: To evaluate the feasibility of mesenchymal stem cells(MSCs) to differentiate into corneal epithelial cells after being seeded on the decellularized small incision lenticule extraction(SMILE)-derived lenticules. METHODS: The fresh lenticules procured from patients undergoing SMILE for the correction of myopia were decellularized. The MSCs were subsequently cultivated on those denuded lenticules. The MSCs without lenticules were used as a control. The proliferation activity of the MSCs after seeding 24 h was quantitatively determined with the Cell Counting Kit-8(CCK-8) assay. Immunofluorescence staining and quantitative reverse transcription polymerase chain reaction(qRT-PCR) were used to assess the marker expression in differentiated MSCs. RESULTS: The data showed that both fresh and decellularized lenticules could significantly promote the proliferation of MSCs, compared to that in control(P=0.02 for fresh lenticules, P=0.001 for decellularize ones, respectively). The MSCs seeded on both lenticules were positive for cytokeratin 3(CK3) staining. The expression of CK3 increased 5-fold in MSCs seeded on fresh lenticules and 18-fold on decellularized ones, compared to that in control. There was a significant difference in the expression of CK3 in MSCs seeded on fresh and decellularized lenticules(P<0.001). The expression of CK8 and CK18 was similar in pure MSCs and MSCs seeded on fresh lenticules(P>0.05), while the expression of these markers was decreased in MSCs seeded on decellularized ones. CONCLUSION: These results suggest that the decellularized lenticules might be more suitable for MSCs to differentiate into corneal epithelial cells, which offersthe prospect of a novel therapeutic modality of SMILEderived lenticules in regenerative corneal engineering.

Umbilical cord-derived mesenchymal stem cells preconditioned with isorhamnetin:potential therapy for burn wounds

BACKGROUND Impaired wound healing can be associated with different pathological states.Burn wounds are the most common and detrimental injuries and remain a major health issue worldwide.Mesenchymal stem cells(MSCs)possess the ability to regenerate tissues by secreting factors involved in promoting cell migration,proliferation and differentiation,while suppressing immune reactions.Preconditioning of MSCs with small molecules having cytoprotective properties can enhance the potential of these cells for their use in cell-based therapeutics.AIM To enhance the therapeutic potential of MSCs by preconditioning them with isorhamnetin for second degree burn wounds in rats.METHODS Human umbilical cord MSCs(hU-MSCs)were isolated and characterized by surface markers,CD105,vimentin and CD90.For preconditioning,hU-MSCs were treated with isorhamnetin after selection of the optimized concentration(5μmol/L)by cytotoxicity analysis.The migration potential of these MSCs was analyzed by the in vitro scratch assay.The healing potential of normal,and preconditioned hU-MSCs was compared by transplanting these MSCs in a rat model of a second degree burn wound.Normal,and preconditioned MSCs(IH+MSCs)were transplanted after 72 h of burn injury and observed for 2 wk.Histological and gene expression analyses were performed on day 7 and 14 after cell transplantation to determine complete wound healing.RESULTS The scratch assay analysis showed a significant reduction in the scratch area in the case of IH+MSCs compared to the normal untreated MSCs at 24 h,while complete closure of the scratch area was observed at 48 h.Histological analysis showed reduced inflammation,completely remodeled epidermis and dermis without scar formation and regeneration of hair follicles in the group that received IH+MSCs.Gene expression analysis was time dependent and more pronounced in the case of IH+MSCs.Interleukin(IL)-1β,IL-6 and Bcl-2 associated X genes showed significant downregulation,while transforming growth factorβ,vascular endothelial growth factor,Bcl-2 and matrix metallopeptidase 9 showed significant upregulation compared to the burn wound,showing increased angiogenesis and reduced inflammation and apoptosis.CONCLUSION Preconditioning of hU-MSCs with isorhamnetin decreases wound progression by reducing inflammation,and improving tissue architecture and wound healing.The study outcome is expected to lead to an improved cell-based therapeutic approach for burn wounds.

Mesenchymal stem cell therapy in patients with small bowel transplantation:Single center experience

AIM:To study the effects of mesenchymal stem cell(MSC) therapy on the prevention of acute rejection and graft vs host disease following small bowel transplantation.METHODS: In our transplantation center, 6 isolated intestinal transplants have been performed with MSC therapy since 2009. The primary reasons for transplants were short gut syndrome caused by surgical intestine resection for superior mesenteric artery thrombosis(n = 4), Crohn's disease(n = 1) and intestinal aganglionosis(n = 1). Two of the patients were children. At the time of reperfusion, the first dose of MSCs cultured from the patient's bone marrow was passedinto the transplanted intestinal artery at a dose of 1000000 cells/kg. The second and third doses of MSCs were given directly into the mesenteric artery through the arterial anastomosis using an angiography catheter on day 15 and 30 post-transplant.RESULTS: The median follow-up for these patients was 10.6 mo(min: 2 mo-max: 30 mo). Three of the patients developed severe acute rejection. One of these patients did not respond to bolus steroid therapy. Although the other two patients did respond to antirejection treatment, they developed severe fungal and bacterial infections. All of these patients died in the 2nd and 3rd months post-transplant due to sepsis. The remaining patients who did not have acute rejection had good quality of life with no complications observed during the follow-up period. In addition, their intestinal grafts were functioning properly in the 13th, 25th and 30th month post-transplant. The patients who survived did not encounter any problems related to MSC transplantation.CONCLUSION: Although this is a small case series and not a randomized study, it is our opinion that small bowel transplantation is an effective treatment for intestinal failure, and MSC therapy may help to prevent acute rejection and graft vs host disease following intestinal transplantation.

Review: Neuronal Differentiation Protocols of Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are self-renewing cells found in almost all postnatal organs and tissues in the perivascular region. These cells present multiple characteristics that make them candidates to be applied in cell therapy for neurodegenerative diseases, such as their secretory action, migration to the lesion area, and immunomodulatory potential. These cells have a high capacity for mesodermal differentiation;however, numerous studies have shown that MSCs can also differentiate into neurons. However, despite positive results in multiple trials in which undifferentiated MSCs transplanted into animal models of neurodegenerative diseases, some studies suggest that the therapeutic effects obtained are enhanced by the use of MSCs differentiated towards the neuronal lineage before transplant. In this sense, there are several methods to induce in vitro reprogramming of MSCs towards the neuronal lineage, including chemical substances, growth factors, cocultures with neural lineage cells, transfection of genes, miRNAs, etc., and small molecules stand out. Therefore, this article compares multiple experimental tests in which these inducers promote neuronal differentiation of MSCs and identify those methods that originate an optimal neuronal differentiation. The analysis includes the percentage of differentiation, maturation, expression of neuronal markers, functionality, and cell survival considering the intrinsic characteristics of the MSCs used as the tissue of origin and the species from which they were isolated.

Construction of hepatocyte growth factor gene recombinant adenovirus vector and its expression in rat bone marrow mesenchymal stem cells

Objective：To construct the adenoviral expression vector system containing human hepatocyte growth factor （hHGF） cDNA, and to further study the transduction efficiency and the expression of HGF in mesenchymal stem cells （MSCs）. Methods：The HGF cDNA was amplificated from the expression plasmid pCMV-HGF, and was subcloned into the adenovirus shuttle plasmid pDC316-IRESEGFP vector containing a green fluorescence protein （GFP） reporter gene. Virus Ad-HGF was produced by homologous recombination in HEK293 package cells. Bone marrow derived MSCs were harvested and cultured, and then were transduced with Ad-HGF. The efficiency of Ad-HGF transduction was assessed by FACS analysis using GFP gene expression. And HGF/MSCs were generated. The HGF concentrations in supernatants of HGF/MSCs were determined by ELISA using anti-human HGF monoclonal antibody. Results： The recombinant, named pDC316-HGF-IRES-eGFP, was digested with restriction enzyme, and the DNA sequencing of HGF was identical to the report in Genebank and did not reveal any mutation. GFP expression could be observed on the second day after packing of the linearized pAd-HGF in HEK293 cells and 7.15 × 10^10pfu/ml titer of Ad-HGF was obtained. Forty-eight hours after transduction, 96.89% of HGF/MSCs were GFP positive. Peak concentration levels of hHGF（103ng/mL） in the cultured supernatants were detected on day 2 post-transduction, and the adenovirus-mediated expression of HGF by MSCs was maintained for at least 2 weeks in vivo. Conclusion：Our data demonstrated that the adenovirus expression＇vector system pDC316-HGF-IRES-EGFP has been constructed successfully, and their effective expressions also have been obtained in MSCs. This will provide material basis for the next study on liver regeneration after small-for-size liver transplantation.

An inhibitor of HIF-α subunit expression suppresses hypoxiainduced dedifferentiation of human NSCLC into cancer stem cell-like cells

AIM:To investigate whether hypoxia induces dedifferentiation of non-small cell lung cancer(NSCLC) cells and whether a hypoxia-inducible factor(HIF) inhibitor is able to suppress the process.METHODS:Human lung adenocarcinoma A549 cells and squamous carcinoma QG56 cells were cultured under normoxic(21%O_2) or hypoxic(4%or 1%O_2) conditions.The expression of the following genes were examined by reverse transcription-polymerase chain reaction,Western blotting and/or immunofluorescence:HIF-1α and HIF-2αsubunits;differentiation marker genes,namely surfactant protein C(SP-C)(type Ⅱ alveolar cell marker),CC10(type I alveolar cell marker) and aquaporin 5(AQP5)(Clara cell marker);and stem cell-associated genes,namely CD133,0CT4,and Musashi-1(MSI1).The tumor sphere-forming ability of the cells was evaluated by culturing them in serum-free growth factor-rich medium containing epidermal growth factor(EGF) and fibroblast growth factor(FGF).CD133 expression in hypoxic regions in A549 tumors was examined by double-immunostaining of tissue cryosections with an anti-2-nitroimidazole EF5 antibody and an anti-CD133 antibody.The metastatic ability of A549 cells was examined macroscopically and histologically after injecting them into the tail vein of immunocompromised mice.RESULTS:A549 cells primarily expressed SP-C,and QG56 cells expressed CC10 and AQP5.Exposure of A549 cells to hypoxia resulted in a marked downregulation of SP-C and upregulation of CD133,OCT4,and MSI1 in a time-dependent manner.Moreover,hypoxia mimetics,namely desferrioxamine and cobalt chloride,elicited similar effects.Ectopic expression of the constitutively active HIF-la subunit also caused the downregulation of SP-C and upregulation of CD133 and MSI1 but not OCT4,which is a direct target of HIF-2.Hypoxia enhanced the sphere-forming activity of A549 cells in serum-free medium containing EGF and FGF.Similarly,hypoxia downregulated the expression of CC10 and AQP5 genes and upregulated CD133,OCT4,and MSI1 genes in QG56 cells.TX-402(3-amino-2-quinoxalinecarbonitrile 1,4-dioxide),which is a small molecule inhibitor of the expression of HIF-1α and HIF-2αsubunits under hypoxic conditions,inhibited the upregulation of SP-C'and hypoxia-induced down-regulation of CD133,OCT4,and MSI1.Notably,TX-402 significantly suppressed the hypoxia-enhanced lung-colonizing ability of A549 cells.CONCLUSION:Hypoxia induces the de-differentiation of NSCLC cells into cancer stem cell-like cells,and HIF inhibitors are promising agents to prevent this process.