Bone Marrow Aspirate Concentrate versus Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells for Combined Cartilage Regeneration Procedure in Patients Undergoing High Tibial Osteotomy: A Systematic Review and Meta-Analysis.

Background and objectives: Cartilage regeneration using mesenchymal stem cells (MSCs) has been attempted to improve articular cartilage regeneration in varus knee osteoarthritis (OA) patients undergoing high tibial osteotomy (HTO). Bone marrow aspirate concentrate (BMAC) and human umbilical cord blood-derived MSCs (hUCB-MSCs) have been reported to be effective. However, whether BMAC is superior to hUCB-MSCs remains unclear. This systematic review and meta-analysis aimed to determine the clinical efficacy of cartilage repair procedures with BMAC or hUCB-MSCs in patients undergoing HTO. Materials and Methods: A systematic search was conducted using three global databases, PubMed, EMBASE, and the Cochrane Library, for studies in which the clinical outcomes after BMAC or hUCB-MSCs were used in patients undergoing HTO for varus knee OA. Data extraction, quality control, and meta-analysis were performed. To compare the clinical efficacy of BMAC and hUCB-MSCs, reported clinical outcome assessments and second-look arthroscopic findings were analyzed using standardized mean differences (SMDs) with 95% confidence intervals (CIs). Results: The present review included seven studies of 499 patients who received either BMAC (BMAC group, n = 169) or hUCB-MSCs (hUCB-MSC group, n = 330). Improved clinical outcomes were found in both BMAC and hUCB-MSC groups; however, a significant difference was not observed between procedures (International Knee Documentation Committee score; p = 0.91, Western Ontario and McMaster Universities OA Index; p = 0.05, Knee Society Score (KSS) Pain; p = 0.85, KSS Function; p = 0.37). On second-look arthroscopy, the hUCB-MSC group showed better International Cartilage Repair Society Cartilage Repair Assessment grade compared with the BMAC group (p < 0.001). Conclusions: Both BMAC and hUCB-MSCs with HTO improved clinical outcomes in varus knee OA patients, and there was no difference in clinical outcomes between them. However, hUCB-MSCs were more effective in articular cartilage regeneration than BMAC augmentation.

The efficiency of human umbilical cord mesenchymal stem cells as a salvage treatment for steroid-refractory acute graft-versus-host disease.

Acute graft-versus-host disease (aGVHD) is a life-threatening complication after hematopoietic stem cell transplantation (HSCT) and is primarily treated with steroids. However, there is no standard treatment for steroid-refractory acute graft-versus-host disease (SR-aGVHD). Although mesenchymal stem cells (MSCs) have proven effective for SR-aGVHD, few reports have focused on human umbilical cord blood-derived MSCs (hUCB-MSCs). Here, we report on the efficiency of hUCB-MSCs as the salvage therapy for SR-aGVHD in 54 patients. The overall response rate (ORR) reached 59.3% (32/54) 28 days later. Twenty-four patients achieved complete remission (CR), and 8 achieved partial remission (PR). The median follow-up time after the initiation of hUCB-MSC treatment was 19.3 (0.6-59.0) months. The probability of overall survival (OS) and progression-free survival (PFS) was 60.9% (47.4-74.4%, 95% CI) and 58.8% (45.3-72.3%, 95% CI), respectively, while that of GVHD/relapse-free survival (GRFS) was only 30.8% (17.86-43.74%, 95% CI). Multivariate analysis revealed that response on Day 28 was an independent favorable prognostic factor (OS, P < 0.001; PFS, P < 0.001; GRFS, P = 0.001), but an age of ≥ 18 years suggested an unfavorable long-term prognosis (OS, P < 0.001; PFS, P < 0.001; GRFS, P = 0.003). In addition, liver involvement was adversely associated with PFS (P = 0.021) and GRFS (P = 0.009). An infused MNC ≥ 8.66 × 108/kg was also detrimental to GRFS (P = 0.031). Collectively, our results support hUCB-MSCs as an effective treatment for SR-aGVHD.

Cartilage Regeneration Using Human Umbilical Cord Blood Derived Mesenchymal Stem Cells: A Systematic Review and Meta-Analysis.

Background and Objectives: Human umbilical-cord-blood-derived mesenchymal stem cells (hUCB-MSCs) have recently been used in clinical cartilage regeneration procedures with the expectation of improved regeneration capacity. However, the number of studies using hUCB-MSCs is still insufficient, and long-term follow-up results after use are insufficient, indicating the need for additional data and research. We have attempted to prove the efficacy and safety of hUCB-MSC treatment in a comprehensive analysis by including all subjects with knee articular cartilage defect or osteoarthritis who have undergone cartilage repair surgery using hUCB-MSCs. We conducted a meta-analysis and demonstrated efficacy and safety based on a systematic review. Materials and Methods: This systematic review was conducted following the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines. For this study, we searched the PubMed, Embase, Web of Science, Scopus, and Cochrane Library literature databases up to June 2022. A total of seven studies were included, and quality assessment was performed for each included study using the Newcastle−Ottawa Quality Assessment Scale. Statistical analysis was performed on the extracted pooled clinical outcome data, and subgroup analyses were completed. Results: A total of 570 patients were included in the analysis. In pooled analysis, the final follow-up International Knee Documentation Committee (IKDC) score showed a significant increase (mean difference (MD), −32.82; 95% confidence interval (CI), −38.32 to −27.32; p < 0.00001) with significant heterogeneity (I2 = 93%, p < 0.00001) compared to the preoperative score. The Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) scores at final follow-up were significantly decreased (MD, 30.73; 95% CI, 24.10−37.36; p < 0.00001) compared to the preoperative scores, with significant heterogeneity (I2 = 95%, p < 0.00001). The visual analog scale (VAS) score at final follow-up was significantly decreased (MD, 4.81; 95% CI, 3.17−6.46; p < 0.00001) compared to the preoperative score, with significant heterogeneity (I2 = 98%, p < 0.00001). Two studies evaluated the modified Magnetic Resonance Observation of Cartilage Repair Tissue (M-MOCART) score and confirmed sufficient improvement. In a study analyzing a group treated with bone marrow aspiration concentrate (BMAC), there was no significant difference in clinical outcome or M-MOCART score, and the post-treatment International Cartilage Repair Society (ICRS) grade increased. Conclusion: This analysis demonstrated the safety, efficacy, and quality of repaired cartilage following hUCB-MSC therapy. However, there was no clear difference in the comparison with BMAC. In the future, comparative studies with other stem cell therapies or cartilage repair procedures should be published to support the superior effect of hUCB-MSC therapy to improve treatment of cartilage defect or osteoarthritis.

Human umbilical cord blood-derived MSCs trans-differentiate into endometrial cells and regulate Th17/Treg balance through NF-κB signaling in rabbit intrauterine adhesions endometrium.

PURPOSE: The fundamental cause of intrauterine adhesions (IUAs) is the destruction and reduction in stem cells in endometrial basal layer, resulting in endometrial reconstruction very difficult. The purpose of this study was to investigate the effects and underlying mechanism of human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) on the endometrial reconstruction after transplantation. METHODS: hUCB-MSCs were isolated and identified by flow cytometry, osteogenic, adipogenic and chondrogenic differentiation assays. The rabbit IUA models were established and set five groups (control, 14/28th day after surgery, estrogen and hUCB-MSCs treatment). The number of endometrial glands and the fibrosis rate were evaluated using HE and Masson staining, respectively. Endometrial proliferation, angiogenesis and inflammation were evaluated by immunohistochemical staining of ER, Ki-67and TGF-ß1, respectively. Single-cell RNA sequencing (scRNA-seq) was applied to explore the cell differentiation trajectory after hUCB-MSCs transplanted into IUA endometrium. Finally, molecular mechanism of hUCB-MSCs repairing damaged endometrium was investigated by RNA sequencing, qRT-PCR and Western blot assays. RESULTS: After transplantation of the hUCB-MSCs, the increase in endometrial gland number, estrogen receptor (ER) and Ki-67 expression, and the decrease in fibrosis rate and TGF-ß expression (P < 0.05), suggested the endometrial repair, angiogenesis and inflammatory suppression. The therapeutic effect of hUCB-MSCs was significantly improved compared with 28th day after surgery and estrogen group. ScRNA-seq demonstrated that the transplanted hUCB-MSCs can trans-differentiate into endometrial cells: epithelial, fibroblast and macrophage. RNA sequencing of six IUA samples combined with qRT-PCR and Western blot assays further revealed that hUCB-MSCs may regulate Th17/Treg balance through NF-κB signaling, thus inhibiting the immune response of damaged endometrium. CONCLUSIONS: Our study demonstrated that hUCB-MSCs can repair damaged endometrium through trans-differentiation, immunomodulatory capacities and NF-κB signaling, suggesting the treatment value of hUCB-MSCs in IUA.

A potential role of mesenchymal stem cells derived from human umbilical cord blood in ameliorating psoriasis-like skin lesion in the rats.

BACKGROUND: Psoriasis is a common autoimmune inflammatory skin disease, with no clear cause, treated with topical agents and phototherapy, conventional immunosuppressant drugs and biologic agents. Stem cell therapy has generated significant interest in regenerative medicine. The aim of this study was to use mesenchymal stem cell (MSC) therapy compared to the topical application of the standard conventional corticosteroid cream. MATERIALS AND METHODS: Forty male adult albino rats were used, divided into four groups, 10 rats each: group I (control), group II (psoriasis-like lesions induced by usage of Aldara cream), group III (treated with betamethasone) and group IV (treated with MSCs). Specimens were stained with haematoxylin and eosin, Masson's trichrome, immune-histochemical technique for CD4, CD8 and CD31. Ultra-sections were prepared for transmission electron microscope (TEM) examination. RESULTS: Mesenchymal stem cells demonstrated efficacy in reduction of disease severity in the form of uniform epidermal thickness covered by a very thin keratin layer. Normally arranged layers of epidermal layers, with a clear border demarcation, were seen between the epidermis and the dermis with apparently intact basement membrane. TEM showed absence of gaps between the tightly connected cells of the basal layer and the resting basement membrane. CONCLUSIONS: Application of MSCs raises hope for developing a new, safe and effective therapy for psoriatic patients, avoiding the side effects of betamethasone.

Exosomal miR-22-3p from human umbilical cord blood-derived mesenchymal stem cells protects against lipopolysaccharid-induced acute lung injury.

BACKGROUND: Mesenchymal stem cells (MSCs) are widely applied in various clinical disorders, including acute lung injury (ALI). We aimed to investigate the effects of human umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs)-derived exosomal microRNA-22-3p (miR-22-3p) on lipopolysaccharid (LPS)-induced ALI via regulating frizzled class receptor 6 (FZD6). METHODS: Rat lung cells were selected to construct the LPS-induced ALI cell model. The LPS-treated cells were transfected with restored miR-22-3p and depleted FZD6 for investigating their roles in ALI. Human UCB-MSCs were cultured and exosomes were extracted. Rat lung cells were co-cultured with exosomes that had been transfected with restored miR-22-3p and upregulated FZD6 to detect their roles in inflammatory reaction, oxidative stress, cell proliferation activity and apoptosis. The ALI rat model was established through LPS inhalation and the rats were respectively treated. Then, the pathology, apoptosis and expression of the NF-κB signaling pathway-related factors in rat lung tissues were determined. RESULTS: miR-22-3p expression was reduced and FZD6 expression was enhanced in LPS-treated rat lung cells while exosomes raised miR-22-3p expression and decreased FZD6 expression. In LPS-treated cells, up-regulating miR-22-3p or depleting FZD6 reduced inflammatory reaction and oxidative stress response, raised rat lung cell proliferation activity and inhibited cell apoptosis rate. In the in vivo ALI model, exosomes suppressed pathological changes, apoptosis and NF-κB expression in LPS-treated rats. Upregulated miR-22-3p further attenuated ALI. CONCLUSION: Our study highlights the potential of UCB-MSC-exosomal miR-22-3p in preventing ALI. This study may provide further insights into ALI therapy.

High tibial osteotomy with human umbilical cord blood-derived mesenchymal stem cells implantation for knee cartilage regeneration.

BACKGROUND: High tibial osteotomy (HTO) is a well-established method for the treatment of medial compartment osteoarthritis of the knee with varus deformity. However, HTO alone cannot adequately repair the arthritic joint, necessitating cartilage regeneration therapy. Cartilage regeneration procedures with concomitant HTO are used to improve the clinical outcome in patients with varus deformity. AIM: To evaluate cartilage regeneration after implantation of allogenic human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) with concomitant HTO. METHODS: Data for patients who underwent implantation of hUCB-MSCs with concomitant HTO were evaluated. The patients included in this study were over 40 years old, had a varus deformity of more than 5°, and a full-thickness International Cartilage Repair Society (ICRS) grade IV articular cartilage lesion of more than 4 cm2 in the medial compartment of the knee. All patients underwent second-look arthroscopy during hardware removal. Cartilage regeneration was evaluated macroscopically using the ICRS grading system in second-look arthroscopy. We also assessed the effects of patient characteristics, such as trochlear lesions, age, and lesion size, using patient medical records. RESULTS: A total of 125 patients were included in the study, with an average age of 58.3 ± 6.8 years (range: 43-74 years old); 95 (76%) were female and 30 (24%) were male. The average hip-knee-ankle (HKA) angle for measuring varus deformity was 7.6° ± 2.4° (range: 5.0-14.2°). In second-look arthroscopy, the status of medial femoral condyle (MFC) cartilage was as follows: 73 (58.4%) patients with ICRS grade I, 37 (29.6%) with ICRS grade II, and 15 (12%) with ICRS grade III. No patients were staged with ICRS grade IV. Additionally, the scores [except International Knee Documentation Committee (IKDC) at 1 year] of the ICRS grade I group improved more significantly than those of the ICRS grade II and III groups. CONCLUSION: Implantation of hUCB-MSCs with concomitant HTO is an effective treatment for patients with medial compartment osteoarthritis and varus deformity. Regeneration of cartilage improves the clinical outcomes for the patients.

Implantation of allogenic umbilical cord blood-derived mesenchymal stem cells improves knee osteoarthritis outcomes: Two-year follow-up.

INTRODUCTION: Clinical outcomes after the implantation of allogenic human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) in osteoarthritic knees have been rarely reported. Our study aimed to investigate clinical outcomes of osteoarthritic patients who underwent hUCB-MSC implantation. METHODS: In this case series (level of evidence: 4), from January 2014 to December 2015, 128 patients with full-thickness cartilage lesions (International Cartilage Repair Society grade 4 and Kellgren-Lawrence grade ≤3) who underwent hUCB-MSC implantation were retrospectively evaluated with a minimum of 2-year follow-up. After removing the sclerotic subchondral bone with an arthroscopic burr, 4-mm-diameter holes were created at 2-mm intervals, and hyaluronic acid and hUCB-MSCs were subsequently mixed and implanted in the holes and other articular defect sites.Clinical outcomes were evaluated preoperatively, 1 year postoperatively, and 2 years postoperatively (minimum) using visual analog scale (VAS), Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC), and International Knee Documentation Committee (IKDC) scores. To assess clinical outcomes, patients were divided into two or three groups according to the lesion size, lesion location, number of lesions, body mass index, and age; statistical analyses were performed using these data. RESULTS: The mean (±standard deviation) VAS, WOMAC, and IKDC scores at 1 and 2 years after surgery including hUCB-MSC implantation improved significantly compared to the preoperative scores (P < 0.001). There were significant differences in the lesion location (P < 0.05). Medial femoral condyle lesions resulted in worse outcomes compared with lateral femoral condyle and trochlea lesions. No adverse reactions or postoperative complications were noted. CONCLUSIONS: Implantation of hUCB-MSCs is effective for treating knee osteoarthritis based on a follow-up lasting a minimum of 2 years.

Preconditioning with interleukin-1 beta and interferon-gamma enhances the efficacy of human umbilical cord blood-derived mesenchymal stem cells-based therapy via enhancing prostaglandin E2 secretion and indoleamine 2,3-dioxygenase activity in dextran sulfate sodium-induced colitis.

Preconditioning with inflammatory cytokines has improved mesenchymal stem cells characteristics, including differentiation and immunomodulating functions. In this study, we developed a preconditioning combination strategy using interleukin-1beta (IL-1ß) and interferon-gamma (IFN-γ) to enhance the immuneregulatory ability of human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs). Our results showed that hUCB-MSCs preconditioned with IL-1ß and IFN-γ (primed hUCB-MSCs) created a statistically significant decrease in peripheral blood mononuclear cell proliferation, indicating that their immunosuppressive ability was increased. The secretion of PGE2, cyclooxygenase 2 mRNA expression, and indoleamine 2,3-dioxygenase (IDO) mRNA expression in primed hUCB-MSCs was significantly higher than those in the untreated hUCB-MSCs or the IL-1ß or IFN-γ only treated hUCB-MSCs. When inhibitors of IDO and PGE2 were treated, peripheral blood mononuclear cell proliferation, which is inhibited by primed hUCB-MSCs, was recovered. We found that Th1 T cell differentiation was also inhibited by PGE2 and IDO in the primed hUCB-MSCs, and Tregs differentiation was increased by PGE2 and IDO in the primed hUCB-MSCs. Furthermore, the primed hUCB-MSCs as well as supernatants increase CD4+ T cells migration. We demonstrated the therapeutic effects of primed hUCB-MSCs in dextran sulfate sodium-induced colitis model. In conclusion, we have demonstrated that primed hUCB-MSCs simultaneously enhance PGE2 and IDO and greatly improve the immunoregulatory capacity of MSCs, and we have developed an optimal condition for pretreatment of MSCs for the treatment of immune diseases. Our results raise the possibility that the combination of PGE2 and IDO could be therapeutic mediators for controlling immunosuppression of MSCs.

Traditional two-dimensional mesenchymal stem cells (MSCs) are better than spheroid MSCs on promoting retinal ganglion cells survival and axon regeneration.

The loss of retinal ganglion cells (RGCs) is one of the common pathological features associated with optic nerve diseases leading to blindness. The aims of our study were to compare the neuroprotection of two forms of human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) on RGCs and axon regeneration after optic nerve crush (ONC) in vivo, and to investigate the molecular mechanism. The effects of intravitreally transplanted hUCB-MSCs cultured in two-dimensional (2D-MSCs) and spheroids (3D-MSCs) were assessed by the survival of RGCs, regenerating axons, and flash visual evoked potentials (fVEP); the level of signal factors secreted by transplanted MSCs in vitreous and the marker protein levels of JAK/STAT3, PI3K/Akt/mTOR and MAPK/ERK pathways were detected using Bead-Based analysis and Western blot, respectively. We found that RGCs began to lose at day 3 after ONC, rapidly decreased at day 7, and flattened at day 14. The neuroprotection of transplanted 2D-MSCs was much stronger than that of 3D-MSCs. The transplanted 2D-MSCs could survive at least 2 weeks without differentiation and keep the characters of MSCs, which secreted multiple tropic factors and accompanied by activation of JAK/STAT3 and MAPK/ERK signaling pathways, top three most abundant factors: stem cell growth factor- ß (SCGF-ß), hepatocyte growth factor (HGF), and monocyte chemoattractant protein-1 (MCP-1). These results indicate that intravitreal injection of 2D-MSCs is a promising therapeutic strategy for retinal pathological diseases characterized by the loss of RGCs and open the door for the application of SCGF-ß, HGF, and MCP-1 in the treatment of optic nerve diseases.

Therapeutic effect of long-interval repeated intravenous administration of human umbilical cord blood-derived mesenchymal stem cells in DBA/1 mice with collagen-induced arthritis.

Rheumatoid arthritis (RA) is a common inflammatory chronic disease. It has been reported that mesenchymal stem cells (MSCs) have the effect of immune suppression in collagen-induced arthritis (CIA) mice model. However, the in vivo therapeutic effect from the long-interval repeated intravenous administration of human umbilical cord blood-derived (hUCB)-MSCs had not been investigated in CIA mice model. This study was undertaken to investigate the effects of long-interval repeated intravenous administration of hUCB-MSCs at different doses in CIA mice model. Mice were intravenously injected with three different doses of hUCB-MSCs once every 2 weeks for three times. RA severity was assessed by clinical joint score and histologic analysis including hematoxylin and eosin staining, safranin-O staining, and toluidine blue staining. We used real-time polymerase chain reaction and flow cytometry to quantify differences in inflammatory cytokines and Tregs. Mice treated with hUCB-MSCs showed significant improvement in clinical joint score. Histologic analysis revealed that hUCB-MSCs definitely reduced joint inflammation, cartilage damage, and formation of pannus in multimedium and multihigh groups. These hUCB-MSCs also significantly decreased IL-1 beta protein levels in multimedium and multihigh groups and IL-6 protein levels in all hUCB-MSCs-treated groups. Furthermore, mRNA levels of IL-1 beta and IL-6 were decreased significantly in all hUCB-MSCs-treated groups, whereas the expression of anti-inflammatory cytokine IL-10 was increased in the multihigh group. Tregs known as suppressor T cells were also significantly increased in the multihigh group. Our findings suggest that long-interval repeated intravenous administration of hUCB-MSCs has therapeutic effects by improving symptoms of RA in CIA mice model in a dose-dependent manner.

Effect of growth differentiation factor-15 secreted by human umbilical cord blood-derived mesenchymal stem cells on amyloid beta levels in in vitro and in vivo models of Alzheimer's disease.

Alzheimer's disease (AD), which is the most common progressive neurodegenerative disease, causes learning and memory impairment. The pathological progress of AD can derive from imbalanced homeostasis of amyloid beta (Aß) in the brain. In such cases, microglia play important roles in regulating the brain Aß levels. In the present study, we found that human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) can increase, through paracrine action, the ability of microglial cells to clear Aß. In order to identify the associated paracrine factors, a secretome of hUCB-MSCs co-cultured with Aß-treated BV2 microglial cells was analyzed using a human cytokine protein array. As a result, growth differentiation factor-15 (GDF-15) was identified as a predominant candidate, and its association with Aß clearance by microglial cells was investigated in vitro and in a 5XFAD mouse model. When Aß-treated BV2 cells were treated with exogenous recombinant GDF-15, the Aß levels in the culture medium decreased. Moreover, GDF-15 injection in the brain parenchyma of 5XFAD mice also led to decrease in Aß plaques. In contrast, co-culture of BV2 cells and hUCB-MSCs treated with GDF-15-specific siRNA did not influence the Aß levels in the culture medium. To elucidate how these phenomena are related, we confirmed that GDF-15 specifically increases insulin-degrading enzyme (IDE) expression in microglial cells through TGFß receptor type II (TGFßRII), both in vitro and in vivo. These findings suggest that hUCB-MSCs promote the Aß clearance ability of microglial cells through regulation of GDF-15 secretion, thus elucidating a therapeutic mechanism for AD.

[Cancer stem-like cell-derived exosomes promotes the proliferation and invasion of human umbilical cord blood-derived mesenchymal stem cells].

OBJECTIVE: To investigate the effect of Piwil2-induced cancer stem-like cell (Piwil2-iCSC)-derived exosomes on the proliferation,migration and invasion of human umbilical cord blood-derived mesenchymal stem cells (hucMSCs). METHODS: Piwil2-iCSC-derived exosomes were isolated by ultracentrifugation and identified using transmission electron microscopy,nanoparticle tracking analysis and Western blotting.Exosome uptake assay was used to identify the pathway that Piwil2-iCSCderived exosomes utilized.HucMSCs were divided into control group,PBS intervention group and exosome intervention group,and CCK-8 assay,wound healing assay,Transwell assay,Western blotting and cell karyotype analysis were used to observe the proliferation,migration,invasion,expression levels of MMP2 and MMP9 proteins,and chromosome structure of hucMSCs. RESULTS: The diameter of Piwil2-iCSC-derived exosomes ranged from 50 nm to 100 nm,and most of them were oval or spherical capsules rich in CD9,CD63 and Piwil2 proteins.Exosomal uptake assay showed that the exosomes executed theirs functions after entering the cells.Compared with the control cells and PBS-treated cells,hucMSCs treated with the exosomes showed significantly increased number of proliferating cells (P<0.05) with accelerated healing rate (P<0.05 at 24 h;P<0.01 at 48 h),increased invasive cells (P<0.01),enhanced protein expressions of MMP2(P<0.05 vs PBS group;P<0.01 vs control group) and MMP9(P<0.05),but their karyotype still remained 46XY without any abnormalities. CONCLUSIONS: Piwil2-iCSC-derived exosomes can promote the proliferation,migration and invasion but does not cause cancer-like heterogeneity changes in hucMSCs.

Cancer stem-like cell-derived exosomes promotes the proliferation and invasion of human umbilical cord blood-derived mesenchymal stem cells / 南方医科大学学报

OBJECTIVE@#To investigate the effect of Piwil2-induced cancer stem-like cell (Piwil2-iCSC)-derived exosomes on the proliferation,migration and invasion of human umbilical cord blood-derived mesenchymal stem cells (hucMSCs).@*METHODS@#Piwil2-iCSC-derived exosomes were isolated by ultracentrifugation and identified using transmission electron microscopy,nanoparticle tracking analysis and Western blotting.Exosome uptake assay was used to identify the pathway that Piwil2-iCSCderived exosomes utilized.HucMSCs were divided into control group,PBS intervention group and exosome intervention group,and CCK-8 assay,wound healing assay,Transwell assay,Western blotting and cell karyotype analysis were used to observe the proliferation,migration,invasion,expression levels of MMP2 and MMP9 proteins,and chromosome structure of hucMSCs.@*RESULTS@#The diameter of Piwil2-iCSC-derived exosomes ranged from 50 nm to 100 nm,and most of them were oval or spherical capsules rich in CD9,CD63 and Piwil2 proteins.Exosomal uptake assay showed that the exosomes executed theirs functions after entering the cells.Compared with the control cells and PBS-treated cells,hucMSCs treated with the exosomes showed significantly increased number of proliferating cells (<0.05) with accelerated healing rate (<0.05 at 24 h;<0.01 at 48 h),increased invasive cells (<0.01),enhanced protein expressions of MMP2(<0.05 PBS group;<0.01 control group) and MMP9(<0.05),but their karyotype still remained 46XY without any abnormalities.@*CONCLUSIONS@#Piwil2-iCSC-derived exosomes can promote the proliferation,migration and invasion but does not cause cancer-like heterogeneity changes in hucMSCs.

Optimization of culture conditions for rapid clinical-scale expansion of human umbilical cord blood-derived mesenchymal stem cells.

BACKGROUND: Mesenchymal stem cells (MSCs) have broad-spectrum therapeutic effects in various diseases, and thus have many clinical applications. However, it is difficult to produce sufficient numbers of MSCs for clinical use, and improved culture systems are required. Here, we report the effects of calcium (Ca2+) and hypoxia on the proliferation of human umbilical cord blood-derived MSCs (hUCB-MSCs). In addition, we determined the optimal conditions of these two factors for the large-scale culture of hUCB-MSCs. METHODS: hUCB-MSCs were maintained under hypoxic conditions (3% O2) with 1.8 mM Ca2+ during long-term culture, and their proliferation was evaluated. To characterize the underlying mechanisms, the effects on hypoxia-inducible factor (HIF)-1α and the extracellular signal-regulated kinase (ERK) signaling pathways were investigated. The therapeutic effects in a mouse emphysema model were analyzed and compared with those of naive MSCs. RESULTS: The proliferation of Ca2+/hypoxia-treated hUCB-MSCs was increased compared with that observed using either calcium or hypoxia culture alone, without loss of stem cell marker expression or differentiation ability. The enhancement of the proliferation capacity of hUCB-MSCs by the synergistic effects of Ca2+ and hypoxia was dependent on the expression of HIF-1α and the ERK signaling pathway. The proliferation of Ca2+/hypoxia-treated hUCB-MSCs resulted in a delayed senescence phenotype and increased the expression levels of stemness genes such as Oct4 and Nanog compared to those observed in conventional culture conditions. In addition, Ca2+/hypoxia-treated MSCs transplantation in the mouse emphysema model showed the same therapeutic effects as observed with naive MSCs. CONCLUSIONS: These findings suggest that a Ca2+/hypoxia-based expansion system has applications for the large-scale production of MSCs for therapeutic purposes.

Adenovirus vector-mediated ex vivo gene transfer of brain-derived neurotrophic factor (BDNF) tohuman umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) promotescrush-injured rat sciatic nerve regeneration.

This study was designed toinvestigate the efficacy of adenovirus vector-mediated brain-derived neurotrophic factor (BDNF) ex vivo gene transfer to human umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) in a rat sciatic nerve crush injury model. BDNF protein and mRNA expression after infection was checked through an enzyme-linked immunosorbent assay (ELISA) and quantitative real-time polymerase chain reaction (qRT-PCR). Male Sprague-Dawley rats (200-250g, 6 weeks old) were distributed into threegroups (n=20 each): the control group, UCB-MSC group, and BDNF-adenovirus infected UCB-MSC (BDNF-Ad+UCB-MSC) group. UCB-MSCs (1×106cells/10µl/rat) or BDNF-Ad+UCB-MSCs (1×106cells/10µl/rat)were transplantedinto the rats at the crush site immediately after sciatic nerve injury. Cell tracking was done with PKH26-labeled UCB-MSCs and BDNF-Ad+UCB-MSCs (1×106cells/10µl/rat). The rats were monitored for 4 weeks post-surgery. Results showed that expression of BDNF at both the protein and mRNA levels was higher inthe BDNF-Ad+UCB-MSC group compared to theUCB-MSC group in vitro.Moreover, BDNF mRNA expression was higher in both UCB-MSC group and BDNF-Ad+ UCB-MSC group compared tothe control group, and BDNF mRNA expression in theBDNF-Ad+UCB-MSC group was higher than inboth other groups 5days after surgeryin vivo. Labeled neurons in the dorsal root ganglia (DRG), axon counts, axon density, and sciatic function index were significantly increased in the UCB-MSC and BDNF-Ad+ UCB-MSCgroupscompared to the controlgroup four weeksaftercell transplantation. Importantly,the BDNF-Ad+UCB-MSCgroup exhibited more peripheral nerve regeneration than the other two groups.Our results indicate thatboth UCB-MSCs and BDNF-Ad+UCB-MSCscan improve rat sciatic nerve regeneration, with BDNF-Ad+UCB-MSCsshowing a greater effectthan UCB-MSCs.

Downregulation of Melanoma Cell Adhesion Molecule (MCAM/CD146) Accelerates Cellular Senescence in Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells.

UNLABELLED: Therapeutic applications of mesenchymal stem cells (MSCs) for treating various diseases have increased in recent years. To ensure that treatment is effective, an adequate MSC dosage should be determined before these cells are used for therapeutic purposes. To obtain a sufficient number of cells for therapeutic applications, MSCs must be expanded in long-term cell culture, which inevitably triggers cellular senescence. In this study, we investigated the surface markers of human umbilical cord blood-derived MSCs (hUCB-MSCs) associated with cellular senescence using fluorescence-activated cell sorting analysis and 242 cell surface-marker antibodies. Among these surface proteins, we selected the melanoma cell adhesion molecule (MCAM/CD146) for further study with the aim of validating observed expression differences and investigating the associated implications in hUCB-MSCs during cellular senescence. We observed that CD146 expression markedly decreased in hUCB-MSCs following prolonged in vitro expansion. Using preparative sorting, we found that hUCB-MSCs with high CD146 expression displayed high growth rates, multilineage differentiation, expression of stemness markers, and telomerase activity, as well as significantly lower expression of the senescence markers p16, p21, p53, and senescence-associated ß-galactosidase, compared with that observed in hUCB-MSCs with low-level CD146 expression. In contrast, CD146 downregulation with small interfering RNAs enhanced the senescence phenotype. In addition, CD146 suppression in hUCB-MSCs caused downregulation of other cellular senescence regulators, including Bmi-1, Id1, and Twist1. Collectively, our results suggest that CD146 regulates cellular senescence; thus, it could be used as a therapeutic marker to identify senescent hUCB-MSCs. SIGNIFICANCE: One of the fundamental requirements for mesenchymal stem cell (MSC)-based therapies is the expansion of MSCs during long-term culture because a sufficient number of functional cells is required. However, long-term growth inevitably induces cellular senescence, which potentially causes poor clinical outcomes by inducing growth arrest and the loss of stem cell properties. Thus, the identification of markers for evaluating the status of MSC senescence during long-term culture may enhance the success of MSC-based therapy. This study provides strong evidence that CD146 is a novel and useful marker for predicting senescence in human umbilical cord blood-derived MSCs (hUCB-MSCs), and CD146 can potentially be applied in quality-control assessments of hUCB-MSC-based therapy.

Mesenchymal cells condensation-inducible mesh scaffolds for cartilage tissue engineering.

Mesenchymal cells condensation is crucial in chondrogenic development. However current tissue-engineered scaffolds for chondrogenesis pay little attention to this phenomenon. In this study, we fabricate poly(l-lactide-co-glycolide) (PLGA)/poly(l-lactide) (PLLA) microfiber scaffolds and coat them with human fibroblast-derived matrix (hFDM) that is a decellularized extracellular matrix (ECM) obtained from in vitro cultured human lung fibroblasts (WI-38). Those scaffolds were then conjugated with heparin via EDC chemistry and subsequently immobilized with transforming growth factor (TGF)-ß1. The amount of TGF-ß1 was quantitatively measured and the release profile showed a continuous release of TGF-ß1 for 4 weeks. Human umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) were seeded in four different scaffolds; control, fibronectin (FN)-coated, hFDM-coated, hFDM/TGF-ß1 and subjected to chondrogenic differentiation in vitro for up to 28 days. Both hFDM and hFDM/TGF-ß1 groups exhibited significantly more synthesis of glycosaminoglycan (GAG) and much better upregulation of chondrogenic markers expression. Interestingly, MSCs condensation that led to cell aggregates was clearly observed with time in the two hFDM-coated groups and the quantitative difference was obvious compared to the control and FN group. A mechanistic study in gene and protein level indicated that focal adhesion kinase (FAK) was involved at the early stage of cell adhesion and cell-cell contact-related markers, N-cadherin and neural cell adhesion molecule (NCAM), were highly up-regulated at later time point. In addition histological analysis proved that hFDM/TGF-ß1 group was the most effective in forming neocartilage tissue in a rabbit articular cartilage defect model. Taken together, this study demonstrates not only the positive effect of hFDM on chondrogenesis of MSCs and cartilage repair but also provides an important insight toward the significance of in vitro mesenchymal condensation on chondrogenic development of MSCs.

Electro-acupuncture at Conception and Governor vessels and transplantation of umbilical cord blood-derived mesenchymal stem cells for treating cerebral ischemia/reperfusion injury.

Mesenchymal stem cell transplantation is a novel means of treating cerebral ischemia/reperfusion, and can promote angiogenesis and neurological functional recovery. Acupuncture at Conception and Governor vessels also has positive effects as a treatment for cerebral ischemia/reperfusion. Therefore, we hypothesized that electro-acupuncture at Conception and Governor vessels plus mesenchymal stem cell transplantation may have better therapeutic effects on the promotion of angiogenesis and recovery of neurological function than either treatment alone. In the present study, human umbilical cord blood-derived mesenchymal stem cells were isolated, cultured, identified and intracranially transplanted into the striatum and subcortex of rats at 24 hours following cerebral ischemia/reperfusion. Subsequently, rats were electro-acupunctured at Conception and Governor vessels at 24 hours after transplantation. Modified neurological severity scores and immunohistochemistry findings revealed that the combined interventions of electro-acupuncture and mesenchymal stem cell transplantation clearly improved neurological impairment and up-regulated vascular endothelial growth factor expression around the ischemic focus. The combined intervention provided a better outcome than mesenchymal stem cell transplantation alone. These findings demonstrate that electro-acupuncture at Conception and Governor vessels and mesenchymal stem cell transplantation have synergetic effects on promoting neurological function recovery and angiogenesis in rats after cerebral ischemia/reperfusion.

Effect of preemptive treatment with human umbilical cord blood-derived mesenchymal stem cells on the development of renal ischemia-reperfusion injury in mice.

Human umbilical cord blood-derived mesenchymal stem cells (HUCB-MSCs) have been studied in several models of immune-mediated disease because of their unique immunomodulatory properties. We hypothesized that HUCB-MSCs could suppress the inflammatory response in postischemic kidneys and attenuate early renal injury. In 8- to 10-wk-old male C57BL/6 mice, bilateral ischemia-reperfusion injury (IRI) surgery was performed, and 1 × 10(6) HUCB-MSCs were injected intraperitoneally 24 h before surgery and during reperfusion. Renal functional and histological changes, HUCB-MSC trafficking, leukocyte infiltration, and cytokine expression were analyzed. Renal functional decline and tubular injury after IRI were attenuated by HUCB-MSC treatment. PKH-26-labeled HUCB-MSCs trafficked into the postischemic kidney. Although numbers of CD45-positive leukocytes in the postischemic kidney were comparable between groups, the expression of interferon-γ in the postischemic kidney was suppressed by HUCB-MSC treatment. The rapid decrease in intrarenal VEGF after IRI was markedly mitigated by HUCB-MSC treatment. In inflammatory conditions simulated in a cell culture experiment, VEGF secretion from HUCB-MSCs was substantially enhanced. VEGF inhibitor abolished the renoprotective effect of HUCB-MSCs after IRI. Flow cytometry analysis revealed the decreased infiltration of natural killer T cells and increased number of regulatory T cells in postischemic kidneys. In addition, these effects of HUCB-MSCs on kidney infiltrating mononuclear cells after IRI were attenuated by VEGF inhibitor. HUCB-MSCs attenuated renal injury in mice in the early injury phase after IRI, mainly by humoral effects and secretion of VEGF. Our results suggest a promising role for HUCB-MSCs in the treatment of renal IRI.