Expansion of human umbilical cord derived mesenchymal stem cells in regenerative medicine

BACKGROUND Stem cells are undifferentiated cells that possess the potential for self-renewal with the capacity to differentiate into multiple lineages.In humans,their limited numbers pose a challenge in fulfilling the necessary demands for the regeneration and repair of damaged tissues or organs.Studies suggested that mesenchymal stem cells(MSCs),necessary for repair and regeneration via transplantation,require doses ranging from 10 to 400 million cells.Furthermore,the limited expansion of MSCs restricts their therapeutic application.AIM To optimize a novel protocol to achieve qualitative and quantitative expansion of MSCs to reach the targeted number of cells for cellular transplantation and minimize the limitations in stem cell therapy protocols.METHODS Human umbilical cord(hUC)tissue derived MSCs were obtained and re-cultured.These cultured cells were subjected to the following evaluation pro-cedures:Immunophenotyping,immunocytochemical staining,trilineage differentiation,population doubling time and number,gene expression markers for proliferation,cell cycle progression,senescence-associatedβ-galactosidase assay,human telomerase reverse transcriptase(hTERT)expression,mycoplasma,cytomegalovirus and endotoxin detection.RESULTS Analysis of pluripotent gene markers Oct4,Sox2,and Nanog in recultured hUC-MSC revealed no significant differences.The immunophenotypic markers CD90,CD73,CD105,CD44,vimentin,CD29,Stro-1,and Lin28 were positively expressed by these recultured expanded MSCs,and were found negative for CD34,CD11b,CD19,CD45,and HLA-DR.The recultured hUC-MSC population continued to expand through passage 15.Proliferative gene expression of Pax6,BMP2,and TGFb1 showed no significant variation between recultured hUC-MSC groups.Nevertheless,a significant increase(P<0.001)in the mitotic phase of the cell cycle was observed in recultured hUC-MSCs.Cellular senescence markers(hTERT expression andβ-galactosidase activity)did not show any negative effect on recultured hUC-MSCs.Additionally,quality control assessments consistently confirmed the absence of mycoplasma,cytomegalovirus,and endotoxin contamination.CONCLUSION This study proposes the development of a novel protocol for efficiently expanding stem cell population.This would address the growing demand for larger stem cell doses needed for cellular transplantation and will significantly improve the feasibility of stem cell based therapies.

Therapeutic utility of human umbilical cord-derived mesenchymal stem cells-based approaches in pulmonary diseases:Recent advancements and prospects

Pulmonary diseases across all ages threaten millions of people and have emerged as one of the major public health issues worldwide.For diverse disease con-ditions,the currently available approaches are focused on alleviating clinical symptoms and delaying disease progression but have not shown significant therapeutic effects in patients with lung diseases.Human umbilical cord-derived mesenchymal stem cells(UC-MSCs)isolated from the human UC have the capacity for self-renewal and multilineage differentiation.Moreover,in recent years,these cells have been demonstrated to have unique advantages in the treatment of lung diseases.We searched the Public Clinical Trial Database and found 55 clinical trials involving UC-MSC therapy for pulmonary diseases,including coronavirus disease 2019,acute respiratory distress syndrome,bron-chopulmonary dysplasia,chronic obstructive pulmonary disease,and pulmonary fibrosis.In this review,we summarize the characteristics of these registered clinical trials and relevant published results and explore in depth the challenges and opportunitiesfaced in clinical application.Moreover,the underlying mole-cular mechanisms involved in UC-MSC-based therapy for pulmonary diseases are also analyzed in depth.In brief,this comprehensive review and detailed analysis of these clinical trials can be expected to provide a scientific reference for future large-scale clinical application.

Human umbilical cord mesenchymal stem cell-derived exosomes loaded into a composite conduit promote functional recovery after peripheral nerve injury in rats

Complete transverse injury of peripheral nerves is challenging to treat.Exosomes secreted by human umbilical cord mesenchymal stem cells are considered to play an important role in intercellular communication and regulate tissue regeneration.In previous studies,a collagen/hyaluronic acid sponge was shown to provide a suitable regeneration environment for Schwann cell proliferation and to promote axonal regeneration.This three-dimensional(3D)composite conduit contains a collagen/hyaluronic acid inner sponge enclosed in an electrospun hollow poly(lactic-co-glycolic acid)tube.However,whether there is a synergy between the 3D composite conduit and exosomes in the repair of peripheral nerve injury remains unknown.In this study,we tested a comprehensive strategy for repairing long-gap(10 mm)peripheral nerve injury that combined the 3D composite conduit with human umbilical cord mesenchymal stem cell-derived exosomes.Repair effectiveness was evaluated by sciatic functional index,sciatic nerve compound muscle action potential recording,recovery of muscle mass,measuring the cross-sectional area of the muscle fiber,Masson trichrome staining,and transmission electron microscopy of the regenerated nerve in rats.The results showed that transplantation of the 3D composite conduit loaded with human umbilical cord mesenchymal stem cell-derived exosomes promoted peripheral nerve regeneration and restoration of motor function,similar to autograft transplantation.More CD31-positive endothelial cells were observed in the regenerated nerve after transplantation of the loaded conduit than after transplantation of the conduit without exosomes,which may have contributed to the observed increase in axon regeneration and distal nerve reconnection.Therefore,the use of a 3D composite conduit loaded with human umbilical cord mesenchymal stem cell-derived exosomes represents a promising cell-free therapeutic option for the treatment of peripheral nerve injury.

Hypoxia and inflammatory factor preconditioning enhances the immunosuppressive properties of human umbilical cord mesenchymal stem cells

BACKGROUND Mesenchymal stem cells(MSCs)have great potential for the treatment of various immune diseases due to their unique immunomodulatory properties.However,MSCs exposed to the harsh inflammatory environment of damaged tissue after intravenous transplantation cannot exert their biological effects,and therefore,their therapeutic efficacy is reduced.In this challenging context,an in vitro preconditioning method is necessary for the development of MSC-based therapies with increased immunomodulatory capacity and transplantation efficacy.AIM To determine whether hypoxia and inflammatory factor preconditioning increases the immunosuppressive properties of MSCs without affecting their biological characteristics.METHODS Umbilical cord MSCs(UC-MSCs)were pretreated with hypoxia(2%O_(2))exposure and inflammatory factors(interleukin-1β,tumor necrosis factor-α,interferon-γ)for 24 h.Flow cytometry,polymerase chain reaction,enzyme-linked immunosorbent assay and other experimental methods were used to evaluate the biological characteristics of pretreated UC-MSCs and to determine whether pretreatment affected the immunosuppressive ability of UC-MSCs in coculture with immune cells.RESULTS Pretreatment with hypoxia and inflammatory factors caused UC-MSCs to be elongated but did not affect their viability,proliferation or size.In addition,pretreatment significantly decreased the expression of coagulationrelated tissue factors but did not affect the expression of other surface markers.Similarly,mitochondrial function and integrity were retained.Although pretreatment promoted UC-MSC apoptosis and senescence,it increased the expression of genes and proteins related to immune regulation.Pretreatment increased peripheral blood mononuclear cell and natural killer(NK)cell proliferation rates and inhibited NK cell-induced toxicity to varying degrees.CONCLUSION In summary,hypoxia and inflammatory factor preconditioning led to higher immunosuppressive effects of MSCs without damaging their biological characteristics.

Dissecting molecular mechanisms underlying ferroptosis in human umbilical cord mesenchymal stem cells:Role of cystathionineγ-lyase/hydrogen sulfide pathway

BACKGROUND Ferroptosis can induce low retention and engraftment after mesenchymal stem cell(MSC)delivery,which is considered a major challenge to the effectiveness of MSC-based pulmonary arterial hypertension(PAH)therapy.Interestingly,the cystathionineγ-lyase(CSE)/hydrogen sulfide(H_(2)S)pathway may contribute to mediating ferroptosis.However,the influence of the CSE/H_(2)S pathway on ferroptosis in human umbilical cord MSCs(HUCMSCs)remains unclear.AIM To clarify whether the effect of HUCMSCs on vascular remodelling in PAH mice is affected by CSE/H_(2)S pathway-mediated ferroptosis,and to investigate the functions of the CSE/H_(2)S pathway in ferroptosis in HUCMSCs and the underlying mechanisms.METHODS Erastin and ferrostatin-1(Fer-1)were used to induce and inhibit ferroptosis,respectively.HUCMSCs were transfected with a vector to overexpress or inhibit expression of CSE.A PAH mouse model was established using 4-wk-old male BALB/c nude mice under hypoxic conditions,and pulmonary pressure and vascular remodelling were measured.The survival of HUCMSCs after delivery was observed by in vivo bioluminescence imaging.Cell viability,iron accumulation,reactive oxygen species production,cystine uptake,and lipid peroxidation in HUCMSCs were tested.Ferroptosis-related proteins and S-sulfhydrated Kelchlike ECH-associating protein 1(Keap1)were detected by western blot analysis.RESULTS In vivo,CSE overexpression improved cell survival after erastin-treated HUCMSC delivery in mice with hypoxiainduced PAH.In vitro,CSE overexpression improved H_(2)S production and ferroptosis-related indexes,such as cell viability,iron level,reactive oxygen species production,cystine uptake,lipid peroxidation,mitochondrial membrane density,and ferroptosis-related protein expression,in erastin-treated HUCMSCs.In contrast,in vivo,CSE inhibition decreased cell survival after Fer-1-treated HUCMSC delivery and aggravated vascular remodelling in PAH mice.In vitro,CSE inhibition decreased H_(2)S levels and restored ferroptosis in Fer-1-treated HUCMSCs.Interestingly,upregulation of the CSE/H_(2)S pathway induced Keap1 S-sulfhydration,which contributed to the inhibition of ferroptosis.CONCLUSION Regulation of the CSE/H_(2)S pathway in HUCMSCs contributes to the inhibition of ferroptosis and improves the suppressive effect on vascular remodelling in mice with hypoxia-induced PAH.Moreover,the protective effect of the CSE/H_(2)S pathway against ferroptosis in HUCMSCs is mediated via S-sulfhydrated Keap1/nuclear factor erythroid 2-related factor 2 signalling.The present study may provide a novel therapeutic avenue for improving the protective capacity of transplanted MSCs in PAH.

Zinc enhances the cell adhesion,migration,and self-renewal potential of human umbilical cord derived mesenchymal stem cells

BACKGROUND Zinc(Zn)is the second most abundant trace element after Fe,present in the human body.It is frequently reported in association with cell growth and proliferation,and its deficiency is considered to be a major disease contributing factor.AIM To determine the effect of Zn on in vitro growth and proliferation of human umbilical cord(hUC)-derived mesenchymal stem cells(MSCs).METHODS hUC-MSCs were isolated from human umbilical cord tissue and characterized based on immunocytochemistry,immunophenotyping,and tri-lineage differentiation.The impact of Zn on cytotoxicity and proliferation was determined by MTT and Alamar blue assay.To determine the effect of Zn on population doubling time(PDT),hUC-MSCs were cultured in media with and without Zn for several passages.An in vitro scratch assay was performed to analyze the effect of Zn on the wound healing and migration capability of hUC-MSCs.A cell adhesion assay was used to test the surface adhesiveness of hUC-MSCs.Transcriptional analysis of genes involved in the cell cycle,proliferation,migration,and selfrenewal of hUC-MSCs was performed by quantitative real-time polymerase chain reaction.The protein expression of Lin28,a pluripotency marker,was analyzed by immunocytochemistry.RESULTS Zn at lower concentrations enhanced the rate of proliferation but at higher concentrations(>100μM),showed concentration dependent cytotoxicity in hUC-MSCs.hUC-MSCs treated with Zn exhibited a significantly greater healing and migration rate compared to untreated cells.Zn also increased the cell adhesion rate,and colony forming efficiency(CFE).In addition,Zn upregulated the expression of genes involved in the cell cycle(CDC20,CDK1,CCNA2,CDCA2),proliferation(transforming growth factorβ1,GDF5,hypoxia-inducible factor 1α),migration(CXCR4,VCAM1,VEGF-A),and self-renewal(OCT4,SOX2,NANOG)of hUC-MSCs.Expression of Lin28 protein was significantly increased in cells treated with Zn.CONCLUSION Our findings suggest that zinc enhances the proliferation rate of hUC-MSCs decreasing the PDT,and maintaining the CFE.Zn also enhances the cell adhesion,migration,and self-renewal of hUC-MSCs.These results highlight the essential role of Zn in cell growth and development.

Enhanced wound healing and hemostasis with exosome-loaded gelatin sponges from human umbilical cord mesenchymal stem cells

BACKGROUND Rapid wound healing remains a pressing clinical challenge,necessitating studies to hasten this process.A promising approach involves the utilization of human umbilical cord mesenchymal stem cells(hUC-MSCs)derived exosomes.The hypothesis of this study was that these exosomes,when loaded onto a gelatin sponge,a common hemostatic material,would enhance hemostasis and accelerate wound healing.AIM To investigate the hemostatic and wound healing efficacy of gelatin sponges loaded with hUC-MSCs-derived exosomes.METHODS Ultracentrifugation was used to extract exosomes from hUC-MSCs.Nanoparticle tracking analysis(NTA),transmission electron microscopy(TEM),and western blot techniques were used to validate the exosomes.In vitro experiments were performed using L929 cells to evaluate the cytotoxicity of the exosomes and their impact on cell growth and survival.New Zealand rabbits were used for skin irritation experiments to assess whether they caused adverse skin reactions.Hemolysis test was conducted using a 2%rabbit red blood cell suspension to detect whether they caused hemolysis.Moreover,in vivo experiments were carried out by implanting a gelatin sponge loaded with exosomes subcutaneously in Sprague-Dawley(SD)rats to perform biocompatibility tests.In addition,coagulation index test was conducted to evaluate their impact on blood coagulation.Meanwhile,SD rat liver defect hemostasis model and full-thickness skin defect model were used to study whether the gelatin sponge loaded with exosomes effectively stopped bleeding and promoted wound healing.RESULTS The NTA,TEM,and western blot experimental results confirmed that exosomes were successfully isolated from hUC-MSCs.The gelatin sponge loaded with exosomes did not exhibit significant cell toxicity,skin irritation,or hemolysis,and they demonstrated good compatibility in SD rats.Additionally,the effectiveness of the gelatin sponge loaded with exosomes in hemostasis and wound healing was validated.The results of the coagulation index experiment indicated that the gelatin sponge loaded with exosomes had significantly better coagulation effect compared to the regular gelatin sponge,and they showed excellent hemostatic performance in a liver defect hemostasis model.Finally,the full-thickness skin defect healing experiment results showed significant improvement in the healing process of wounds treated with the gelatin sponge loaded with exosomes compared to other groups.CONCLUSION Collectively,the gelatin sponge loaded with hUC-MSCs-derived exosomes is safe and efficacious for promoting hemostasis and accelerating wound healing,warranting further clinical application.

Effect of Human Umbilical Cord Mesenchymal Stem Cells on GRP78/ATF4 Pathway in Alzheimer s Disease Model Mice

[Objectives]To study the effect of human umbilical cord mesenchymal stem cells(hUC-MSCs)on GRP78/ATF4 pathway in APP/PS1 mice.[Methods]Twelve 6-month-old female APP/PS1 mice were randomly divided into model group(MOD,n=6)and human umbilical cord mesenchymal stem cell treatment group(MSC,n=6);six 6-month-old C57BL/6N mice were used as control group(CON,n=6).The mice in each group were treated with the fourth generation of human umbilical cord mesenchymal stem cells through tail vein.Four weeks later,the mice in each group were killed.The expression of GFP78 and ATF4 in the cortex of mice in each group was detected by Western blotting and real-time fluorescence quantitative PCR.[Results]The results of immunoblotting and real-time fluorescence quantitative PCR showed that the expression of GRP78 in MOD group was lower than that in CON group and the expression of ATF4 increased.The expression of GRP78 protein in MSC group was higher than that in MOD group,but the expression of ATF4 protein was lower.The results of real-time fluorescence quantitative PCR showed that the mRNA level of GRP78 decreased and the mRNA level of ATF4 increased in MOD group compared with CON group.The mRNA level of GRP78 in MSC group was higher than that in MOD group,while the mRNA level of ATF4 in MSC group was lower than that in MOD group.[Conclusions]Human umbilical cord mesenchymal stem cells can regulate the expression of GRP78/ATF4 pathway in APP/PSI mice,which may be related to the stress level of endoplasmic reticulum in the brain of APP/PS1 mice mediated by human umbilical cord mesenchymal stem cells.

WJSC 6^(th) Anniversary Special Issues(2):Mesenchymal stem cells Umbilical cord-derived mesenchymal stem cells:Their advantages and potential clinical utility

Human umbilical cord(UC)is a promising source of mesenchymal stem cells(MSCs).Apart from their prominent advantages,such as a painless collection procedure and faster self-renewal,UC-MSCs have shown the ability to differentiate into three germ layers,to accumulate in damaged tissue or inflamed regions,to promote tissue repair,and to modulate immune response.There are diverse protocols and culture methods for the isolation of MSCs from the various compartments of UC,such as Wharton’s jelly,vein,arteries,UC lining and subamnion and perivascular regions.In this review,we give a brief introduction to various compartments of UC as a source of MSCs and emphasize the potential clinical utility of UC-MSCs for regenerative medicine and immunotherapy.

Therapeutic and regenerative potential of different sources of mesenchymal stem cells for cardiovascular diseases

Mesenchymalstemcells(MSCs)areidealcandidatesfortreatingmanycardiovasculardiseases.MSCscanmodify the internal cardiac microenvironment to facilitate their immunomodulatory and differentiation abilities,which are essential to restore heart function.MSCs can be easily isolated from different sources,including bone marrow,adipose tissues,umbilical cord,and dental pulp.MSCs from various sources differ in their regenerative and therapeutic abilities for cardiovascular disorders.In this review,we will summarize the therapeutic potential of each MSC source for heart diseases and highlight the possible molecular mechanisms of each source to restore cardiac function.

Transplantation of mesenchymal stem cells from human umbilical cord versus human umbilical cord blood for peripheral nerve regeneration

BACKGROUND： Mesenchymal stem cells （MSCs） appear to be a good alternative to Schwann cells in the treatment of peripheral nerve injury. Fetal stem cells, like umbilical cord blood （UCB） and umbilical cord （UC） stem cells, have several advantages over adult stem cells. OBJECTIVE： To assess the effects of UC-derived MSCs （UCMSCs） and UCB-derived MSCs （UCBMSCs） in repair of sciatic nerve defects. DESIGN, TIME AND SETTING： A randomized controlled animal experiment was performed at the laboratory of Department of Oral and Maxillofacial Surgery, Seoul National University Dental Hospital, from July to December 2009. MATERIALS： UCMSCs were provided by the Research Institute of Biotechnology, Dongguk University. UCBMSCs were provided by the Laboratory of Stem Cells and Tumor Biology, College of Veterinary Medicine, Seoul National University. Dulbecco＇s modified Eagle＇s medium （DMEM） was purchased from Gibco-BRL, USA. METHODS： Seven-week-old Sprague-Dawley rats were randomly and evenly divided into three groups： DMEM, UCBMSCs, and UCMSCs. A 10-mm defect in the left sciatic nerve was constructed in all rats. DMEM （15 μL） containing 1×10^6 UCBMSCs or UCMSCs was injected into the gap between nerve stumps, with the surrounding epineurium as a natural conduit. For the DMEM group, simple DMEM was injected. MAIN OUTCOME MEASURES： At 7 weeks after sciatic nerve dissection, dorsal root ganglia neurons were labeled by fluorogold retrograde labeling. At 8 weeks, electrophysiology and histomorphometry were performed. At 2, 4, 6, and 8 weeks after surgery, sciatic nerve function was evaluated using gait analysis. RESULTS： The UCBMSCs group and the UCMSCs group exhibited similar sciatic nerve function and electrophysiological indices, which were better than the DMEM group, as measured by gait analysis （P 〈 0.05）. Fluorogold retrograde labeling of sciatic nerve revealed that the UCBMSCs group demonstrated a higher number of labeled neurons; however, the differences were not significant. Histomorphometric indices were similar in the UCBMSCs and UCMSCs groups, and total axon counts, particularly axon density （P 〈 0.05）, were significantly greater in the UCBMSCs and UCMSCs groups than in the DMEM group. CONCLUSION： Transplanting either UCBMSCs or UCMSCs into axotomized sciatic nerves could accelerate and promote sciatic nerve regeneration over 8 weeks. Both treatments had similar effects on nerve regeneration.

Differentiation of isolated human umbilical cord mesenchymal stem cells into neural stem cells

AIM：To investigate whether umbilical cord human mesenchymal stem cell（UC-MSC）was able to differentiate into neural stem cell and neuron.·METHODS：The umbilical cords were o btained from pregnant women with their written consent and the approval of the Clinic Ethnics Committee.UC-MSC were isolated by adherent culture in the medium contains 20%fetal bovine serum（FBS）,then they were maintained in the medium contain 10%FBS and induced to neural cells in neural differentiation medium.We investigated whether UC-MSC was able to differentiate into neural stem cell and neuron by using flow cytometry,reverse transcriptase-polymerase chain reaction（RT-PCR）and immunofluorescence（IF）analyzes.·R ESULTS：A substantial number of UC-MSC was harvested using the tissue explants adherent method at about 2wk.Flow cytometric study revealed that these cells expressed common markers of MSCs,such as CD105（SH2）,CD73（SH3）and CD90.After induction of differentiation of neural stem cells,the cells began to form clusters;RT-PCR and IF showed that the neuron specific enolase（NSE）and neurogenic differentiation 1-positive cells reached 87.3%±14.7%and 72.6%±11.8%,respectively.Cells showed neuronal cell differentiation after induced,including neuron-like protrusions,plump cell body,obviously and stronger refraction.RT-PCR and IF analysis showed that microtubule-associated protein 2（MAP2）and nuclear factor-M-positive cells reached 43.1%±10.3%and 69.4%±19.5%,respectively.·CONCLUSION：Human umbilical cord derived MSCs can be cultured and proliferated and differentiate into neural stem cells,which may be a valuable source for cell therapy of neurodegenerative eye diseases.

Experimental treatment of radiation pneumonitis with human umbilical cord mesenchymal stem cells

Objective:To evaluate of the curative effect of human umbilical cord mesenchymal stem cells(hUC-MSCs)on rat acute radiation pneumonitis.Methods:Fourty rats were randomly divided into control group,radiation group,stem cell prevention group,stem cell treatment group and prednisone treatment group.All rats except those in the control group were radiated with X ray to establish the acute radiation pneumonitis damage model.The hUC-MSCs cultured in vitro was administrated to the rats of the prevention group via tail vein(1×10~6 cells/kg BW)24 h before the radiation,while the same administration was performed in the rats of the treatment group 24 h after the radiation.After 24 h post the radiation,the rats in tbe radiation group were given 0.4 mL physiological saline,and those in the prednisone group were given 1 mg/kg prednisone.All rats were,observed and executed 72 h after the radiation to defect lung histological changes.Results:After the administration of hUC-MSCs,the survival status of the rats in the prevention group and treatment group was obviously better than that in the control group.As shown by the histological staining,the morphology,proliferation activity aad bronchial state of lung tissues were better in the prevention group and treatment group than in the control group.Conclusion:The hUC-MSCs have definite therapeutic effects on acute radiation pneumonitis in rats.

An experimental study of preventing and treating acute radioactive enteritis with human umbilical cord mesenchymal stem cells

Objective:To test the curative effect of human umbilical cord-derived mesenchymal stem cells on rat acute radioactive enteritis and thus in provide clinical therapeutic basis for radiation sickness.Methods:Human umbilical cord-derived mesenchymal stem cells were cultivated in vitro and the model of acute radioactive enteritis of rats was established.Then,the umbilical cord mesenchymal stem cells were injected into the rats via tail vein.Visual and histopathiological changes of the experimental rats were observed.Results:After the injection,the rats in the prevention group and treatment group had remarkably better survival status than those in the control group.The histological observations revealed that the former also had better intestinal mucosa structure,more regenerative cells and stronger proliferation activity than the latter.Conclusions:Human umbilical cord-derived mesenchymal stem cells have a definite therapeutic effect on acute radioactive enteritis in rats.

Collagen scaffold combined with human umbilical cord-mesenchymal stem cells transplantation for acute complete spinal cord injury

Currently, there is no effective strategy to promote functional recovery after a spinal cord injury. Collagen scaffolds can not only provide support and guidance for axonal regeneration, but can also serve as a bridge for nerve regeneration at the injury site. They can additionally be used as carriers to retain mesenchymal stem cells at the injury site to enhance their effectiveness. Hence, we hypothesized that transplanting human umbilical cord-mesenchymal stem cells on collagen scaffolds would enhance healing following acute complete spinal cord injury. Here, we test this hypothesis through animal studies and a phase I clinical trial.(1) Animal experiments: Models of completely transected spinal cord injury were established in rats and canines by microsurgery. Mesenchymal stem cells derived from neonatal umbilical cord tissue were adsorbed onto collagen scaffolds and surgically implanted at the injury site in rats and canines;the animals were observed after 1 week–6 months. The transplantation resulted in increased motor scores, enhanced amplitude and shortened latency of the motor evoked potential, and reduced injury area as measured by magnetic resonance imaging.(2) Phase I clinical trial: Forty patients with acute complete cervical injuries were enrolled at the Characteristic Medical Center of Chinese People's Armed Police Force and divided into two groups. The treatment group(n = 20) received collagen scaffolds loaded with mesenchymal stem cells derived from neonatal umbilical cordtissues;the control group(n = 20) did not receive the stem-cell loaded collagen implant. All patients were followed for 12 months. In the treatment group, the American Spinal Injury Association scores and activities of daily life scores were increased, bowel and urinary functions were recovered, and residual urine volume was reduced compared with the pre-treatment baseline. Furthermore, magnetic resonance imaging showed that new nerve fiber connections were formed, and diffusion tensor imaging showed that electrophysiological activity was recovered after the treatment. No serious complication was observed during follow-up. In contrast, the neurological functions of the patients in the control group were not improved over the follow-up period. The above data preliminarily demonstrate that the transplantation of human umbilical cord-mesenchymal stem cells on a collagen scaffold can promote the recovery of neurological function after acute spinal cord injury. In the future, these results need to be confirmed in a multicenter, randomized controlled clinical trial with a larger sample size. The clinical trial was approved by the Ethics Committee of the Characteristic Medical Center of Chinese People's Armed Police Force on February 3, 2016(approval No. PJHEC-2016-A8). All animal experiments were approved by the Ethics Committee of the Characteristic Medical Center of Chinese People's Armed Police Force on May 20, 2015(approval No. PJHEC-2015-D5).

Stem cell transplantation for treatment of cerebral ischemia in rats Effects of human umbilical cord blood stem cells and human neural stem cells

BACKGROUND： Exogenous neural stem cell transplantation promotes neural regeneration. However, various types of stem cells transplantation outcomes remain controversial. OBJECTIVE： To explore distribution, proliferation and differentiation of human neural stem cells （hNSCs） and human umbilical cord blood stem cells （hUCBSCs） following transplantation in ischemic brain tissue of rats, and to compare therapeutic outcomes between hNSCs and hUCBSCs. DESIGN, TIME AND SETTING： Randomized controlled animal studies were performed at the Experimental Animal Center of Nanjing Medical University and Central Laboratory of Second Affiliated Hospital of Nanjing Medical University of China from September 2008 to April 2009. MATERIALS： hNSCs were harvested from brain tissue of 10 13 week old fetuses following spontaneous abortion, and hUCBSCs were collected from umbilical cord blood of full-term newborns at the Second Affiliated Hospital of Nanjing Medical University of China. hNSCs and hUCBSCs were labeled by 5-bromodeoxyuridine （BrdU） prior to transplantation. METHODS： Rat models of cerebral ischemia were established by the suture method. A total of 60 healthy male Sprague Dawley rats aged 7-9 weeks were randomly assigned to hNSC transplantation, hUCBSC transplantation and control groups. The rat models in the hNSC transplantation, hUCBSC transplantation and control groups were infused with hNSC suspension, hUCBSC suspension and saline via the caudal vein, respectively. MAIN OUTCOME MEASURES： The distribution, proliferation and differentiation of hNSCs and hUCBSCs in ischemic brain tissue were observed using immunohistochemical methods. Neurological function in rats was assessed using the neurological severity score. RESULTS： The number of BrdU-positive cells was significantly greater in the hNSC transplantation group compared with hUCBSC transplantation group at 14 days following transplantation （P 〈 0.05） The number of BrdU-positive cells reached a peak at 28 days following transplantation. Nestin-positive, glial fibrillary acidic protein-positive, cyclic nucleotide 3＇ phosphohydrolase-positive and neuron specific enolase-positive cells were visible following transplantation. No significant difference was determined in the constituent ratio of various cells between hNSC and hUCBSC transplantation groups （P 〉 0.05）. The neurological severity score was significantly decreased in rats at 21 days following transplantation （P 〈 0.05）. No significant difference was detected in neurological severity score between hNSC and hUCBSC transplantation groups at various time points （P 〉 0.05）. CONCLUSION： The transplanted hNSCs and hUCBSCs can migrate into ischemic brain tissue, proliferate and differentiate into neuron-like, astrocyte-like and oligodendrocyte-like cells, and improve neurological function in rats with cerebral ischemia.

Ability of human umbilical cord mesenchymal stem cells to repair chemotherapy-induced premature ovarian failure

BACKGROUND Premature ovarian insufficiency(POI)and premature ovarian failure(POF)have become one of the major problems threatening women of childbearing age.Studies have shown that stem cells transplanted from bone marrow,umbilical cord,peripheral blood and amniotic fluid can migrate and proliferate to the ovary,promote ovarian function repair,increase the number of follicles and granulosa cells at all levels of ovary,improve endocrine function,and can differentiate into oocytes in specific ovarian environment to restore fertility to some extent.AIM To study the ability of human umbilical cord mesenchymal stem cells(hUCMSCs)to repair ovarian injury after chemotherapy.METHODS A total of 110 female BALB/c mice(aged 7-8 wk old)with body masses of 16.0-20.0 g were selected.The mice were fed until 12 wk of age,and cyclophosphamide was administered by intraperitoneal injection for 14 consecutive days to induce premature ovarian failure in mice.Seventy-five mice with estrous cycle disorder were screened and randomly divided into 3 groups according to their body weight:model group,positive control group and hUCMSC group,and each group had 25 mice.Another 25 mice were used as negative controls.The mice in the hUCMSC group were injected with hUCMSCs in the tail vein,and the mice in the positive control group were given an oestradiol valerate solution and a medroxyprogesterone acetate solution in the tail vein.On the 1^st,15^th,30^th,45^th,and 60^th days after intravenous administration,vaginal smears were made to monitor the estrous cycles of the mice.The ovaries were weighed,and pathological sections were made to observe the morphology of the follicles;blood samples were collected to monitor the concentration of sex hormones(oestradiol and follicle-stimulating hormone).RESULTS The estrous cycles of the model group mice were disrupted throughout the experiment.Mice in the hUCMSC group and the positive control group resumed normal estrous cycles.The ovarian weight of the model group mice continued to decline.The ovarian weight of the hUCMSC group mice and the positive control group mice decreased first and then gradually increased,and the ovarian weight of the hUCMSC group mice was heavier than that of the positive control group mice.The difference was statistically significant(P<0.05).Compared with the negative control group,the model group experienced a decrease in oestradiol and an increase in follicle-stimulating hormone,and the difference was statistically significant(P<0.05).Compared with the model group,the hUCMSC and positive control groups experienced a slight increase in oestradiol and a decrease in follicle-stimulating hormone;the difference was statistically significant(P<0.05).The pathological examination revealed that the mouse ovaries from the model group were atrophied,the volume was reduced,the cortical and medullary structures were disordered,the number of follicles at all stages was significantly reduced,the number of atretic follicles increased,the number of primordial follicles and corpus luteum significantly decreased,and the corpus luteum had an irregular shape.Compared with those of the model group,the lesions of the hUCMSC and positive control groups significantly improved.CONCLUSION hUCMSCs can repair ovarian tissue damaged by chemotherapy to a certain extent,can improve the degree of apoptosis in ovarian tissue,and can improve the endocrine function of mouse ovaries.

Human umbilical cord mesenchymal stem cells to treat spinal cord injury in the early chronic phase: study protocol for a prospective, multicenter, randomized, placebo-controlled, single-blinded clinical trial

Human umbilical cord mesenchymal stem cells(hUC-MSCs)support revascularization,inhibition of inflammation,regulation of apoptosis,and promotion of the release of beneficial factors.Thus,they are regarded as a promising candidate for the treatment of intractable spinal cord injury(SCI).Clinical studies on patients with early chronic SCI(from 2 months to 1 year post-injury),which is clinically common,are rare;therefore,we will conduct a prospective,multicenter,randomized,placebo-controlled,single-blinded clinical trial at the Third Affiliated Hospital of Sun Yat-sen University,West China Hospital of Sichuan University,and Shanghai East Hospital,Tongji University School of Medicine,China.The trial plans to recruit 66 early chronic SCI patients.Eligible patients will undergo randomization at a 2:1 ratio to two arms:the observation group and the control group.Subjects in the observation group will receive four intrathecal transplantations of stem cells,with a dosage of 1×106/kg,at one calendar month intervals.Subjects in the control group will receive intrathecal administrations of 10 mL sterile normal saline in place of the stem cell transplantations.Clinical safety will be assessed by the analysis of adverse events and laboratory tests.The American Spinal Injury Association(ASIA)total score will be the primary efficacy endpoint,and the secondary efficacy outcomes will be the following:ASIA impairment scale,International Association of Neural Restoration-Spinal Cord Injury Functional Rating Scale,muscle tension,electromyogram,cortical motor and cortical sensory evoked potentials,residual urine volume,magnetic resonance imaging–diffusion tensor imaging,T cell subtypes in serum,neurotrophic factors and inflammatory factors in both serum and cerebrospinal fluid.All evaluations will be performed at 1,3,6,and 12 months following the final intrathecal administration.During the entire study procedure,all adverse events will be reported as soon as they are noted.This trial is designed to evaluate the clinical safety and efficacy of subarachnoid transplantation of hUC-MSCs to treat early chronic SCI.Moreover,it will establish whether cytotherapy can ameliorate local hostile microenvironments,promote tracking fiber regeneration,and strengthen spinal conduction ability,thus improving overall motor,sensory,and micturition/defecation function in patients with early chronic SCI.This study was approved by the Stem Cell Research Ethics Committee of the Third Affiliated Hospital of Sun Yat-sen University,China(approval No.[2018]-02)on March 30,2018,and was registered with ClinicalTrials.gov(registration No.NCT03521323)on April 12,2018.The revised trial protocol(protocol version 4.0)was approved by the Stem Cell Research Ethics Committee of the Third Affiliated Hospital of Sun Yat-sen University,China(approval No.[2019]-10)on February 25,2019,and released on ClinicalTrials.gov on April 29,2019.

Human umbilical cord mesenchymal stem cells ameliorate liver fibrosis in vitro and in vivo: From biological characteristics to therapeutic mechanisms

Liver fibrosis is a wound-healing response to chronic injuries, characterized by the excessive accumulation of extracellular matrix or scar tissue within the liver;in addition, its formation is associated with multiple cytokines as well as several cell types and a variety of signaling pathways. When liver fibrosis is not well controlled, it can progress to liver cirrhosis, but it is reversible in principle. Thus far, no efficient therapy is available for treatment of liver fibrosis. Although liver transplantation is the preferred strategy, there are many challenges remaining in this approach, such as shortage of donor organs, immunological rejection, and surgical complications. Hence, there is a great need for an alternative therapeutic strategy. Currently, mesenchymal stem cell (MSC) therapy is considered a promising therapeutic strategy for the treatment of liver fibrosis;advantageously, the characteristics of MSCs are continuous self-renewal, proliferation, multipotent differentiation, and immunomodulatory activities. The human umbilical cord-derived (hUC)-MSCs possess not only the common attributes of MSCs but also more stable biological characteristics, relatively easy accessibility, abundant source, and no ethical issues (e.g., bone marrow being the adult source), making hUC-MSCs a good choice for treatment of liver fibrosis. In this review, we summarize the biological characteristics of hUC-MSCs and their paracrine effects, exerted by secretion of various cytokines, which ultimately promote liver repair through several signaling pathways. Additionally, we discuss the capacity of hUC-MSCs to differentiate into hepatocyte-like cells for compensating the function of existing hepatocytes, which may aid in amelioration of liver fibrosis. Finally, we discuss the current status of the research field and its future prospects.

Migration capacity of human umbilical cord mesenchymal stem cells towards glioma in vivo

High-grade glioma is the most common malignant primary brain tumor in adults. The poor prognosis of glioma, combined with a resistance tQ currently available treatments, necessitates the develop- ment of more effective tumor-selective therapies. Stem cell-based therapies are emerging as novel cell-based delivery vehicle for therapeutic agents. In the present study, we successfully isolated human umbilical cord mesenchymal stem cells by expiant culture. The human umbilical cord mes- enchymal stem cells were adherent to plastic surfaces, expressed specific surface phenotypes of mesenchymal stem cells as demonstrated by flow cytometry, and possessed multi-differentiation potentials in permissive induction media in vitro. Furthermore, human umbilical cord mesenchymal stem cells demonstrated excellent glioma-specific targeting capacity in established rat glioma models after intratumoral injection or contralateral ventricular administration in vivo. The excellent glioma-specific targeting ability and extensive intratumoral distribution of human umbilical cord mesenchymal stem cells indicate that they may serve as a novel cellular vehicle for delivering therapeutic molecules in glioma therapy.