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.

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.

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.

Human umbilical cord mesenchymal stem cells derivedexosomes on VEGF-A in hypoxic-induced mice retinal astrocytes and mice model of retinopathy of prematurity

AIM:To observe the effect of human umbilical cord mesenchymal stem cells(hUCMSCs)secretions on the relevant factors in mouse retinal astrocytes,and to investigate the effect of hUCMSCs on the expression of vascular endothelial growth factor-A(VEGF-A)and to observe the therapeutic effect on the mouse model of retinopathy of prematurity(ROP).METHODS:Cultured hUCMSCs and extracted exosomes from them and then retinal astrocytes were divided into control group and hypoxia group.MTT assay,flow cytometry,reverse transcription-polymerase chain reaction(RT-PCR)and Western blot were used to detect related indicators.Possible mechanisms by which hUCMSCs exosomes affect VEGF-A expression in hypoxia-induced mouse retinal astrocytes were explored.At last,the efficacy of exosomes of UCMSCs in a mouse ROP model was explored.Graphpad6 was used to comprehensively process data information.RESULTS:The secretion was successfully extracted from the culture supernatant of hUCMSCs by gradient ultracentrifugation.Reactive oxygen species(ROS)and hypoxia inducible factor-1α(HIF-1α)of mice retinal astrocytes under different hypoxia time and the expression level of VEGF-A protein and VEGF-A mRNA increased,and the ROP cell model was established after 6h of hypoxia.The secretions of medium and high concentrations of hUCMSCs can reduce ROS and HIF-1α,the expression levels of VEGF-A protein and VEGF-A mRNA are statistically significant and concentration dependent.Compared with the ROP cell model group,the expression of phosphatidylinositol 3-kinase(PI3K)/protein kinase B(AKT)/mammalian target of rapamycin(mTOR)signal pathway related factors in the hUCMSCs exocrine group is significantly decreased.The intravitreal injection of the secretions of medium and high concentrations of hUCMSCs can reduce VEGF-A and HIF-1αin ROP model tissues.HE staining shows that the number of retinal neovascularization in ROP mice decreases with the increase of the dose of hUCMSCs secretion.CONCLUSION:In a hypoxia induced mouse retinal astrocyte model,hUCMSCs exosomes are found to effectively reduce the expression of HIF-1αand VEGF-A,which are positively correlated with the concentration of hUCMSCs exosomes.HUCMSCs exosomes can effectively reduce the number of retinal neovascularization and the expression of HIF-1αand VEGF-A proteins in ROP mice,and are positively correlated with drug dosage.Besides,they can reduce the related factors on the PI3K/AKT/mTOR signaling pathway.

Neuronal-like differentiation of single versus multiple treatments with human amnion-derived mesenchymal stem cells induced by basic fibroblast growth factor

BACKGROUND： Cultures from multiple portions of umbilical cord blood mesenchymal stem cells have been shown to undergo more rapid proliferation and attachment than single portions. OBJECTIVE： To observe growth of basic fibroblast growth factor （bFGF）-induced cultures of human amnion-derived mesenchymal stem cells （AMSCs） and differentiation into neuronal-like cells. DESIGN, TIME AND SETTING： Comparative observation. The study was performed at the Laboratory of Microbiology and Immunology, Basic Medical School of Zhengzhou University from January to May 2008. METHODS： Amnia from full-term, uterine-incision delivery were donated by 12 healthy women. AMSCs were obtained by cell separation and culture techniques, and were passaged and induced by bFGF. From the third passage, a total of 1 mLAMSCs, at a density of 1.0 × 10^4/mL, was separately harvested from six samples, which served as group A. A total of 1 mL AMSCs, at a density of 1.0 × 10^4/mL, was harvested separately from the remaining six samples, which served a group B. A total of 0.5 mL from the six samples of group A and 0.5 mL from the six samples of grot, B were combined to form group C. MAIN OUTCOME MEASURES： Differences in cell quantity among the three groups were compare by cell quantification and 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide （MTT） analysis. Expression of a glial cell marker, neuron-specific enolase, and nestin was detected in the three groups by immunocytochemistry. RESULTS： Cell quantification and MTT analysis of live cells, as well as AMSC absorbance, were significantly greater in group C compared with groups A and B at 18 days of culture （P 〈 0.05）, anc no significant difference was observed between groups A and B. Glial fibrillary acidic protein, neuron-specific enolase, and nestin were expressed in all groups following bFGF induction. CONCLUSION： Mixed AMSC cultures promoted proliferation, and bFGF-induced AMSCs differentiated into neuronal-like cells.

Human umbilical cord mesenchymal stem cells as treatment of adjuvant rheumatoid arthritis in a rat model

AIM:To investigate the effect of human umbilical cord stem cells,both mesenchymal and hematopoietic(CD34+),in the treatment of arthritis.METHODS:Mesenchymal stem cells(MSCs) and hematopoietic(CD34+) stem cells(HSC) were isolated from human umbilical cord blood obtained from the umbilical cord of healthy pregnant donors undergoing fullterm normal vaginal delivery.MSC,HSC,methotrexate(MTX) and sterile saline were injected intra-articularly into the rat hindpaw with complete freunds adjuvant(CFA) induced arthritis after the onset of disease(day 34),when arthritis had become well established(arthritis score ≥ 2).Arthritic indices were evaluated and the levels of interleukin(IL)-1,tumor necrosis factor(TNF)-α and interferon(IFN)-γ and anti-inflammatory cytokine IL-10 in serum were determined using enzyme-linked immunosorbent assay.Animals of all groups were sacrificed 34 d after beginning treatment,except positive control(PC) which was sacrificed at 10,21 and 34 d for microscopic observation of disease progression.We used hematoxylin,eosin and Masson's trichrome stains for histopathological examination of cartilage and synovium.RESULTS:The mean arthritis scores were similar in all groups at 12 and 34 d post immunization,with no statistical significant difference.Upon the injection of stem cells(hematopoietic and mesenchymal),the overall arthritis signs were significantly improved around 21 d after receiving the injection and totally disappeared at day 34 post treatment in MSC group.Mean hindpaw diameter(mm) in the MSC rats was about half that of the PC and MTX groups(P = 0.007 and P = 0.021,respectively) and 0.6 mm less than the HSC group(P = 0.047),as indicated by paw swelling.Associated with these findings,serum levels of TNF-α,IFN-γ and IL-1 decreased significantly in HSC and MSC groups compared to PC and MTX groups(P < 0.05),while the expression of IL-10 was increased.Histopathological examination with H and E stain revealed that the MTX treated group showed significant reduction of leucocytic infiltrate and hypertrophy of the synovial tissue with moderate obliteration of the joint cavity.Stem cells treated groups(both hematopoietic CD34+ and mesenchymal),showed significant reduction in leucocytic infiltrate and hypertrophy of the synovial tissue with mild obliteration of the joint cavity.With Masson's trichrome,stain sections from the PC group showed evidence of vascular edema of almost all vessels within the synovium in nearly all arthritic rats.Vacuoles were also visible in the outer vessel wall.The vessel became hemorrhagic and finally necrotic.In addition,there was extensive fibrosis completely obliterating the joint cavity.The mean color area percentage of collagen in this group was 0.324 ± 0.096,which was significantly increased when compared to the negative control group.The mean color area percentage of collagen in hematopoietic CD34+ and mesenchymal groups was 0.176 ± 0.0137 and 0.174 ± 0.0197 respectively,which showed a marked decrement compared to the PC group,denoting a mild increase in synovial tissue collagen fibers.CONCLUSION:MSC enhance the efficacy of CFAinduced arthritis treatment,most likely through the modulation of the expression of cytokines and amelioration of pathological changes in joints.

Neuroprotective effects of human bone marrow mesenchymal stem cells against cerebral ischemia are mediated in part by an anti-apoptotic mechanism

Transplantation of human bone marrow mesenchymal stem cells(hMSCs) stands as a potent stroke therapy, but its exact mechanism remains unknown. This study investigated the anti-apoptotic mechanisms by which hMSCs exert neuroprotective effects on cerebral ischemia. Primary mixed cultures of rat neurons and astrocytes were cultured and exposed to oxygen-glucose deprivation. A two-hour period of "reperfusion" in standard medium and normoxic conditions was allowed and immediately followed by hMSCs and/or Bcl-2 antibody treatment. Cell viability of primary rat neurons and astrocytes was determined by 3-(4,5-dimethylthianol-2-yl)-2,5 diphenyl tetrazolium bromide and trypan blue exclusion methods. hMSC survival and differentiation were characterized by immunocytochemistry, while the concentration of Bcl-2 in the supernatant was measured by enzyme-linked immunosorbent assay to reveal the secretory anti-apoptotic function of hMSCs. Cultured hMSCs expressed embryonic-like stem cell phenotypic markers CXCR4, Oct4, SSEA4, and Nanog, as well as immature neural phenotypic marker Nestin. Primary rat neurons and astrocytes were protected from oxygen-glucose deprivation by hMSCs, which was antagonized by the Bcl-2 antibody. However, Bcl-2 levels in the supernatants did not differ between hMSCand non-treated cells exposed to oxygen-glucose deprivation. Neuroprotective effects of hMSCs against cerebral ischemia were partially mediated by the anti-apoptotic mechanisms. However, further studies are warranted to fully elucidate this pathway.

Magnetic resonance evaluation of human mesenchymal stem cells in corpus cavernosa of rats and rabbits

Aim： To investigate whether the biological process of superparamagnetic iron oxide （SPIO）-labeled human mesenchymal stem cells （hMSCs） may be monitored non-invasively by using in vivo magnetic resonance （MR） imaging with conventional 1.5-T system examinations in corpus cavernosa of rats and rabbits. Methods： The labeling efficiency and viability of SP10-labeled hMSCs were examined with Prussian blue and Tripan blue, respectively. After SPIO-labeled hMSCs were transplanted to the corpus cavernosa of rats and rabbits, serial T2-weighted MR images were taken and histological examinations were carried out over a 4-week period. Results： hMSCs loaded with SPIO compared to unlabeled cells had a similar viability. For SPIO-labeled hMSCs more than lx 105 concentration in vitro, MR images showed a decrease in signal intensity. MR signal intensity at the areas of SPIO-labeled hMSCs in the rat and rabbit corpus cavernosa decreased and was confined locally. After injection of SPIO-labeled hMSCs into the corpus cavernosum, MR imaging demonstrated that hMSCs could be seen for at least 12 weeks after injection. The presence of iron was confirmed with Prussian blue staining in histological sections. Conclusion： SP10-labeled hMSCs in corpus cavernosa of rats and rabbits can be evaluated non-invasively by molecular MR imaging. Our findings suggest that MR imaging has the ability to test the long-term therapeutic potential of hMSCs in animals in the setting of erectile dysfunction.

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.

Human amnion tissue injected with human umbilical cord mesenchymal stem cells repairs damaged sciatic nerves in rats

Human umbilical cord mesenchymal stem cells,incorporated into an amnion carrier tubes,were assessed for nerve regeneration potential in a rat nerve defect model.Damaged nerves were exposed to human amnion carriers containing either human umbilical cord mesenchymal stem cell （cell transplantation group）or saline（control group）.At 8,12,16 and 20 weeks after cell implantation,the sciatic functional index was higher in the cell transplantation group compared with the control group.Furthermore,electrophysiological examination showed that threshold stimulus and maximum stimulus intensity gradually decreased while compound action potential amplitude gradually increased.Hematoxylin-eosin staining showed that regenerating nerve fibers were arranged in nerve tracts in the cell transplantation group and connective tissue between nerve tracts and amnion tissue reduced over time.Gastrocnemius muscle cell diameter,wet weight and restoration ratio were increased.These data indicate that transplanted human umbilical cord mesenchymal stem cells,using the amnion tube connection method,promote restoration of damaged sciatic nerves in rats.

HNF-4α determines hepatic differentiation of human mesenchymal stem cells from bone marrow

AIM: To investigate the differentiation status and key factors to facilitate hepatic differentiation of human bone-marrow-derived mesenchymal stem cells (MSCs). METHODS: Human MSCs derived from bone marrow were induced into hepatocyte-like cells following a previously published protocol. The differentiation status of the hepatocyte-like cells was compared with various human hepatoma cell lines. Overexpression of hepatocyte nuclear factor (HNF)-4α was mediated by adenovirus infection of these hepatocyte-like cells. The expression of interesting genes was then examined by either re-verse transcription-polymerase chain reaction (RT-PCR) or real-time RT-PCR methods. RESULTS: Our results demonstrated that the differentiation status of hepatocyte-like cells induced from human MSCs was relatively similar to poorly differentiated human hepatoma cell lines. Interestingly, the HNF-4 isoform in induced MSCs and poorly differentiated human hepatoma cell lines was identified as HNF4γ instead of HNF-4α. Overexpression of HNF-4α in induced MSCs significantly enhanced the expression level of hepatic-specific genes, liver-enriched transcription factors, and cytochrome P450 (P450) genes. CONCLUSION: Overexpression of HNF-4α improves the hepatic differentiation of human MSCs from bone marrow and is a simple way of providing better cell sources for clinical applications.

5-Azacytidine induces changes in electrophysiological properties of human mesenchymal stem cells

Previously, mouse bone marrow-derived stem cells （MSC） treated with the unspecific DNA methyltransferase inhibitor 5-azacytidine were reported to differentiate into cardiomyocytes. The aim of the present study was to investigate the efficiency of a similar differentiation strategy in human mononuclear cells obtained from healthy bone marrow donors. After 1-3 passages, cultures were exposed for 24 h to 5-azacytidine （3 μM） followed by 6 weeks of further culture. Drug treatment did not induce expression of myogenic marker MyoD or cardiac markers Nkx2.5 and GATA-4 and did not yield beating cells during follow-up. In patch clamp experiments, approximately 10-15% of treated and untreated cells exhibited L-type Ca^2＋ currents. Almost all cells showed outwardly rectifying K^＋ currents of rapid or slow activation kinetics. Mean current amplitude at ＋60 mV doubled after 6 weeks of treatment compared with time-matched controls. Membrane capacitance of treated cells was significantly larger than in controls 2 weeks after treatment and remained high after 6 weeks, Expression levels of mRNAs for the K^＋ channels Kv 1,1, Kv 1,5, Kv2,1, Kv4,3 and KCNMA 1 and for the Ca^2＋ channel Cav 1.2 were not affected by 5-azacytidine. Treatment with potassium channel blockers tetraethylammonium and clofilium at concentrations shown previously to inhibit rapid or slowly activating K^＋ currents of hMSC inhibited proliferation of these cells. Our results suggest that despite the absence of differentiation ofhMSC into cardiomyocytes, treatme.nt with 5-azacytidine caused profound changes in current density.

Neural differentiation of human Wharton＇s jelly-derived mesenchymal stem cells improves the recovery of neurological function after transplantation in ischemic stroke rats

Human Wharton＇s jelly-derived mesenchymal stem cells（h WJ-MSCs）have excellent proliferative ability,differentiation ability,low immunogenicity,and can be easily obtained.However,there are few studies on their application in the treatment of ischemic stroke,therefore their therapeutic effect requires further verification.In this study,h WJ-MSCs were transplanted into an ischemic stroke rat model via the tail vein 48 hours after transient middle cerebral artery occlusion.After 4 weeks,neurological functions of the rats implanted with h WJ-MSCs were significantly recovered.Furthermore,many h WJ-MSCs homed to the ischemic frontal cortex whereby they differentiated into neuron-like cells at this region.These results confirm that h WJ-MSCs transplanted into the ischemic stroke rat can differentiate into neuron-like cells to improve rat neurological function and behavior.

Cardiomyocyte-like differentiation of human bone marrow mesenchymal stem cells after exposure to 5-azacytidine in vitro

Objective To investigate the potential of adult mesenchymal stem cells (MSCs) derived from human bone marrow to undergo cardiomyogenic differentiation after exposure to 5-azacytidine (5-aza) in vitro. Methods A small bone marrow aspirate was taken from the iliac crest of human volunteers, and hMSCs were isolated by 1.073g/mL Percoll and propagated in the right cell culturing medium as previously described. The phenotypes of hMSCs were characterized with the use of flow cytometry. The hMSCs were cultured in cell culture medium (as control) and medium mixed with 5-aza for cellular differentiation. We examined by immunohistochemistry at 21 days the inducement of desmin, cardiac-specific cardiac troponin I (cTnI), GATA 4 and connexin-43 respectively. Results The hMSCs are fibroblast-like morphology and express CD44+ CD29+ CD90+ / CD34- CD45- CD31- CD11a. After 5-aza treatment, 20-30% hMSCs connected with adjoining cells and coalesced into myotube structures after 14days. Twenty-one days after 5-aza treatment, immunofluorescence showed that some cells expressed desmin,GATA4, cTnI and connexin-43 in 5,10 μmol/L 5-aza groups, but no cardiac specific protein was found in neither 3μmol/L 5-aza group nor in the control group. The ratio of cTnI positively stained cells in 10 μmol/L group was higher than that in 5 μmol/L group (65.3 ± 4.7% vs 48.2 ± 5.4%, P ＜ 0.05). Electron microscopy revealed that myofilaments were formed. The induced cells expressed cardiac-myosin heavy chain (MyHC) gene by reverse transcription-polymerase chain reaction (RT-PCR). Conclusions Theses findings suggest that hMSCs from adult bone marrow can be differentiated into cardiac-like muscle cells with 5-aza inducement in vitro and the differentiation is in line with the 5-aza concentration. (J Geriatr Cardiol 2004;1(2) :101-107. )

Conditions to improve expansion of human mesenchymal stem cells based on rat samples

AIM: To improve the isolation and expansion of human marrow-derived mesenchymal stem cells (MSCs) based on rat samples. METHODS: Based on the fact that rat MSCs are relatively easy to obtain from a small aspirate, bone marrow-derived MSCs from rat were cultured and characterized to set up the different protocols used in this study. Then, accordingly, almost the same protocols were performed on human healthy bone marrow samples, after obtaining approval of the ethics committee and gaining informed consent. We used different protocols and culture conditions, including the type of basal media and the culture composition. The MSCs were characterized by immunophenotyping and differentiation. RESULTS: There was no difference in morphology and proliferation capacity between different culture media at the first passage. During the 5-7th passages, the cells gradually lost their morphology and proliferation potential on Dulbecco’s modified Eagle’s medium (DMEM) high glucose and α modified Eagle’s medium. Although the cells expanded rapidly for up to 10 passages on DMEM low glucose containing 10% to 15% fetal calf serum (FCS), their proliferation was arrested without change in morphology and differentiation capacity at the third passage on 5% FCS. Flow cytometric analysis and functional tests confirmed that more than 90% of marrow cells which were isolated and expanded by our selective protocols were MSCs. CONCLUSION: We improved the isolation and expansion of human bone marrow derived MSCs, based on rat sample experiments, for further experimental and clinical use.

The genomic landscapes of histone H3-Lys9 modifications of gene promoter regions and expression profiles in human bone marrow mesenchymal stem cells

Mesenchymal stem cells （MSCs） of nonembryonic origins possess the proliferation and multi-lineage differentiation potentials. It has been established that epigenetic mechanisms could be critical for determining the fate of stem cells, and MSCs derived from different origins exhibited different expression profiles individually to a certain extent. In this study, ChiP-on-chip was used to generate genome-wide histone H3-Lys9 acetylation and dimethylation profiles at gene promoters in human bone marrow MSCs. We showed that modifications of histone H3-Lys9 at gene promoters correlated well with mRNA expression in human bone marrow MSCs. Functional analysis revealed that many key cellular pathways in human bone marrow MSC self-renewal, such as the canonical signaling pathways, cell cycle pathways and cytokine related pathways may be regulated by H3-Lys9 modifications. These data suggest that gene activation and silencing affected by H3-Lys9 acetylation and dimethylation, respectively, may be essential to the maintenance of human bone marrow MSC self-renewal and multi-potency.

Human umbilical cord blood-derived mesenchymal stem cells promote regeneration of crush-injured rat sciatic nerves

Several studies have demonstrated that human umbilical cord blood-derived mesenchymal stem cells can promote neural regeneration following brain injury. However, the therapeutic effects of human umbilical cord blood-derived mesenchymal stem cells in guiding peripheral nerve regeneration remain poorly understood. This study was designed to investigate the effects of human umbilical cord blood-derived mesenchymal stem cells on neural regeneration using a rat sciatic nerve crush injury model. Human umbilical cord blood-derived mesenchymal stem cells （1 ~ 106） or a PBS control were injected into the crush-injured segment of the sciatic nerve. Four weeks after cell injection, brain-derived neurotrophic factor and tyrosine kinase receptor B mRNA expression at the lesion site was increased in comparison to control. Furthermore, sciatic function index, Fluoro Gold-labeled neuron counts and axon density were also significantly increased when compared with control. Our results indicate that human umbilical cord blood-derived mesenchvmal stem cells promote the functinnal r~.RcJv^rv nf P.n I^h-inillr^4 ~r^i~tit, n^r~e

Enhancement of mouse germ cell-associated genes expression by injection of human umbilical cord mesenchymal stem cells into the testis of chemical-induced azoospermic mice

Various methods are currently under investigation to preserve fertility in males treated with high-dose chemotherapy and radiation for malignant and nonmalignant disorders. Human umbilical cord mesenchymal stem cells （HUC-MSCs）, which possess potent immunosuppressive function and secrete various cytokines and growth factors, have the potential clinical applications. As a potential alternative, we investigate whether injection of HUC-MSCs into the interstitial compartment of the testes to promote spermatogenic regeneration efficiently. HUC-MSCs were isolated from different sources of umbilical cords and injected into the interstitial space of one testis from 10 busulfan-treated mice （saline and HEK293 cells injections were performed in a separate set of mice） and the other testis remained uninjected. Three weeks after MSCs injection, Relative quantitative reverse transcription polymerase chain reaction was used to identify the expression of 10 of germ cell associated, which are all related to meiosis, demonstrated higher levels of spermatogenic gene expression （2-8 fold） in HUC-MSCs injected testes compared to the contralateral uninjected testes （five mice）. Protein levels for germ cell-specific genes, miwi, vasa and synaptonemal complex protein （Scp3） were also higher in MSC-treated testes compared to injected controls 3 weeks after treatment. However, no different expression was detected in saline water and HEK293 cells injection control group. We have demonstrated HUC-MSCs could affect mouse germ cell-specific genes expression. The results also provide a possibility that the transplanted HUC-MSCs may promote the recovery of spermatogenesis. This study provides further evidence for preclinical therapeutic effects of HUC-MSCs, and explores a new approach to the treatment of azoospermia.