Proportion Of Mesenchymal Stem Cells Research Articles

CHARACTERIZING CELLULAR COMPONENTS OF BONE MARROW ASPIRATE CONCENTRATE: A ONE-STEP CELL THERAPY FOR ANKLE CARTILAGE DEFECTS

AbstractObjectivesBone marrow aspirate concentrate (BMAC), together with fibrin glue (Tisseel, Baxter, UK) and Hyaluronic acid (HA) were used as a one-step cell therapy treating patients with ankle cartilage defects in our hospital. This therapy was proven to be safe, with patients demonstrating a significant improvement 12 months post-treatment. Enriched mesenchymal stem cells (MSCs) in BMAC are suggested inducers of cartilage regeneration, however, currently there is no point-of-care assessment for BMAC quality; especially regarding the proportion of MSCs within. This study aims to characterise the cellular component of CCR-generated BMAC using a point-of-care device, and to investigate if the total nucleated cell (TNC) count and patient age are predictive of MSC concentration.MethodsDuring surgery, 35ml of bone marrow aspirate (BMA) was collected from each patients’ iliac crest under anaesthesia, and BMAC was obtained via a commercial kit (Cartilage Regeneration kit, CCR, Innotec®, UK). BMAC was then mixed with thrombin (B+T) for injection with HA and fibrinogen. In our study, donor-matched BMA, BMAC and B+T were obtained from consented patients (n=12, age 41 ± 16years) undergoing surgery with BMAC therapy. TNC, red blood cell (RBC) and platelet (PLT) counts were measured via a haematology analyser (ABX Micros ES 60, Horiba, UK), and the proportion of MSCs in BMA, BMAC and B+T were assessed via colony forming unit-fibroblast (CFU-F) assays. Significant differences data in matched donors were tested using Friedman test. All data were shown as mean ± SD.ResultsMean TNC counts in BMA and BMAC were not significantly different (14.0 ± 4.4 million/ml and 19.4 ± 32.9 million/ml, respectively, P>0.9999). However, TNC counts were significantly lower in B+T compared to BMAC (9.7 ± 24.5 million/ml and 19.4 ± 32.9 million/ml, respectively, P=0.0167). Similarly, PLT counts were decreased in B+T compared to BMAC (40.7 ± 30.7 million/ml and 417.5 ± 365.5 million/ml, respectively, P<0.0001), however, PLTs were significantly concentrated in BMAC compared to BMA (417.5 ± 365.5 million/ml and 114.8 ± 61.6 million/ml, respectively, P=0.0429). RBC counts were significantly decreased in BMAC and B+T compared to BMA (P=0.0322 and P<0.0001, respectively). Higher concentration of MSCs were observed in BMAC compared to BMA (0.006% ± 0.01% and 0.00007% ± 0.0001%, respectively, P=0.0176). Similar to TNCs and PLTs, the proportion of MSCs significantly decreased in B+T compared to BMAC (0.0004% ± 0.001% and 0.006% ± 0.01%, respectively, P=0.0023). Furthermore, patient age and TNC counts did not correlate with MSC concentration (Spearman's Rank test, P=0.3266 and P=0.4880, respectively).ConclusionsBMAC successfully concentrated PLTs, but BMAC preparations were highly variable. Mixing BMAC and thrombin however, as described in the CCR protocol, resulted in a dramatic reduction in TNCs, PLTs and MSCs. TNC counts and patient age could not be used to predict the MSC proportion in the BMAC based on current data. Future work aims to look at the biomolecule profile of BMAC plasma, and to correlate them to patient clinical outcomes.Declaration of Interest(a) fully declare any financial or other potential conflict of interest

Comparison of adhesion of thawed and cultured synovial mesenchymal stem cells to the porcine meniscus and the relevance of cell surface microspikes

BackgroundPlacement of a cultured synovial mesenchymal stem cell (MSC) suspension on a repaired meniscus for 10 min accelerated meniscus repair. Upon placement of the MSC suspension on the meniscus, microspikes projecting from the MSC surface trap meniscus fibers and promote MSC adhesion. Thawed cryopreserved MSCs are preferred materials for meniscus repair, as they can be transplanted without additional culture. However, the adhesion ability of thawed cryopreserved MSCs is unknown. Here, we compared the proportion of cultured versus thawed MSCs adhering to a porcine meniscus immediately and 10 min after placement. We also investigated the relationship between adhesion and the number of microspikes on the synovial MSCs.MethodsSynovial MSCs were prepared from the knees of four donors with osteoarthritis. The “cultured MSCs” were thawed MSCs that were re-cultured and suspended in PBS for transplantation. A similarly prepared suspension was cryopreserved, thawed again, suspended in PBS, and used without further culture as the “thawed MSCs.” MSCs with at least three microspikes in SEM images were defined as microspike-positive MSCs. Porcine meniscus surfaces were abraded, cut into a cylindrical shape, and treated with MSC suspension. Non-adherent cells were counted immediately and again 10 min after placement to calculate the adhesion proportion.ResultsThe proportion of microspike-positive MSCs was significantly higher in thawed (53 ± 3%) than in cultured (28 ± 5%) MSC suspensions. MSC adhesion to the meniscus was significantly better for the thawed than for the cultured MSC suspensions immediately after placement on the meniscus, but no differences were detected after 10 min. The proportion of MSCs with microspikes in the cell suspension was significantly correlated with the proportion of adhered MSCs immediately after the placement, but not 10 min later. Addition of FBS to the cryopreservation medium promoted a concentration-dependent increase in the proportion of microspike-positive cells.ConclusionsThawed MSCs adhered better than cultured MSCs immediately after placement, but adhesion was similar for both MSC preparations after 10 min. Immediately after placement, the presence of microspikes was associated with better adhesion of synovial MSCs to the meniscus.

P336 Can Mesenchymal Stem Cell (MSC) apoptosis be used as a biomarker for treatment success in perianal fistulising Crohn’s Disease (pCD)? Findings from a prospective pilot study

Abstract Background Mesenchymal Stem Cells (MSC) demonstrated promising results in patients with refractory perianal fistulising Crohn’s Disease (pCD) in the ADMIRE-CD trial, however their use is limited by treatment cost. Studies of Graft vs. Host Disease (GvHD) patients receiving MSCs for steroid refractory disease have shown that in vitro Peripheral Blood Mononuclear Cell (PBMC) induced MSC apoptosis correlates with clinical response. We conducted a pilot prospective study to investigate the clinical and radiological outcome of MSC treatment and determine whether in vitro MSC apoptosis correlates with clinical response in patients with pCD. Methods Patient eligibility and MSC administration mirrored that in ADMIRE-CD. Prior to treatment, patient PBMCs were isolated and co-cultured with MSCs and the proportion of apoptotic MSCs identified using flow cytometry (Annexin V+, 7AAD- MSCs). Patients were followed up at, 6-monthly intervals and clinical indices including the Perianal Disease Activity Index (PDAI) and Fistula Drainage Assessment (FDA) were recorded in addition to MRI response. Clinical response categories were determined according to ADMIRE-CD as well as new criteria accounting for radiological healing (Table 1). Results Ten patients underwent MSC treatment (M=4, median age, 35, range, 22–73 years). At final follow up (median, 24 months, range, 18–30 months), there was a significant improvement in mean FDA (baseline, 1.3, final follow up, 0.5, p<0.01) but not in PDAI. The apoptosis assay was performed in, 9 patients. A greater proportion of MSC apoptosis was demonstrated when co-cultured with pCD patient PBMCs than with PBMCs from healthy controls (Figure, 1). Patients with a percentage of apoptotic MSCs greater than the group median (22.3%) were classed as apoptosishigh. At final follow up, apoptosishigh patients were also those who demonstrated a response to MSC treatment according to both St Mark’s (Figure, 2) and ADMIRE CD criteria (Figure 3). Conclusion The ability of patient PBMCs to induce the highest proportions of MSC apoptosis correlates with clinical response to MSC treatment in this small sample of patients. An adequately powered prospective study is required to determine the potential use of MSC apoptosis as a biomarker of clinical response.

Efficient mesenchymal stem cell conversion to corneal keratocytes by the use of specific biomaterial and Cell Rev™ mSC diffKera media

Background & Aim Corneal blindness is a prevalent cause of vision loss, affecting approximately 4.9 million people worldwide (Flaxman et al, 2017). The demand for donor replacement corneas often exceeds the supply, requiring alternative treatment options. To achieve corneal function while minimizing the risks of donor rejection, we propose an autologous biomimetic cornea whereby patient-derived mesenchymal stem cells (MSCs) are converted to corneal-like cells on scaffolds that closely resemble the structure of the human cornea. We hypothesize that the effectiveness of cell differentiation depends on the biomaterials used and the cultured media composition. The more closely the environment resembles that of the human cornea, the higher the likelihood that a greater proportion of MSCs will differentiate into corneal keratocytes. Methods, Results & Conclusion Here, we investigated MSC differentiation into corneal keratocytes using various medium formulations on our electrospun scaffolds. Polycaprolactone-bovine dermis type I atelocollagen (PCL-atelocollagen) and polycaprolactone (PCL) solutions were electrospun to obtain the aligned nanofibrous scaffolds. MSCs were cultured on the scaffolds for 12 days in 3 different types of media: the expansion media described by Lynch & Ahearne (2007); keratocyte differentiation media used in the same study; and our Cell Rev™ mSC diffKera media. Immunofluorescence staining for keratocyte markers such as aldehyde dehydrogenase 1 and lumican was conducted on cells fixed on the scaffolds at days 6 and 12. The results confirmed that a greater proportion of MSCs differentiated into keratocytes in Cell Rev™ mSC diffKera media compared with the media used by Lynch & Ahearne (Figure 1). In addition, electrospun PCL-atelocollagen scaffolds are likely to support keratocyte differentiation better than electrospun PCL scaffolds (control) because corneal stromal collagen fibrils comprise mainly type I collagen. These findings demonstrate that the use of our electrospun PCL-atelocollagen scaffolds and Cell Rev™ mSC diffKera formulation effectively enhances MSC differentiation into keratocytes, and represents a novel step for the creation of artificial corneas. Corneal blindness is a prevalent cause of vision loss, affecting approximately 4.9 million people worldwide (Flaxman et al, 2017). The demand for donor replacement corneas often exceeds the supply, requiring alternative treatment options. To achieve corneal function while minimizing the risks of donor rejection, we propose an autologous biomimetic cornea whereby patient-derived mesenchymal stem cells (MSCs) are converted to corneal-like cells on scaffolds that closely resemble the structure of the human cornea. We hypothesize that the effectiveness of cell differentiation depends on the biomaterials used and the cultured media composition. The more closely the environment resembles that of the human cornea, the higher the likelihood that a greater proportion of MSCs will differentiate into corneal keratocytes. Here, we investigated MSC differentiation into corneal keratocytes using various medium formulations on our electrospun scaffolds. Polycaprolactone-bovine dermis type I atelocollagen (PCL-atelocollagen) and polycaprolactone (PCL) solutions were electrospun to obtain the aligned nanofibrous scaffolds. MSCs were cultured on the scaffolds for 12 days in 3 different types of media: the expansion media described by Lynch & Ahearne (2007); keratocyte differentiation media used in the same study; and our Cell Rev™ mSC diffKera media. Immunofluorescence staining for keratocyte markers such as aldehyde dehydrogenase 1 and lumican was conducted on cells fixed on the scaffolds at days 6 and 12. The results confirmed that a greater proportion of MSCs differentiated into keratocytes in Cell Rev™ mSC diffKera media compared with the media used by Lynch & Ahearne (Figure 1). In addition, electrospun PCL-atelocollagen scaffolds are likely to support keratocyte differentiation better than electrospun PCL scaffolds (control) because corneal stromal collagen fibrils comprise mainly type I collagen. These findings demonstrate that the use of our electrospun PCL-atelocollagen scaffolds and Cell Rev™ mSC diffKera formulation effectively enhances MSC differentiation into keratocytes, and represents a novel step for the creation of artificial corneas.

Emulsified Fat Grafting Accelerates Tissue Expansion: An Experimental Study in a Rat Model.

Tissue expansion has been applied in tissue repair and reconstruction of large soft tissue defects. Stromal vascular fraction (SVF) transplantation is a promising treatment in raising expansion efficiency. However, the clinical utilization of SVF is limited because of its conventional collagenase-based production. The aim of this study was to evaluate the effect of emulsified fat (EF), SVF obtained by using mechanical method, on accelerating tissue expansion. The microstructure of EF fragments and the proportion of mesenchymal stem cells (MSCs; CD45-/CD34+) in EF were detected. Wistar rats were divided into the following 3 groups randomly: the 1-mL EF group, the 0.5-mL EF group, and the control group. The tissue expansion was carried out twice a week to maintain the capsule pressure at 60 mm Hg. After 4 weeks, inflation volume and histological changes, which includes collagen content, cell proliferation, and capillary density, were observed to evaluate the effect of EF on tissue expansion. Mechanical emulsification effectively destroyed the mature adipocytes in adipose tissue. The proportion of MSCs population in the EF fragments was 12.40 ± 0.86%. After expansion, the inflation volume and the levels of collagen deposition, cell proliferation, and capillary density of the expanded tissue in the 1-mL EF group were significantly higher than that in the 0.5-mL EF group and the control group (P < 0.05). However, all these regenerative indicators in the 0.5-mL EF group showed no statistical difference from the control group (P > 0.05). The thickness of epidermal and dermal layers showed no significant difference among the 3 groups (P > 0.05). Our findings suggested that EF grafting can be used as a new alternative to increase tissue expansion efficiency.

Treatment of Periodontal Bone Defects with Stem Cells from Inflammatory Dental Pulp Tissues in Miniature Swine.

Containing a certain proportion of mesenchymal stem cells, inflammatory dental tissue showed great tissue regeneration potential in recent years. However, whether it is applicable to promote tissue regeneration in vivo remains to be elucidated. Therefore, we evaluated the feasibility of stem cells from inflammatory dental pulp tissues (DPSCs-IPs) to reconstruct periodontal defects in miniature pigs. The autologous pig DPSCs-IPs were first cultured, appraised and loaded onto β-tricalcium phosphate (β-TCP). The compounds were then engrafted into an artificially-created periodontal defect. Three months later, the extent of periodontal regeneration was evaluated. Clinical examination, radiological examination and immunohistochemical staining were used to assess periodontal regeneration. The data collectively showed that DPSCs-IPs from miniature pigs expressed moderate to high levels of STRO-1 and CD146 as well as low levels of CD34 and CD45. DPSCs-IPs have osteogentic, adipogenic and chondrogenic differentiation abilities. DPSCs-IPs were engrafted onto β-TCP and regenerated bone to repair periodontal defects by 3months' post-surgical reconstruction. Autologous DPSCs-IPs may be a feasible means of periodontal regeneration in miniature pigs.

Regeneration of esophagus using a scaffold-free biomimetic structure created with bio-three-dimensional printing.

Various strategies have been attempted to replace esophageal defects with natural or artificial substitutes using tissue engineering. However, these methods have not yet reached clinical application because of the high risks related to their immunogenicity or insufficient biocompatibility. In this study, we developed a scaffold-free structure with a mixture of cell types using bio-three-dimensional (3D) printing technology and assessed its characteristics in vitro and in vivo after transplantation into rats. Normal human dermal fibroblasts, human esophageal smooth muscle cells, human bone marrow-derived mesenchymal stem cells, and human umbilical vein endothelial cells were purchased and used as a cell source. After the preparation of multicellular spheroids, esophageal-like tube structures were prepared by bio-3D printing. The structures were matured in a bioreactor and transplanted into 10-12-week-old F344 male rats as esophageal grafts under general anesthesia. Mechanical and histochemical assessment of the structures were performed. Among 4 types of structures evaluated, those with the larger proportion of mesenchymal stem cells tended to show greater strength and expansion on mechanical testing and highly expressed α-smooth muscle actin and vascular endothelial growth factor on immunohistochemistry. Therefore, the structure with the larger proportion of mesenchymal stem cells was selected for transplantation. The scaffold-free structures had sufficient strength for transplantation between the esophagus and stomach using silicon stents. The structures were maintained in vivo for 30 days after transplantation. Smooth muscle cells were maintained, and flat epithelium extended and covered the inner surface of the lumen. Food had also passed through the structure. These results suggested that the esophagus-like scaffold-free tubular structures created using bio-3D printing could hold promise as a substitute for the repair of esophageal defects.

Bone Mesenchymal Stem Cell-Enriched β-Tricalcium Phosphate Scaffold Processed by the Screen-Enrich-Combine Circulating System Promotes Regeneration of Diaphyseal Bone Non-Union

Bone non-union after fracture, considered a therapeutic challenge for orthopedics, always needs a reversion surgery, including autograft transplantation (AGT). However, adverse events related to autograft harvest cannot be ignored. Our group designed a novel system called the bone marrow stem cell Screen-Enrich-Combine Circulating System (SECCS) by seeding mesenchymal stem cells (MSCs) into β-tricalcium phosphate (β-TCP) during surgery to thereafter rapidly process bioactive bone implantation. In this retrospective case-control study, 30 non-union patients who accepted SECCS therapy and 20 non-union patients who accepted AGT were enrolled. By SECCS therapy, the MSC-enriched β-TCP particles were implanted into the non-union gap. During the enrichment procedure, a significant proportion of MSCs were screened and enriched from bone marrow into porous β-TCP particles, and the cells possessed the capacity for three-line differentiation and were CD90+/CD105+/CD34-/CD45-. Approximately 82.0±10.7% of MSCs were enriched from 60 mL bone marrow without damaging cell viability, and approximately 11,444.0±6,018 MSCs were transplanted per patient. No implant-related infections occurred in any case. After 9 months of follow-up, 27 patients (90%) in the SECCS group acquired clinical union, compared with 18 patients (90%) in the AGT group (clinical union time, P = 0.064), and postoperative radiographic union score at 9 months post-operation was similar between the two groups. In conclusion, the SECCS could concentrate a large proportion of MSCs from bone marrow to acquire enough effective cells for therapy without in vitro cell culture. Bone substitutes processed by SECCS demonstrated encouraging promotion of bone regeneration and showed a satisfactory clinical curative effect for diaphyseal bone non-union, which was non-inferior to AGT.

Mesenchymal stem cells participate in oral mucosa carcinogenesis by regulating T cell proliferation

Recent evidences suggested that Mesenchymal stem cells (MSCs) may be involved in tumor formation by modulating of the tumor microenvironment, but it is still unclear the potential of MSCs in the malignant transformation of oral mucosa. Using a chemically-induced oral carcinogenesis model by 4-nitroquinoline-1-oxide (4NQO), we generated precancerous lesions and cancerous lesions in the oral cavity of rats. Flow cytometric analysis on lesions derived single cell suspension revealed an increase in the proportion of MSCs and a decreased proportion of T cell during oral mucosa malignancy. Moreover, MSCs showed increased immunosuppression capacity on T cell proliferation during mucosa malignancy. At last, we demonstrated that higher frequency of lesions resident MSCs was correlated with more Ki67 expression in the lesion, which indicated higher cellular proliferative status in the lesions. Our study demonstrated that MSCs may play an important role in oral mucosa malignant transformation through regulating T cell proliferation.

Mesenchymal Stem Cells Gene Signature in High-Risk Myeloma Bone Marrow Linked to Suppression of Distinct IGFBP2-Expressing Small Adipocytes

Introduction:Certain studies suggest that multiple myeloma (MM) induces expansion of bone marrow (BM) mesenchymal stem cells (MSCs), but others showed induction of MSC senescence. MM cells suppress MSC lineages such as osteoblasts, while their effects on adipocytes remain to be elucidated. Recent studies identified “regulated” adipocytes that are smaller in size than large adipocytes that are constitutively present (Scheller et al, Nature Commun 2015), and that function as an endocrine tissue and regulators of hematopoiesis (Cawthorn et al, Cell Metab 2014), suggesting that BM adipocytes are functionally heterogeneous.We established a MSC gene signature in whole bone biopsies and showed that it gradually changes in different disease stages and is associated with outcome (Schinke et al, CCR 2018).The aim of the study was to identify changes in expression of MSC genes in BM of patients with high risk MM and in focal lesions (FLs), and to elucidate whether these changes reflect altered proportion and function of MSCs and their lineages.Methods:MSC gene expression in whole bone biopsies from normal donors (n=68), and patients with MGUS/SMM (n=90) and MM (n=531) was analyzed using global gene expression profiles. Unexpanded single MSCs from normal donors (n=3; 175 single MSCs) and MM patients (n=3; 162 single MSCs) were sorted by FACSAria and expression of mesenchymal cell, proliferation and senescence markers were analyzed by qRT-PCR using Fluidigm Biomark HD. Functionally, single MSCs were tested for their ability to multiply using supportive serum and MSC-conditioned media. Cell senescence was analyzed by SA-βGal staining. IGFBP2 and adiponectin protein were detected using immunohistochemistry (IHC). Numbers of IGFBP2+ cells were analyzed in biopsies from patients with MGUS/SMM, low risk (LR) and high risk (HR) MM (10 biopsies/group). Cultured MSCs were differentiated to adipocytes by treatment with dexamethasone, insulin and indomethacin for 3-4 weeks. The effects of recombinant IGF1 and IGFBP2 on MM cell growth were performed on BM-dependent MM lines (n=3) cultured in serum-free conditions for 48 hrs.Results:We compared MSC gene expression levels in random interstitial BM biopsies of patients with LR and HR MM, and in paired biopsy samples from random BM and FLs; 41 of the 345 MSC genes were differentially expressed in both comparisons. Most overexpressed genes were related to angiogenesis and ECM, including several collagen genes (e.g., COL4A1, POSTN, and HSPG2). Several underexpressed genes were associated with adipocytes, including IGFBP2 and aldo/keto reductases.To unravel whether these differences in gene expression reflect changes in the proportion of MSCs we tested expression of genes associated with MSCs, osteoblastogenesis, adipogenesis, proliferation and senescence in unexpanded singleMSCs. MM single MSCs had significant reduction in expression of KI67 and increased expression of the senescence marker, CDKN2A/p16, whereas expression of other tested genes were modestly differentially expressed between the two groups. Functionally, unexpanded MM MSCs had increased SA-βGal expression, and only 65±8% of single MSCs divided at least once compared to 90±4% of their normal donor counterparts (p<0.03), suggesting that the in vivo MSC gene expression in MM reflects modifications in mesenchymal cell lineages that encompass most of the mesenchymal compartment in bone.Since IGFBP2 is involved in adipogenesis and bone homeostasis we traced the source of this factor using IHC. Double staining for IGFBP2 and adiponectin revealed that IGFBP2 was mainly expressed by small adipocytes. The number of IGFBP2+ cells was higher in BM biopsies from patients with MGUS/SMM than in those from patients with MM (p<0.02), and the number was lower in biopsies from patients with HR MM than with LR MM (p<0.01).Normal MSCs differentiated to adipocytes produced a high level of IGFBP2, while co-culturing MSCs with MM cells inhibits their differentiation to adipocytes by 6 folds (p<0.001) and reduced expression of IGFBP2 and adiponectin. Recombinant IGFBP2 effectively blocked IGF1-mediated growth of MM cells, indicating its role in controlling IGF1 bioavailability.Conclusions:Our data demonstrate that MM MSCs are less proliferative and that IGFBP2+ small adipocytes are a distinct mesenchymal cell population suppressed by MM, and their depletion may contribute to disease progression. DisclosuresEpstein:University of Arkansas for Medical Sciences: Employment. Davies:Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; MMRF: Honoraria; ASH: Honoraria; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy; TRM Oncology: Honoraria. Morgan:Takeda: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Janssen: Research Funding.

Current Strategies to Generate Human Mesenchymal Stem Cells In Vitro.

Mesenchymal stem cells (MSCs) are heterogeneous multipotent stem cells that are involved in the development of mesenchyme-derived evolving structures and organs during ontogeny. In the adult organism, reservoirs of MSCs can be found in almost all tissues where MSCs contribute to the maintenance of organ integrity. The use of these different MSCs for cell-based therapies has been extensively studied over the past years, which highlights the use of MSCs as a promising option for the treatment of various diseases including autoimmune and cardiovascular disorders. However, the proportion of MSCs contained in primary isolates of adult tissue biopsies is rather low and, thus, vigorous ex vivo expansion is needed especially for therapies that may require extensive and repetitive cell substitution. Therefore, more easily and accessible sources of MSCs are needed. This review summarizes the current knowledge of the different strategies to generate human MSCs in vitro as an alternative method for their applications in regenerative therapy.

Incorporating Refractory Period in Mechanical Stimulation Mitigates Obesity-Induced Adipose Tissue Dysfunction in Adult Mice.

ObjectiveTo determine whether inclusion of a refractory period between bouts of low magnitude mechanical stimulation (LMMS) can curb obesity-induced adipose tissue dysfunction and sequelae in adult mice.MethodsA diet-induced obesity model with 45kcal% fat diet was employed with intention-to-treat. C57BL/6J mice were weight-matched into four groups: low fat diet (LFD, n=8), high fat diet (HF, n=8), high fat diet with one bout of 30min LMMS (HFv, n=9), and high fat diet with two bouts of 15min LMMS with 5-hour separation (refractory period, RHFv, n=9). 2-week of diet was followed by 6-week of diet+LMMS.ResultsHF and HFv continued gaining body weight and visceral adiposity throughout the experiment, which was mitigated in RHFv. HF and HFv had increased adipocyte hypertrophy, immune cell infiltration (B-cells, T cells, and macrophages) into adipose tissue, adipose tissue inflammation (TNF-α gene expression), and decreased proportion of mesenchymal stem cells in adipose tissue, as compared to LFD, all of which was rescued in RHFv. Glucose intolerance, sequelae of adipose tissue dysfunction, were elevated in HF and HFv, but not in RHFv, as compared to LFD.ConclusionIncorporating a 5-hour refractory period between bouts of LMMS attenuates obesity-induced adipose tissue dysfunction and mitigates glucose intolerance.

Gelatin Directly Enhances Neurogenic Differentiation Potential in Bone Marrow-Derived Mesenchymal Stem Cells Without Stimulation of Neural Progenitor Cell Proliferation

Gelatin has been reported to induce generation of mesenchymal stem cells (MSCs) with enhanced potential of differentiation into neuronal lineage cells. However, the presence of various cell types besides MSCs in bone marrow has raised doubts about the effects of gelatin. In the following report, we determined whether gelatin can directly enhance neurogenic differentiation potential in MSCs without proliferation of neural progenitor cells (NPCs). MSCs comprised a high proportion of bone marrow-derived primary cells (BMPCs) and gelatin induced significant increases in MSC proliferation during primary culture, and the proportion of MSCs was maintained at more than 99% throughout the subculture. However, NPCs comprised a low percentage of BMPCs and a decrease in proliferation was detected despite gelatin treatment during the primary culture, and the proportion of subcultured NPCs gradually decreased. In a similar manner, MSCs exposed to gelatin during primary culture showed more enhanced neurogenic differentiation ability than those not exposed to gelatin. Together, these results demonstrate that gelatin directly enhances neurogenic differentiation in bone marrow-derived MSCs without stimulating NPC proliferation.

Isolation and Identification of Human Umbilical Cord and Placenta-derived Stem Cells and Their Component Analysis

Clinical transplantation evidence has been indicated that umbilical cord blood (UCB) can be useful in the hematopoietic reconstitution in the children, but can not be well in the adult hematopoietic transplantation because of the low count. This study was to evaluate a new method for collecting stem cells from human placenta and umbilical cord, and to comparatively analyze the similarity and difference of quality and quantity of the cells. The UCB was collected, in same time the placental tissue was sterily collected; the umbilical placenta was collected by perfusing the blood (UPB) cord arterial and venous vascules with 0.9% saline; the mesenchymal stem cells (MSC) from same source of umbilical cord and placental tissues were isolated and cultured. The cell colony assay and flow cytometry were performed to determine the proliferation capacities and cell markers of UMSC and PMSC. The total nuclear cells (NC) and hematopoietic stem cells (CD34(+)) from UPB and UCB were (17.45 ± 16.86) × 10(8), (6.9 ± 4.61) × 10(8) and (2.97 ± 2.25) × 10(6), (1.91 ± 1.7) × 10(6), respectively. Furthermore, the UPB contained more early precursor of hematopoietic stem cells (CD33(+) CD34(-)) (6.2 ± 13.5) × 10(5), (0.2 ± 0.8) × 10(5) ; and high proportion of MSC to NC (25.21 ± 18.69, 0.05 ± 0.10)%, respectively in 62 samples. There were no difference of the MSC level in UPB and UCB, as well as in the morphology and cell markers. UPB has rich hematopoietic stem cells and mesenchymal stem cells. Placenta may offer another source for hematopoietic stem cells in research of hematopoietic stem cells and regeneration medicine.

Effect of donor age on the proportion of mesenchymal stem cells derived from anterior cruciate ligaments.

The characteristics of anterior cruciate ligament (ACL)-derived mesenchymal stem cells (MSCs), such as proportion and multilineage potential, can be affected by donor age. However, the qualitative and quantitative features of ACL MSCs isolated from younger and older individuals have not yet been compared directly. This study assessed the phenotypic and functional differences in ACL-MSCs isolated from younger and older donors and evaluated the correlation between ACL-MSC proportion and donor age. Torn ACL remnants were harvested from 36 patients undergoing ACL reconstruction (young: 29.67 ± 10.92 years) and 33 undergoing TKA (old: 67.96 ± 5.22 years) and the proportion of their MSCs were measured. The mean proportion of MSCs was slightly higher in older ACL samples of the TKA group than of the younger ACL reconstruction group (19.69 ± 8.57% vs. 15.33 ± 7.49%, p = 0.024), but the proportions of MSCs at passages 1 and 2 were similar. MSCs from both groups possessed comparable multilineage potentiality, as they could be differentiated into adipocytes, osteocytes, and chondrocytes at similar level. No significant correlations were observed between patient age and MSC proportions at passages 0–2 or between age and MSC proportion in both the ACL reconstruction and TKA groups. Multiple linear regression analysis found no significant predictor of MSC proportion including donor age for each passage. Microarray analysis identified several genes that were differentially regulated in ACL-MSCs from old TKA patients compared to young ACL reconstruction patients. Genes of interest encode components of the extracellular matrix (ECM) and may thus play a crucial role in modulating tissue homeostasis, remodeling, and repair in response to damage or disease. In conclusion, the proportion of freshly isolated ACL-MSC was higher in elderly TKA patients than in younger patients with ACL tears, but their phenotypic and multilineage potential were comparable.

Ultrasound-Targeted Microbubble Destruction Improves the Migration and Homing of Mesenchymal Stem Cells after Myocardial Infarction by Upregulating SDF-1/CXCR4: A Pilot Study.

Mesenchymal stem cell (MSC) therapy shows considerable promise for the treatment of myocardial infarction (MI). However, the inefficient migration and homing of MSCs after systemic infusion have limited their therapeutic applications. Ultrasound-targeted microbubble destruction (UTMD) has proven to be promising to improve the homing of MSCs to the ischemic myocardium, but the concrete mechanism remains unclear. We hypothesize that UTMD promotes MSC homing by upregulating SDF-1/CXCR4, and this study was aimed at exploring this potential mechanism. We analyzed SDF-1/CXCR4 expression after UTMD treatment in vitro and in vivo and counted the number of homing MSCs in MI areas. The in vitro results demonstrated that UTMD not only led to elevated secretion of SDF-1 but also resulted in an increased proportion of MSCs that expressed surface CXCR4. The in vivo findings show an increase in the number of homing MSCs and higher expression of SDF-1/CXCR4 in the UTMD combined with MSCs infusion group compared to other groups. In conclusion, UTMD can increase SDF-1 expression in the ischemic myocardium and upregulate the expression of surface CXCR4 on MSCs, which provides a molecular mechanism for the homing of MSCs assisted by UTMD via SDF-1/CXCR4 axis.

CXCR4 Receptor Overexpression in Mesenchymal Stem Cells Facilitates Treatment of Acute Lung Injury in Rats

Novel therapeutic regimens for tissue renewal incorporate mesenchymal stem cells (MSCs) as they differentiate into a variety of cell types and are a stem cell type that is easy to harvest and to expand in vitro. However, surface chemokine receptors, such as CXCR4, which are involved in the mobilization of MSCs, are expressed only on the surface of a small proportion of MSCs, and the lack of CXCR4 expression may underlie the low efficiency of homing of MSCs toward tissue damage, which results in a poor curative effect. Here, a rat CXCR4 expressing lentiviral vector was constructed and introduced into MSCs freshly prepared from rat bone marrow. The influence of CXCR4 expression on migration, proliferation, differentiation, and paracrine effects of MSCs was examined in vitro. The in vivo properties of CXCR4-MSCs were also investigated in a model of acute lung injury in rats induced by lipopolysaccharide. Expression of CXCR4 in MSCs significantly enhanced the chemotactic and paracrine characteristics of the cells in vitro but did not affect self-renewal or differentiation into alveolar and vascular endothelial cells. In vivo, CXCR4 improved MSC homing and colonization of damaged lung tissue, and furthermore, the transplanted CXCR4-MSCs suppressed the development of acute lung injury in part by modulating levels of inflammatory molecules and the neutrophil count. These results indicated that efficient mobilization of MSCs to sites of tissue injury may be due to CXCR4, and therefore, increased expression of CXCR4 may improve their therapeutic potential in the treatment of diseases where tissue damage develops.

Identification of mesenchymal stem cells and osteogenic factors in bone marrow aspirate and peripheral blood for spinal fusion by flow cytometry and proteomic analysis.

BackgroundAn in vivo animal study and a prospective clinical study have indicated that bone marrow aspirate (BMA) augments spinal arthrodesis. However, there is no quantified data to explain why fusion rate can be augmented by BMA in lumbar posterolateral fusion.MethodsTo analyze the proportion of mesenchymal stem cells (MSCs) and osteogenic factors in human BMA and peripheral blood (PB) of the same patient. Autologous BMA and PB from the patients were analyzed by flow cytometry (FACS) using cell markers for MSCs. The osteogenic potential of MSCs was determined by alkaline phosphatase (ALP) activity and calcium level quantification. Proteomics were used for the qualitative and quantitative mapping of the whole proteome from BMA and PB plasma. The mass-to-charge ratio was calculated by time-of-flight mass spectrometry (TOF-MS). The overexpression of protein was confirmed using Western blot analysis.ResultsThe proportion of MSCs (CD34−/CD29+/CD105+) was higher in the BMA than that in the PB. Colony-forming cell (CFC) assays suggested that fewer colonies were formed in PB cultures than in BMA culture. There was no significant difference in the osteogenic potential of the MSCs between the PB and BMA. Proteomic mass spectrometry assays suggested that the levels of catalase (osteoclast inhibitor) and glutathione peroxidase 3 (osteogenic biomarker) were higher in the BMA than those in the PB, and this was confirmed by Western blot analysis.ConclusionsThe proportions of MSCs and osteogenic factors were higher in the BMA than in the PB. This may explain why fusion rate can be augmented by BMA in lumbar posterolateral fusion.

Evaluation of CD24 as a marker to rapidly define the mesenchymal stem cell phenotype and its differentiation in human nucleus pulposus

Background Recent studies have indicated that human nucleus pulposus contain mesenchymal stem cells (NP-MSCs). However, the immunophenotypic variation of NP-MSCs in vitro was unclear. The present study was conducted to address the immunophenotypic variation of mesenchymal stem cells in nucleus pulposus under continuous proliferation in vitro and show the difference between mesenchymal stem cells and nucleus pulposus cell. Methods Tissue samples were obtained from thoracolumbar burst fracture patients and degenerative disc disease patients who underwent discectomy and fusion procedures. Flow cytometric and laser scanning confocal microscope (LSCM) were used to detect the variation of mesenchymal stem cells in nucleus pulposus which were expressing CD105 and CD24 in condition with or without transforming growth factor β1 (TGF-β1). Results More than 90% of the analyzed primary cells of mesenchymal stem cells in nucleus pulposus fulfilled the general immunophenotyping criteria for MSCs, such as CD44, CD105 and CD29, but the marker of mature NP cells characterized as CD24 was negative. In continuous cultures, the proportion of mesenchymal stem cells which were expressing CD44, CD105 and CD29 in nucleus pulposus gradually decreased. The mesenchymal stem cells in nucleus pulposus cells were positive for CD105 and CD29, with slight positivity for CD44. The CD24 expression gradually increased in proliferation. Bi-parametric flow cytometry and laser scanning confocal microscopy confirmed the presence of cells which were expressing CD105 and CD24 independently, and only a small part of cells expressed both CD105 and CD24 simultaneously. TGF-β1 could stimulate mesenchymal stem cells in nucleus pulposus to express CD24. Conclusions Non-degenerative and degenerative NP contains mesechymal stem cells. The variation of CD24 can be used as a marker to identify the NP-MSCs differentiation into NP-like cells.

Donor age negatively affects the immunoregulatory properties of both adipose and bone marrow derived mesenchymal stem cells

PurposeAge negatively impacts the biologic features of mesenchymal stem cells (MSCs), including decreased expansion kinetics and differentiation potential. Clinically, donor-age may be within a wide spectrum; therefore, investigation of the role of donor's age on immunoregulatory potential is of critical importance to translate stem cell therapies from bench to bedside. MethodsAdipose and bone marrow derived MSCs (ASCs and BMSCs) were isolated in parallel from Lewis and Brown Norway rats of young (less than 4-week old) and senior groups (older than 15-month). The presentation of cells and time required for growth to 90% confluence was recorded. FACS sorting based on the expression of CD90 and CD29 double positive and CD45 CD11 double negative quantified the proportions of MSCs. After expansion, ASCs and BMSCs from different age groups were co-cultured in mixed lymphocyte reaction (MLR; Lewis vs. Brown Norway) assays. The suppression of CD3+CD4+ and CD3+CD8+ T cell populations by different sources of MSCs were compared. ResultsThe kinetics of cell growth was slower in old animals (17.3±2days) compared with young animals (8.8±3days), and cell morphology was irregular and enlarged in the senior groups. The yield of MSCs by FACS sorting was significantly higher in young groups compared to senior groups (p<0.02). With regard to immunoregulatory potential, senior ASCs failed to induce any CD3+CD4+ T cell suppression (p>0.05). In addition, young BMSCs-induced suppression was more prominent than seniors (p<0.05). ConclusionsDonor age should be taken into consideration when using recipient MSC of either bone marrow or adipose origin in clinical applications.