Protective activity of adipose-derived stem cell extracellular vesicles in ischemia and/or reperfusion

Increasing evidence of the significant clinical value of protection against ischemia/reperfusion injury has contributed to the realization of the independent importance of this approach in improving prognosis and reducing cardiovascular mortality.Extracellular vesicles(EVs)derived by adipose mesenchymal stem cells may mediate the paracrine effects of stem cells and provide regenerative and anti-inflammatory properties,which are enhanced byγ-aminobutyric acid.The protective effects on cardiac myocytes may result from the EV embarked by miR-21-5p,which is a target for thioredoxin-interacting protein,regulating the formation of thioredoxin-interacting protein-thioredoxin complexes and subsequently enhancing the antioxidant activity of thioredoxin.It has been found thatγ-aminobutyric acid governs myocardial bioenergetics through suppressing inflammation and supporting mitochondrial structure.Finally,stem cell-based cell-free therapy based on adipose-derived stem cell EVs is considered a promising approach to individualized management of ischemia-induced cardiomyopathy.

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.

Outcomes of combined mitochondria and mesenchymal stem cellsderived exosome therapy in rat acute respiratory distress syndrome and sepsis

BACKGROUND The treatment of acute respiratory distress syndrome(ARDS)complicated by sepsis syndrome(SS)remains challenging.AIM To investigate whether combined adipose-derived mesenchymal-stem-cells(ADMSCs)-derived exosome(EXAD)and exogenous mitochondria(mitoEx)protect the lung from ARDS complicated by SS.METHODS In vitro study,including L2 cells treated with lipopolysaccharide(LPS)and in vivo study including male-adult-SD rats categorized into groups 1(sham-operated-control),2(ARDS-SS),3(ARDS-SS+EXAD),4(ARDS-SS+mitoEx),and 5(ARDS-SS+EXAD+mitoEx),were included in the present study.RESULTS In vitro study showed an abundance of mitoEx found in recipient-L2 cells,resulting in significantly higher mitochondrial-cytochrome-C,adenosine triphosphate and relative mitochondrial DNA levels(P<0.001).The protein levels of inflammation[interleukin(IL)-1β/tumor necrosis factor(TNF)-α/nuclear factor-κB/toll-like receptor(TLR)-4/matrix-metalloproteinase(MMP)-9/oxidative-stress(NOX-1/NOX-2)/apoptosis(cleaved-caspase3/cleaved-poly(ADP-ribose)polymerase)]were significantly attenuated in lipopolysaccharide(LPS)-treated L2 cells with EXAD treatment than without EXAD treatment,whereas the protein expressions of cellular junctions[occluding/β-catenin/zonula occludens(ZO)-1/E-cadherin]exhibited an opposite pattern of inflam-mation(all P<0.001).Animals were euthanized by 72 h post-48 h-ARDS induction,and lung tissues were harvested.By 72 h,flow cytometric analysis of bronchoalveolar lavage fluid demonstrated that the levels of inflam-matory cells(Ly6G+/CD14+/CD68+/CD11b/c+/myeloperoxidase+)and albumin were lowest in group 1,highest in group 2,and significantly higher in groups 3 and 4 than in group 5(all P<0.0001),whereas arterial oxygen-saturation(SaO2%)displayed an opposite pattern of albumin among the groups.Histopathological findings of lung injury/fibrosis area and inflammatory/DNA-damaged markers(CD68+/γ-H2AX)displayed an identical pattern of SaO2%among the groups(all P<0.0001).The protein expressions of inflammatory(TLR-4/MMP-9/IL-1β/TNF-α)/oxidative stress(NOX-1/NOX-2/p22phox/oxidized protein)/mitochondrial-damaged(cytosolic-cytochrome-C/dynamin-related protein 1)/autophagic(beclin-1/Atg-5/ratio of LC3B-II/LC3B-I)biomarkers exhibited a similar manner,whereas antioxidants[nuclear respiratory factor(Nrf)-1/Nrf-2]/cellular junctions(ZO-1/E-cadherin)/mitochondrial electron transport chain(complex I-V)exhibited an opposite manner of albumin among the groups(all P<0.0001).CONCLUSION Combined EXAD-mitoEx therapy was better than merely one for protecting the lung against ARDS-SS induced injury.

Immunomodulatory effects of mesenchymal stem cells derived from adipose tissues in a rat orthotopic liver transplantation model

BACKGROUND: Acute rejection after liver transplantation is usually treated with large doses of immunosuppressants with severe toxic and side-effects, so it is imperative to find a safe and effective method for preventing and treating rejection. This study was designed to confirm the immunomodulatory effects of rat mesenchymal stem cells (MSCs) in vitro and investigate the tolerogenic features in a rat model of allogeneic liver transplantation. METHODS: MSCs were isolated from adipose tissue of Sprague-Dawley (SD) rats and cultured. In vitro, MSCs were added into a mixed lymphocyte culture (MLC) system to study the inhibitory effects of MSCs on the proliferation of T lymphocytes in Wistar rats. By using SD and Wistar rats as liver donors and recipients, an orthotopic liver transplantation model was established and the rats were divided into a MSC-treated group and a blank control group. On postoperative day 7, all rats were sacrificed, and the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL), interleukin-2 (IL-2) and interleukin-10 (IL-10) were measured. The pathological changes of liver tissue and apoptosis of hepatocytes were also assessed. RESULTS: In in vitro MLC, T lymphocyte proliferation in Wistar rats was significantly inhibited by 48.44%. In the MSC-treated group, the levels of ALT, AST, TBIL, IL-2 and IL-10 were 134.2 +/- 45.0 U/L, 162.5 +/- 30.5 U/L, 30.6 +/- 5.4 mu mol/L, 187.35 +/- 18.26 mu g/L and 193.95 +/- 37.62 mu g/L, and those in the blank control group were 355.6 +/- 54.3 U/L, 296.4 +/- 71.2 U/L, 145.7 +/- 28.6 +/- mol/L, 295.73 +/- 57.15 mu g/L and 75.12 +/- 11.23 mu g/L, respectively, with statistically significant differences (P<0.05). Pathological examination revealed that the rejection in the MSC-treated group was clearly alleviated compared with that in the blank control group. TUNEL indicated that the apoptosis of hepatocytes in the MSC-treated group was milder than that in the blank control group (P<0.05). CONCLUSION: Adipose-derived MSCs clearly inhibit recipient-derived T lymphocyte proliferation in MLC and significantly alleviate acute rejection following orthotopic liver transplantation in rats.

Hepatogenic differentiation of human mesenchymal stem cells from adipose tissue in comparison with bone marrow mesenchymal stem cells

AIM： To investigate and compare the hepatogenic transdifferentiation of adipose tissue-derived stem cells （ADSC） and bone marrow-derived mesenchymal stem cells （BMSC） in vitro. Transdifferentiation of BMSC into hepatic cells in vivo has been described. Adipose tissue represents an accessible source of ADSC, with similar characteristics to BMSC. METHODS： BMSCs were obtained from patients undergoing total hip arthroplasty and ADSC from human adipose tissue obtained from lipectomy. Cells were grown in medium containing 15% human serum. Cultures were serum deprived for 2 d before cultivating under similar pro-hepatogenic conditions to those of liver development using a 2-step protocol with sequential addition of growth factors, cytokines and hormones. Hepatic differentiation was RT-PCR-assessed and liver-marker genes were immunohistochemically analysed.RESULTS： BMSC and ADSC exhibited a fibroblastic morphology that changed to a polygonal shape when cells differentiated. Expression of stem cell marker Thyl decreased in differentiated ADSC and BMSC. However, the expression of the hepatic markers, albumin and CYPs increased to a similar extent in differentiated BMSC and ADSC. Hepatic gene activation could be attributed to increased liver-enriched transcription factors （C/EBPβ and HNF4α）, as demonstrated by adenoviral expression vectors.CONCLUSION： Mesenchymal stem cells can be induced to hepatogenic transdifferentiation in vitro. ADSCs have a similar hepatogenic differentiation potential to BMSC, but a longer culture period and higher proliferation capacity. Therefore, adipose tissue may be an ideal source of large amounts of autologous stem cells, and may become an alternative for hepatocyte regeneration, liver cell transplantation or preclinical drug testing.

Adipose-derived mesenchymal stromal/stem cells: An update on their phenotype in vivo and in vitro

Adipose tissue is a rich, ubiquitous and easily acces-sible source for multipotent stromal/stem cells and has, therefore, several advantages compared to other sourc-es of mesenchymal stromal/stem cells. Several studies have tried to identify the origin of the stromal/stem cell population within adipose tissue in situ. This is a complicated attempt because no marker has currently been described which unambiguously identifies native adipose-derived stromal/stem cells(ASCs). Isolated and cultured ASCs are a non-uniform preparation consisting of several subsets of stem and precursor cells. Cultured ASCs are characterized by their expression of a panel of markers(and the absence of others), whereas their in vitro phenotype is dynamic. Some markers were ex-pressed de novo during culture, the expression of some markers is lost. For a long time, CD34 expression was solely used to characterize haematopoietic stem and progenitor cells, but now it has become evident that it is also a potential marker to identify an ASC subpopula-tion in situ and after a short culture time. Nevertheless, long-term cultured ASCs do not express CD34, perhaps due to the artificial environment. This review gives an update of the recently published data on the origin and phenotype of ASCs both in vivo and in vitro. In addition, the composition of ASCs(or their subpopula-tions) seems to vary between different laboratories andpreparations. This heterogeneity of ASC preparationsmay result from different reasons. One of the main problems in comparing results from different laborato-ries is the lack of a standardized isolation and culture protocol for ASCs. Since many aspects of ASCs, suchas the differential potential or the current use in clinical trials, are fully described in other recent reviews, this review further updates the more basic research issues concerning ASCs' subpopulations, heterogeneity andculture standardization.

Osteogenic potential: comparison between bone marrow and adipose-derived mesenchymal stem cells

Bone tissue engineering(BTE) is now a promising re-search issue to improve the drawbacks from traditional bone grafting procedure such as limited donor sources and possible complications. Stem cells are one of the major factors in BTE due to the capability of self re-newal and multi-lineage differentiation. Unlike embry-onic stem cells, which are more controversial in ethical problem, adult mesenchymal stem cells are considered to be a more appropriate cell source for BTE. Bone marrow mesenchymal stem cells(BMSCs) are the ear-liest-discovered and well-known stem cell source using in BTE. However, the low stem cell yield requiring long expansion time in vitro, pain and possible morbidities during bone marrow aspiration and poor proliferation and osteogenic ability at old age impede its' clinical ap-plication. Afterwards, a new stem cell source coming from adipose tissue, so-called adipose-derived stemcells(ASCs), is found to be more suitable in clinical ap-plication because of high stem cells yield from lipoaspi-rates, faster cell proliferation and less discomfort and morbidities during harvesting procedure. However, the osteogenic capacity of ASCs is now still debated be-cause most papers described the inferior osteogenesis of ASCs than BMSCs. A better understanding of the osteogenic differences between ASCs and BMSCs is crucial for future selection of cells in clinical application for BTE. In this review, we describe the commonality and difference between BMSCs and ASCs by cell yield, cell surface markers and multiple-differentiation poten-tial. Then we compare the osteogenic capacity in vitro and bone regeneration ability in vivo between BMSCs and ASCs based on the literatures which utilized both BMSCs and ASCs simultaneously in their articles. The outcome indicated both BMSCs and ASCs exhibited the osteogenic ability to a certain extent both in-vitro and in-vivo. However, most in-vitro study papers verified the inferior osteogenesis of ASCs; conversely, in-vivo research reviews revealed more controversies in this issue. We expect the new researchers can have a quick understanding of the progress in this filed and design a more comprehensive research based on this review.

Translational products of adipose tissue-derived mesenchymal stem cells:Bench to bedside applications

With developments in the field of tissue engineering and regenerative medicine,the use of biological products for the treatment of various disorders has come into the limelight among researchers and clinicians.Among all the available biological tissues,research and exploration of adipose tissue have become more robust.Adipose tissue engineering aims to develop by-products and their substitutes for their regenerative and immunomodulatory potential.The use of biodegradable scaffolds along with adipose tissue products has a major role in cellular growth,proliferation,and differentiation.Adipose tissue,apart from being the powerhouse of energy storage,also functions as the largest endocrine organ,with the release of various adipokines.The progenitor cells among the heterogeneous population in the adipose tissue are of paramount importance as they determine the capacity of regeneration of these tissues.The results of adipose-derived stemcell assisted fat grafting to provide numerous growth factors and adipokines that improve vasculogenesis,fat graft integration,and survival within the recipient tissue and promote the regeneration of tissue are promising.Adipose tissue gives rise to various by-products upon processing.This article highlights the significance and the usage of various adipose tissue by-products,their individual characteristics,and their clinical applications.

Effect of adipose-derived mesenchymal stem cells on hepatocellular carcinoma: In vitro inhibition of carcinogenesis

AIM To investigate the effect of adipose-derived mesenchymal stem cells(ADMSCs)and their conditioned media(CM) on hepatocellular carcinoma(HCC) cell tumorigenesis.METHODS The proliferation rate of HepG2 and PLC-PRF-5 HCC cancer cells was measured using the trypan blue exclusion method and confirmed using the cell-counting kit8(commonly known as CCK-8) assay. Apoptosis was detected by flow cytometry using annexin V-FITC. Protein and mRNA expression was quantified by ELISA and real time PCR, respectively. Migration and invasion rates were performed by Transwell migration and invasion assays. Wound healing was examined to confirm the data obtained from the migration assays.RESULTS Our data demonstrated that when co-culturing HCC cell lines with ADMSCs or treating them with ADMSC CM, the HCC cell proliferation rate was significantly inhibited and the apoptosis rate increased. The decreased proliferation rate was accompanied by an upregulation of P53 and Retinoblastoma mRNA and a downregulation of c-Myc and hTERT mRNA levels. More notably, ADMSCs and their CM suppressed the expression of the two important markers of HCC carcinogenicity, alpha-fetoprotein and Des-gamma-carboxyprothrombin. In addition, the migration and invasion levels of HepG2 and PLC-PRF-5 cells significantly decreased, potentially through increased expression of the tissue inhibitor metalloproteinases TIMP-1, TIMP-2 and TIMP-3.CONCLUSION These findings shed new light on a protective and therapeutic role for ADMSCs and their CM in controlling HCC invasiveness and carcinogenesis.

Adipose-derived mesenchymal stem cells accelerate nerve regeneration and functional recovery in a rat model of recurrent laryngeal nerve injury

Medialization thyroplasty or injection laryngoplasty for unilateral vocal fold paralysis cannot restore mobility of the vocal fold. Recent studies have shown that transplantation of mesenchymal stem cells is effective in the repair of nerve injuries. This study investigated wheth- er adipose-derived stem celt transplantation could repair recurrent laryngeal nerve injury. Rat models of recurrent laryngeal nerve injury were established by crushing with micro forceps. Adipose-derived mesenchymal stem cells （ADSCs; 8 ×105） or differentiated Schwann-like adipose-derived mesenchymal stem cells （dADSCs; 8×105） or extracellular matrix were injected at the site of injury. At 2, 4 and 6 weeks post-surgery, a higher density of myelinated nerve fiber, thicker myelin sheath, improved vocal fold movement, better recovery of nerve conduction capacity and reduced thyroarytenoid muscle atrophy were found in ADSCs and dADSCs groups compared with the extracellu- lar matrix group. The effects were more pronounced in the ADSCs group than in the dADSCs group. These experimental results indicated that ADSCs transplantation could be an early interventional strategy to promote regeneration after recurrent laryngeal nerve injury.

Anti-osteoarthritis effect of a combination treatment with human adipose tissue-derived mesenchymal stem cells and thrombospondin 2 in rabbits

BACKGROUND Osteoarthritis(OA),a chronic age-related disease characterized by the slowly progressive destruction of articular cartilage,is one of the leading causes of disability.As a new strategy for treatment of OA,mesenchymal stem cells(MSCs)have the potential for articular cartilage regeneration.Meanwhile,thrombospondin 2(TSP2)promotes the chondrogenic differentiation of MSCs.AIM To investigate whether TSP2 induces chondrogenic differentiation of human adipose-derived MSCs(hADMSCs)and potentiates the therapeutic effects of hADMSCs in OA rabbits.METHODS We investigated the chondrogenic potential of TSP2 in hADMSCs by analyzing the expression of chondrogenic markers as well as NOTCH signaling genes in normal and TSP2 small interfering RNA(siRNA)-treated stem cells.Anterior cruciate ligament transection surgery was performed in male New Zealand white rabbits,and 8 wk later,hADMSCs(1.7×10^6 or 1.7×10^7 cells)were injected into the injured knees alone or in combination with intra-articular injection of TSP2(100 ng/knee)at 2-d intervals.OA progression was monitored by gross,radiological,and histological examinations.RESULTS In hADMSC culture,treatment with TSP2 increased the expression of chondrogenic markers(SOX9 and collagen Ⅱ)as well as NOTCH signaling genes(JAGGED1 and NOTCH3),which were inhibited by TSP2 siRNA treatment.In vivo,OA rabbits treated with hADMSCs or TSP2 alone exhibited lower degree of cartilage degeneration,osteophyte formation,and extracellular matrix loss 8 wk after cell transplantation.Notably,such cartilage damage was further alleviated by the combination of hADMSCs and TSP2.In addition,synovial inflammatory cytokines,especially tumor-necrosis factor-α,markedly decreased following the combination treatment.CONCLUSION The results indicate that TSP2 enhances chondrogenic differentiation of hADMSCs via JAGGED1/NOTCH3 signaling,and that combination therapy with hADMSCs and TSP2 exerts synergistic effects in the cartilage regeneration of OA joints.

Cell viability and extracellular matrix synthesis in a co-culture system of corneal stromal cells and adipose-derived mesenchymal stem cells

AIM：To investigate the impact of adipose-derived mesenchymal stem cells（ADSCs） on cell viability and extracellular matrix（ECM） synthesis of corneal stromal cells（CSCs）. METHODS：ADSCs and CSCs were obtained from the corneas of New Zealand white rabbits and indirectly cocultured in vitro. The proliferative capacity of CSCs in the different groups was assessed by CCK-8 assays. Annexin V-fluorescein isothiocyanate（FITC）/proliferation indices（PI） assays were used to detect the apoptosis of CSCs. The expression levels of matrix metalloproteinase（MMP）, such as MMP1, MMP2, MMP9, and collagens were also evaluated by Western blot. RESULTS：ADSCs significantly promoted proliferation and invasion of CSCs in the indirect co-culture assays. The co-cultural group displayed much higher ability of proliferation, especially under the co-culture conditions of ADSCs for 3d, compared with that CSCs cultured alone. The PI of CSCs in the co-culture system were increased approximately 3-8-fold compared with the control group. A significant change was observed in the proportions of cells at apoptosis（early and late） between the negative control group（6.34% and 2.06%） and the ADCSs-treated group（4.69% and 1.59%）. The expression levels of MMPs were down regulated in the co-culture models. Compared with the control group, the decrease intensities of MMP-1, MMP-2 and MMP-9 in CSCs/ADSCs group were observed, 3.90-fold, 1.09-fold and 3.03-fold, respectively. However, the increase intensities of collagen type（I, II, III, IV, and V） in CSCs were observed in CSCs/ADSCs group, 3.47-fold,4.30-fold, 2.35-fold, 2.55-fold and 2.43-fold, respectively, compared to that in the control group. The expressions of aldehyde dehydrogenase and fibronectin in CSCs were upregulated in the co-culture models.CONCLUSION：ADSCs play a promotive role in CSCs＇ growth and invasion, which may be partially associated with MMPs decrease and collagens increase, resulting in a positive participation in the plasticity and ECM synthesis of CSCs. This provided a new insight into the extensive role of ADSCs in CSCs and a potential molecular target for corneal therapy.

Autologous Adipose-Derived Mesenchymal Stem Cells Embedded in Platelet-Rich Fibrin in Diabetic Foot Ulcers

Context and Aim: Mesenchymal stem cells (MSCs) and platelet-rich fibrin (PRF) have emerged as ideal candidates for advanced therapies of various therapeutically-challenging diseases;however, their regenerative potential in diabetic foot ulcers (DFU) has not been well determined. In this study, we reviewed our clinical experience in mitigating chronic ulcer complications of diabetic foot through a conventional treatment of autologous adipose-derived MSCs embedded in PRF with pure PRF injections. Materials and Methods: The present study was carried out in 10 patients with an open DFU wound selected over a period of 1 year starting from April 2019. Patients were either injected with PRF alone (Group A) or injected with MSCs derived from adipose tissue (ADMSC) embedded in (PRF (Group B). Results: Patients in Group B had a better healing index when compared to Group A. Conclusion: Use of ADMSC embedded in PRF showed promising results to treat DFU.

Ameliorative effects of human adipose tissue-derived mesenchymal stem cells on myelin basic protein-induced experimental autoimmune encephalomyelitis in Lewis rats

Mesenchymal stem cells have been previously shown to exert an immunomodulatory function. The present study sought to investigate the effects of multipotential human adipose tissue-derived mesenchymal stem cells （hAdMSCs） on disease progression and cytokine expression in Lewis rats with experimental autoimmune encephalomyelitis （EAE） induced by myelin basic protein. The duration of EAE paralysis in the group treated on day 7 posfimmunization with 5 × 10^6 hAdMSCs was significantly reduced compared with the vehicle-treated controls and the 1 x 106 hAdMSC- treated group. The duration of EAE paralysis in the groups treated with 5 × 10^6 hAdMSCs on both day 1 and day 7 postimmunization was significantly reduced compared with the vehicle-treated controls and the groups treated with 5 × 10^6 hAdMSCs on both day 7 and day 10 postimmunization. The mRNA expression of interleukin-10 and indoleamine 2, 3-dioxygenase was significantly decreased in the hAdMSC-treated group compared with the vehicle-treated group. These findings suggest that the ameliorative effects of hAdMSCs on EAE symptoms operate in a dose- and time-dependent manner and can be mediated in part by the ample production of anti-inflammatory cytokines.

ISOLATION AND INDUCTION OF DIFFERENTIATION OF SEINE ADIPOSE-DERIVED MESENCHYMAL STEM CELLS

Objectives To establish a method for high yield mesenchymal stem cells collection, as well as a culture method for iden- tifying mesenchymal stem cells from the swine adipose-derived mesenchymal stem cell （ADMSC）. Methods Swine AD- MSCs were isolated from fat tissue with collagenase, followed by induction of differentiation to osteogenic, adipogenic and chondrogrnic cells. The survival curve of the ADMSC at the 37℃ and 38℃ were measured using WST-1 Cell Proliferation Assay Reagent. Result ADMSCs isolated with collagenase from swine neck fat tissue generated a stable uniform appearance af- ter the second generation. The passage period was five days. ADMSC could differentiate into osteogenic, adipogenic or chon- drogrnic cells under different culture conditions. The highest growth rate was achieved at 38℃in this study. Conclusion Swine ADMSCs have the potential to differentiate into osteogenic, adipogenic or chondrogrnic cells, and they may be appropriate for transplantation for both research and clinical purpose.

Clinical and Magnetic Resonance Image Changes of Platelet-Rich Plasma Therapy in Combination with Human Mesenchymal Stem Cells from Autologous Adipose Tissue for Knee Osteoarthritis Treatment

Objective: To evaluate the efficacy based on clinical symptom and on magnetic resonance image of platelet-rich plasma therapy in combination with mesenchymal stem cells from autologous adipose tissue for knee osteoarthritis treatment. Patients and Method: 30 patients including 26 females and 4 males;correspondingly, 60 knee joints were diagnosed with osteoarthritis with stages II - III of Kellgren and Lawrence, their mean age was 58.63 ± 11.11. All were injected with autologous platelet-rich plasma that was extracted by PRP set, APC 30 PRP PRCEDURE PRAK and autologously extracted mesenchymal stem cells from abdominal adipose tissue using the ADI-25-01 ADIPOSEPRCEDURE PRAK of USA. Results: After 12 months: the pain level according to VAS score at the right knee joint was decreased from 6.0 ± 1.28 before treatment to 1.9 ± 0.3;VAS score at the left knee joint was decreased from 6.43 ± 1.19 to 2.25 ± 0.43. Total Lequene score at right knee joint was decreased from 16.04 ± 1.57 before treatment to 4.31 ± 1.04, at left knee joint was decreased from 17.52 ± 1.74 before treatment to 5.15 ± 1.48. Total WOMAC score at right knee joint was decreased from 55.93 ± 5.56 to 10.37 ± 1.56;at left knee joint was decreased from 53.97 ± 5.57 to 10.07 ± 1.59. There were 86.77% joints with cartilage thickness change and the patellar cartilage thickness was increased from 1.56 ± 0.09 mm before treatment to 1.65 ± 0.09 mm. Conclusion: The treatment of knee osteoarthritis by platelet-rich plasma therapyin combination with mesenchymal stem cells from autologous adipose tissue is effective in reducing pain, improving patient's mobility and walking function, reforming articular cartilage thickness on magnetic resonance image.

腺病毒介导SDF-1/NELL-1双基因转染ADSCs复合Nano-n HA支架对犬下颌骨缺损修复的实验研究

目的:构建腺病毒介导的基质细胞衍生因子-1(stromal cell-derived factor-1,SDF-1)和尼尔样-1型分子(Nell-like molecule-l,Nell-1)双基因转染犬ADSCs复合Nano-n HA支架,观察其对犬下颌骨缺损的修复作用。方法:构建携SDF-1及NELL-1目的基因片段的腺病毒表达载体,分组转染犬ADSCs后行体外成骨分化诱导,ELISA法检测目的基因转染ADSCs后结合支架体内外生长各期目的蛋白表达。20只比格犬随机分为5组,A组为空白组(无支架置入),B组为单纯支架组,C组为SDF-1/Nano-n HA组,D组为Nell-1/Nano-n HA组,E组为SDF-1/Nell-1/Nano-n HA组。CM-Dil细胞标记后构建ADSCs-Nano-n HA支架骨组织工程复合体,制备犬双侧下颌骨缺损模型,将不同细胞支架复合体分组植入下颌骨缺损区。术后第4、8、12周取材行大体观察、CT、扫描电镜、细胞示踪实验及组织学检测,比较各组缺损区新骨形成情况,行统计学分析。结果:ADSCs传代培养及成骨诱导分化状态良好,荧光显微镜下观察SDF-1、Nell-1及SDF-1/Nell-1重组腺病毒均能稳定转染ADSCs,各组目的蛋白表达体内外实验表达有显著性差异。通过大体观察及X线、CT扫描、ECM检测发现转染组骨缺损区新骨形成情况优于未转染组,且共转染组成骨速度及质量优于其他各组。组织学染色可见转染组新骨形成及血管生成情况均优于未转染组,且共转染组新生骨小梁面积及骨成熟度均优于其他各组。结论:SDF-1、Nell-1均可转染ADSCs并可稳定表达,目的基因转染ADSCs复合Nano-n HA支架后可显著促进下颌骨缺损的成骨修复,为组织工程修复成骨提供了新路径。

WJSC 6^(th) Anniversary Special Issues(2):Mesenchymal stem cells Mesenchymal stem cells in the treatment of spinal cord injuries:A review

With technological advances in basic research,the intricate mechanism of secondary delayed spinal cord injury(SCI)continues to unravel at a rapid pace.However,despite our deeper understanding of the molecular changes occurring after initial insult to the spinal cord,the cure for paralysis remains elusive.Current treatment of SCI is limited to early administration of high dose steroids to mitigate the harmful effect of cord edema that occurs after SCI and to reduce the cascade of secondary delayed SCI.R ecent evident-based clinical studies have cast doubt on the clinical benefit of steroids in SCI and intense focus on stem cell-based therapy has yielded some encouraging results.An array of mesenchymal stem cells(MSCs)from various sources with novel and promising strategies are being developed to improve function after SCI.In this review,we briefly discuss the pathophysiology of spinal cord injuries and characteristics and the potential sources of MSCs that can be used in the treatment of SCI.We will discuss the progress of MSCs application in research,focusing on the neuroprotective properties of MSCs.Finally,we will discuss the results from preclinical and clinical trials involving stem cell-based therapy in SCI.

Comparative analysis of biological characteristics of adult mesenchymal stem cells with different tissue origins

Objective: To invest the dif erences among mesenchymal stem cells(MSCs) derived from different tissues and their impacts on clinical applications. Methods: In this study, MSCs were isolated from adipose tissue(AD), umbilical cord tissue(UC), and menstrual blood(Men) and comparedtheir biological characteristics in terms of proliferation capacity, passage capacity, colony formation, and surface markers were compared. Results: The stem cells(SCs) obtained from different sources were all characterized as MSCs, but demonstrated some dif erences. Umbilical cord-derived MSCs(UCMSCs) were able to overcome density inhibition. The proliferation rate decreased in the order UCMSCs> Men SCs> ADSCs, while the colony-forming ability decreased in the order Men SCs> ADSCs> UCMSCs. Based on gene-expression data for MSCs from dif erent sources within the same donor, 768 Men SC genes were found that were specii cally upregulated or downregulated compared with bone marrow-derived MSCs and UCMSCs, most of which were involved in cell function-related pathways. In addition, Men SCs appeared to be superior in terms of immune inl ammation, stress response, and neural dif erentiation potentials, but weaker in terms of osteogenic and chondrogenic dif erentiation capacities, compared with UCMSCs and bone marrow-derived MSCs. Conclusions: Men SCs have higher extraction ei ciency, colony-forming ability, and long time passage capacity. Although the proliferation capacity is inferior to UCMSCs.

Similarities and differences between mesenchymal stem/progenitor cells derived from various human tissues

BACKGROUND Mesenchymal stromal/stem cells (MSCs) constitute a promising tool in regenerative medicine and can be isolated from different human tissues. However, their biological properties are still not fully characterized. Whereas MSCs from different tissue exhibit many common characteristics, their biological activity and some markers are different and depend on their tissue of origin. Understanding the factors that underlie MSC biology should constitute important points for consideration for researchers interested in clinical MSC application. AIM To characterize the biological activity of MSCs during longterm culture isolated from: bone marrow (BM-MSCs), adipose tissue (AT-MSCs), skeletal muscles (SMMSCs), and skin (SK-MSCs). METHODS MSCs were isolated from the tissues, cultured for 10 passages, and assessed for: phenotype with immunofluorescence and flow cytometry, multipotency with differentiation capacity for osteo-, chondro-, and adipogenesis, stemness markers with qPCR for mRNA for Sox2 and Oct4, and genetic stability for p53 and c-Myc;27 bioactive factors were screened using the multiplex ELISA array, and spontaneous fusion involving a co-culture of SM-MSCs with BM-MSCs or AT-MSCs stained with PKH26 (red) or PKH67 (green) was performed. RESULTS All MSCs showed the basic MSC phenotype;however, their expression decreased during the follow-up period, as confirmed by fluorescence intensity. The examined MSCs express CD146 marker associated with proangiogenic properties;however their expression decreased in AT-MSCs and SM-MSCs, but was maintained in BM-MSCs. In contrast, in SK-MSCs CD146 expression increased in late passages. All MSCs, except BM-MSCs, expressed PW1, a marker associated with differentiation capacity and apoptosis. BM-MSCs and AT-MSCs expressed stemness markers Sox2 and Oct4 in long-term culture. All MSCs showed a stable p53 and c-Myc expression. BM-MSCs and AT-MSCs maintained their differentiation capacity during the follow-up period. In contrast, SK-MSCs and SM-MSCs had a limited ability to differentiate into adipocytes. BM-MSCs and AT-MSCs revealed similarities in phenotype maintenance, capacity for multilineage differentiation, and secretion of bioactive factors. Because AT-MSCs fused with SM-MSCs as effectively as BM-MSCs, AT-MSCs may constitute an alternative source for BM-MSCs. CONCLUSION Long-term culture affects the biological activity of MSCs obtained from various tissues. The source of MSCs and number of passages are important considerations in regenerative medicine.