Colony-forming Unit-fibroblast Assay Research Articles

Establishment and Evaluation Strategy of an in vitro Cell Model of Bone Marrow Microenvironment Injury in Mouse Acute Graft-Versus-Host Disease

To establish a mesenchymal stem cell（MSC）-based in vitro cell model for the evaluation of mouse bone marrow acute graft-versus-host disease (aGVHD). Female C57BL/6N mice aged 6-8 weeks were used as bone marrow and lymphocyte donors, and female BALB/c mice aged 6-8 weeks were used as aGVHD recipients. The recipient mouse received a lethal dose (8.0 Gy,72.76 cGy/min) of total body γ irradiation, and injected with donor mouse derived bone marrow cells (1×107/mouse) in 6-8 hours post irradiation to establish a bone marrow transplantation (BMT) mouse model (n=20). In addition, the recipient mice received a lethal dose (8.0 Gy,72.76 cGy/min) of total body γ irradiation, and injected with donor mouse derived bone marrow cells (1×107/mouse) and spleen lymphocytes (2×106/mouse) in 6-8 hours post irradiation to establish a mouse aGVHD model (n=20). On the day 7 after modeling, the recipient mice were anesthetized and the blood was harvested post eyeball enucleation. The serum was collected by centrifugation. Mouse MSCs were isolated and cultured with the addition of 2%, 5%, and 10% recipient serum from BMT group or aGVHD group respectively. The colony-forming unit-fibroblast（CFU-F) experiment was performed to evaluate the potential effects of serums on the self-renewal ability of MSC. The expression of CD29 and CD105 of MSC was evaluated by immunofluorescence staining. In addition, the expression of self-renewal-related genes including Oct-4, Sox-2, and Nanog in MSC was detected by real-time fluorescence quantitative PCR（RT-qPCR). We successfully established an in vitro cell model that could mimic the bone marrow microenvironment damage of the mouse with aGVHD. CFU-F assay showed that, on day 7 after the culture, compared with the BMT group, MSC colony formation ability of aGVHD serum concentrations groups of 2% and 5% was significantly reduced （P < 0．05）; after the culture, at day 14, compared with the BMT group, MSC colony formation ability in different aGVHD serum concentration was significantly reduced （P < 0．05）. The immunofluorescence staining showed that, compared with the BMT group, the proportion of MSC surface molecules CD29+ and CD105+ cells was significantly dereased in the aGVHD serum concentration group (P < 0.05), the most significant difference was at a serum concentration of 10% （P < 0．001, P < 0.01）. The results of RT-qPCR detection showed that the expression of the MSC self-renewal-related genes Oct-4, Sox-2, and Nanog was decreased, the most significant difference was observed at an aGVHD serum concentration of 10% （P < 0.01,P < 0．001,P < 0．001）. By co-culturing different concentrations of mouse aGVHD serum and mouse MSC, we found that the addition of mouse aGVHD serum at different concentrations impaired the MSC self-renewal ability, which providing a new tool for the field of aGVHD bone marrow microenvironment damage.

The Cytotoxicity of Dental Restorative Materials on Gingival Stromal Mesenchymal Cells-an In Vitro Study.

Due to their minimally invasive high-quality adhesive, aesthetic and mechanical qualities, composite resins are the most frequently used materials in modern restorative dentistry. However, polymerization shrinkage and cytotoxicity are still unresolved drawbacks associated with these biomaterials. The present study aimed to assess the cytotoxicity of some restorative resin-based materials on gingival mesenchymal stromal cells (gMSCs), assuming that no differences in their behavior will be highlighted. The cytotoxicity of the tested materials was evaluated by comparing the behavior of gMSCs cultured in normal conditions and in association with disc-shaped material samples indirectly through functionality tests (colony-forming unit-fibroblast assay, migratory potential) and directly through the MTT assay. The results were statistically analyzed with the ANOVA test and Tukey's Honest Significant Difference test. According to the MTT assay, there are no statistically significant differences regarding the viability of gMSCs cultured in normal conditions or in the presence of resin-based material samples. On the other hand, the present study identified a significantly reduced number of colonies formed by the gMSCs cultured in association with BF and B discs, compared to that of gMSCs cultured in normal conditions. Also, the migratory potential was significantly lower for control gMSCs when compared to ZE-gMSCs and significantly higher for ZE-gMSCs when compared to BF-gMSCs or BFL-gMSCs. The results of the present study highlight a possible risk of cytotoxicity when using resin based-materials in dental practice, but they cannot be directly extrapolated to in vivo situations.

Isolation and characterization of feline dental pulp stem cells.

The aim of this study was to isolate feline dental pulp stem cells (fDPSCs) and characterize their clonogenic and proliferative abilities, as well as their multipotency, immunophenotype and cytogenetic stability. Dental pulp was isolated by explant culture from two cats <1 year old at post mortem. Their clonogenicity was characterized using a colony-forming unit fibroblast assay, and their proliferative ability was quantified with a doubling time assay in passages 2, 4 and 6 (P2, P4 and P6, respectively). Multipotency was characterized with an in vitro trilineage differentiation assay in P2, and cells were immunophenotyped in P4 by flow cytometry. Chromosomic stability was evaluated by cytogenetic analysis in P2, P4 and P6. The fDPSCs displayed spindle and epithelial-like morphologies. Isolated cells showed a marked clonogenic capacity and doubling time was maintained from P2 to P6. Trilineage differentiation was obtained in one sample, while the other showed osteogenic and chondrogenic differentiation. Immunophenotypic analysis showed fDPSCs were CD45-, CD90+ and CD44+. Structural and numerical cytogenetic aberrations were observed in P2-P4. In this study, fDPSCs from two cats were isolated by explant culture and immunophenotyped. Cells displayed clonogenic and proliferative ability, and multipotency in vitro, and signs of chromosomic instability were observed. Although a larger study is needed to confirm these results, this is the first report of fDPSC isolation and in vitro characterization.

The effect of low-level laser irradiation on the proliferation, osteogenesis, inflammatory reaction, and oxidative stress of human periodontal ligament stem cells under inflammatory conditions

Periodontitis often causes damage to the periodontal tissue and affects the function of human periodontal ligament stem cells (hPDLSCs). Low-level laser therapy (LLLT) has been used for periodontal treatment and can upregulate the proliferation and osteogenesis of hPDLSCs. The purpose of this study was to investigate the effects of LLLT on the proliferation, osteogenic differentiation, inflammatory reaction, and oxidative stress of hPDLSCs in an inflammatory environment (pPDLSCs). We designed one control group and three testing groups (treated with Nd:YAG laser at 4, 8, and 16J/cm2) of hPDLSCs from periodontitis patients who were diagnosed with stable phase periodontitis. Cell proliferation was measured by colony-forming unit fibroblast (CFU-F) assays and 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) assays. The osteogenic capacity of the cells was determined by alkaline phosphatase (ALP) staining, ALP activity assays, Alizarin Red S staining and the mRNA transcript levels of runt-related transcription factor 2 (Runx2), ALP, and osteocalcin (OCN). The effects of LLLT on the secretion of tumor necrosis factor-α (TNF-α) and interleukin (IL)-1β by PDLSCs were measured by enzyme-linked immunosorbent assay (ELISA). We also evaluated the oxidative stress of hPDLSCs and pPDLSCs by measuring the reactive oxygen species (ROS) level, malondialdehyde (MDA) level, and superoxide dismutase (SOD) activity after treatment with LLLT at 4, 8, and 16J/cm2. Our results demonstrated that LLLT could modulate the osteogenic potential of pPDLSCs at 8J/cm2. Inflammatory stimuli induced excess ROS release, and LLLT at 4-8J/cm2 promoted oxidative stress levels in hPDLSCs but decreased the expression of inflammatory cytokines and ROS levels in pPDLSCs. Moreover, LLLT at 16J/cm2 could significantly suppress proliferation and osteogenic differentiation and promote inflammatory cytokines and ROS levels in pPDLSCs. In conclusion, LLLT could regulate proliferation, osteogenesis, inflammatory reaction, and oxidative stress of human periodontal ligament stem cells under inflammatory conditions.

Growth kinetics of caprine bone marrow-derived mesenchymal stem cells at different passages

A study was conducted on cellular growth kinetics of caprine bone marrow mesenchymal stem cells (MSCs) with the help of growth curve, population doubling time (PDT) and colony forming unit fibroblast assay (CFU-f). MSCs were cultured up to 10 passages (from P0 to P10) consequent to their isolation, purification and characterization. After employing statistical analysis it was observed that maximum cellular growth (270.00±4.33 cells/well) and minimum PDT (39.16±4.18hr) were noticed in P3 passage followed by in passage 4 which was 263.33±5.07/well and 39.92±1.36 hr respectively. In a similar fashion maximum number of colonies were also counted inpassages which depicted the maximum proliferation and differentiation ability of cells which was 74.00±3.61 in P3 followed by in P4 (66.67±3.84). The potency was calculated at the samepassages on the basis of CFU-f assay which indicated that cells of P3 and P4 passages are best suited for transplantation, cryopreservation and experimental studies.

Increased proliferation and differentiation capacity of placenta-derived mesenchymal stem cells from women of median maternal age correlates with telomere shortening

Mesenchymal stem cells (MSCs) experience functional decline with systemic aging, resulting in reduced proliferation, increased senescence, and lower differentiation potential. The placenta represents a valuable source of MSCs, but the possible effect of donor age on the properties of placenta-derived mesenchymal stem cells (PDMSCs) has not been thoroughly studied. Thus, the aim of this study was to underscore the effect of maternal age on the biological characteristics and stemness properties of PDMSCs. PDMSCs were isolated from 5 donor age groups (A: 18-21, B: 22-25, C: 26-30, D:31-35 and E: ≥36 years) for comparison of morphological, proliferative and differentiation properties. The pluripotency markers NANOG, OCT4, and SSEA4, as well as multipotency and differentiation markers, showed higher expression in PDMSCs from mothers aged 22-35 years, with up to a 7-fold increase in adipogenesis. Cumulative population doubling, cell growth curves, and colony-forming unit-fibroblast assays revealed higher self-renewal ability in donors 26-30 years old. An increase in the proliferative characteristics of PDMSCs correlated with increased telomere shortening, suggesting that shorter telomere lengths could be related to cellular division rather than aging. A clear understanding of the effect of maternal age on MSC regenerative potential will assist in increasing the effectiveness of future cell therapies.

YAP/TAZ Promote Hematopoietic Regeneration Via Accelerating the Recovery of Bone Marrow Niche

Adult hematopoietic stem cells (HSCs) reside and are protected in a unique bone marrow (BM) microenvironment, termed the HSC niche, which consists mainly of vascular endothelial cells (EC) and EC-associated mesenchymal stromal cells (MSC). Myeloablative stresses, such as ionizing radiation (IR) and chemotherapy, induce not only depletion of hematopoietic cells but also disruption of HSC niche components, as exemplified by dilation and leakiness of BM vasculature and depletion and dysfunction of BM MSCs. These structural and functional changes in the HSC niche restrain efficient hematopoietic recovery, which often compromises the efficacy of HSC transplantation (HSCT) and chemotherapy. YAP/TAZ are the two transcriptional coactivators normally repressed by LATS kinases downstream of the Hippo pathway. Although cumulative evidence has established a critical role of YAP/TAZ activation in tissue regeneration of various solid organs, their role in BM regeneration remains poorly understood.Our quantitive RT-PCR revealed that YAP/TAZ are abundantly expressed in steady-state mouse ECs (CD45 -Ter119 -CD31 +Sca1 +CD105 hi) and MSCs (CD45 -Ter119 -CD31 -PDGFRα +CD51 +LepR +) but scarcely in hematopoietic cells including HSCs (Lin -cKit +Sca1 +Flk2 -CD150 +CD48 -CD34 lo), which was confirmed by reanalysis of the published single cell RNA-seq datasets (GSE128423). Immunofluorescent imaging of BM sections revealed that YAP/TAZ are distributed mainly in the cytoplasm of ECs but evenly in the cytoplasm and nuclei of MSCs, indicating their differential basal activity in these two HSC niche components. Kinetic transcriptome analysis revealed that YAP/TAZ activity is transiently activated in ECs at 24 hours and returns to a basal repressive state by day 3 after sublethal IR. This transient activation of endothelial YAP/TAZ was critical for vascular integrity, as conditional deletion of YAP/TAZ in ECs (Cdh5-Cre ERT2Yap1 f/fTaz f/f) caused 100% lethality of mice within 10 days following sublethal IR. In sharp contrast, the kinetic expression analysis of a YAP/TAZ target gene CTGF indicated their transient inhibition in MSCs after sublethal IR, and the conditional YAP/TAZ deletion in BM MSCs (Ebf3-Cre ERT2Yap1 f/fTaz f/f) led to their reduced colony forming ability when assessed by colony forming unit fibroblast (CFU-F) assay.Recently, we discovered a novel and potent LATS inhibitor GA-003 that selectively induces mouse and human YAP/TAZ activation in vitro (IC 50 against LATS1 = 1.06 ± 0.08 nM). To analyze the effect of pharmacological YAP/TAZ activation on BM regeneration in vivo, we treated mice with intraperitoneal injection of GA-003 (50 mg/kg per day, for 8 days) following sublethal IR. Remarkably, we observed an accelerated recovery of hematopoiesis, with the absolute numbers of BM cellularity, GMP (Lin -cKit +Sca1 -FcγR +CD34 +) and HSC EPCR (Lin -cKit +Sca1 +CD150 +EPCR +) on day 14 increased by 3.50-fold (p=0.0002), 6.49-fold (p=0.0022) and 11.41-fold (p=0.022), respectively in the GA-003-treated group compared to vehicle-treated group. In addition, GA-003 also promoted hematopoietic recovery after 5-FU injection (150 mg/kg) and HSCT. Nonetheless, consistent with the scarce expression of YAP/TAZ in hematopoietic stem and progenitor cells (HSPC), in vitro GA-003 treatment did not enhance HSPC growth, suggesting niche-mediated effects by GA-003. Indeed, in vitro tube formation assay indicated accelerated angiogenesis by GA-003-treated human umbilical vein ECs, and CFU-F assays revealed significant enhancement of colony formation by mouse BM-derived MSCs upon GA-003 treatment. To reveal the effect of GA-003 on the HSC niche components in vivo, we performed whole BM immunofluorescent imaging at various time points following sublethal IR and GA-003 treatment. We observed alleviated vascular dilation and leakiness and earlier restoration of vascular damage in GA-003-treated group compared to vehicle-treated group, which was associated with increased VE-Cadherin expression in ECs. These results suggest that reinforcing YAP/TAZ activity upon myelosuppression promotes HSC niche integrity and recovery and accelerates hematopoietic regeneration.Taken together, our results establish YAP/TAZ as novel regulators of HSC niche and highlight YAP/TAZ as promising therapeutic targets to boost hematopoietic recovery after myeloablative interventions such as chemotherapy and HSCT. DisclosuresAihara: Nissan Chemical Corporation: Current Employment. Iwawaki: Nissan Chemical Corporation: Current Employment. Nishino: Nissan Chemical Corporation: Current Employment. Iwama: Nissan Chemical Corporation: Research Funding.

Colony Forming Potential and Protein Composition of Commercial Umbilical Cord Allograft Products in Comparison With Autologous Orthobiologics

Background: Umbilical cord (UC) connective tissues contain plastic-adherent, colony forming unit–fibroblasts (CFU-Fs) amenable to culture expansion for potential therapeutic use. Recently, UC-derived allograft products have been made available to practitioners in orthopaedics and other specialties, by companies purporting “stem cell”–based healing. However, such marketing claims conflict with existing regulations for these human tissues, generating questions over the cellular and protein composition of current commercially available UC allograft products. Purpose: To evaluate commercial UC allograft products for viable cells, CFU-Fs, and protein makeup. Study Design: Descriptive laboratory study. Methods: Five commercial UC allograft products claiming to contain viable, undescribed “stem cells,” 2 obtained from UC blood (UCB) and 3 from UC tissue (UCT), were analyzed. Image-based methods were used to measure cell concentration and viability, a traditional CFU-F assay was used to evaluate in vitro behavior indicative of a connective tissue progenitor cell phenotype often referred to as mesenchymal stem/stromal cells, and quantitative immunoassay arrays were used to measure a combination of cytokines and growth factors. Bone marrow concentrate (BMC) and plasma derived from the blood and bone marrow of middle-aged individuals served as comparative controls for cell culture and protein analyses, respectively. Results: Viable cells were identified within all 5 UC allograft products, with those derived from UCB having greater percentages of living cells (40%-59%) than those from UCT (1%-22%). Compared with autologous BMC (>95% viability and >300 million living cells), no CFU-Fs were observed within any UC allograft product (<15 million living cells). Moreover, a substantial number of proteins, particularly those within UCB allograft products, were undetectable or present at lower concentrations compared with blood and bone marrow plasma controls. Interestingly, several important growth factors and cytokines, including basic fibroblast growth factor, hepatocyte growth factor, interleukin-1 receptor antagonist, and osteoprotegerin, were most prevalent in 1 or more UCT allograft products as compared with blood and bone marrow plasma. Conclusion: CFU-Fs, often referred to as stem cells, were not found within any of the commercial UC allograft products analyzed, and clinicians should remain wary of marketing claims stating otherwise. Clinical Relevance: Any therapeutic benefit of current UC allograft products in orthopaedic medicine is more likely to be attributed to their protein composition (UCT > UCB) or inclusion of cells without colony forming potential (UCB > UCT).

Low-level laser irradiation enhances the proliferation and osteogenic differentiation of PDLSCs via BMP signaling

The aim of this in vitro study was to evaluate the effects of low-level laser therapy (LLLT) at different energy intensities on proliferation and osteogenesis of periodontal ligament stem cells (PDLSCs). We designed one control group, without irradiation and four testing groups, treated with LLLT (Nd:YAG;1064nm) at 2, 4, 6, and 8J/cm2 for human PDLSCs. Cell proliferation was measured using colony-forming unit fibroblast assay and 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide assay. Osteogenic capacity of cells was determined by alkaline phosphatase (ALP) staining, ALP activity assay, Alizarin Red S staining, and the gene levels of runt-related transcription factor 2 (Runx2), ALP, osteocalcin, and bone morphogenetic protein 2 (BMP2). The effects of LLLT on secretion of TNF-α and IL-1β in PDLSCs were measured by enzyme-linked immunosorbent assay. BMP/Smad pathway was measured through the expression of Smad1/5/8 phosphorylation (P-Smad1/5/8). LDN-193189, an inhibitor of the BMP/Smad pathway, was used to explore the underlying effects of BMP/Smad signaling on the process of LLLT regulating the proliferation and osteogenesis of PDLSCs. Our results demonstrated LLLT could promote the proliferation and osteogenesis of PDLSCs at 2-6J/cm2 and LLLT at 8J/cm2 significantly suppress osteogenic differentiation of PDLSCs. Moreover, LLLT stimulated the secretion of TNFα and IL-β1. Finally, we found the irradiation positively modulates the P-Smad1/5/8 level. When the cells were treated with LDN-193189, the proliferation and osteogenic effects of LLLT on PDLSCs were attenuated. In conclusion, LLLT may upregulate the proliferation and bone formation ability of PDLSCs via the BMP/Smad signaling.

The Effect of 5-Azacitidine Treatment on the Biologic Characteristics of Bone Marrow Mesenchymal Stem Cells in Patients with Myelodysplastic Syndromes

Myelodysplastic syndromes (MDS) are clonal disorders of the hematopoietic stem cell characterized by ineffective bone marrow (BM) hematopoiesis and increased risk for leukemic evolution. An abnormal BM microenvironment has been reported to contribute to BM failure in MDS through defective support of hematopoiesis. The cellular elements of the BM microenvironment derive from a common progenitor cell, namely the mesenchymal stem cell (MSC). We have previously shown that MSCs from MDS patients display impaired proliferative and clonogenic potential and develop chromosomal alterations during passages.It has been shown that DNA methylation occurs at a high frequency in MDS and that gene reactivation by hypo-methylating agents may have a positive impact on patients' outcome. Recent evidence also suggests altered global DNA methylation status in BM-MSCs of MDS patients. The methyl transferase inhibitor 5-Azacitidine (ΑΖΑ) is widely used for the treatment of MDS; however the effect of AZA on patients' BM microenvironment and specifically on the MSC compartment has not been extensively studied. In the present study we investigate the functional, molecular and cytogenetic characteristics of ex vivo expanded BM-MSCs from MDS patients receiving AZA, before and after 6-cycle therapy.MSCs were isolated and expanded from 14 patients with high risk MDS and 10 age- and sex-matched hematologically healthy subjects undergoing surgery for hip replacement, after informed consent. MSCs were expanded and re-seeded for a total of 5 passages (P) and phenotypically characterized by flow cytometry. MSCs were induced to differentiate to adipocytes and osteoblasts. Differentiation was assessed by cytochemical stains and by the expression of adipocyte- and osteocyte-specific genes. MSC growth characteristics were assessed by the population doubling time (PDT) and the methyl-triazolyl-tetrazolium (MTT) assay throughout passages. The clonogenic potential of MSCs was evaluated by the colony forming unit fibroblast assay. Conventional cytogenetic and array comparative genomic hybridization (aCGH) analyses were performed in cultured expanded MSCs at P2.MSCs from MDS patients before and after therapy, displayed normal spindle-shape morphology and immunophenotypic characteristics as shown by the expression of CD90, CD105, CD44, CD29, CD73, and the lack of expression of CD45, CD14 and CD34. MSCs were also able to differentiate into adipocytes and osteoblasts before and after therapy, as evidenced by the Oil Red O and Alizarin Red staining, respectively, and the expression of differentiation specific genes. Cumulative analysis showed that the proliferation potential of patient MSCs, evaluated by the PDT and MTT assays, was statistically significant decreased compared to healthy individuals (P<0.01) but increased significantly following AZA treatment compared to baseline (P<0.05), although not at the level of the healthy controls. A paired analysis of patient samples before and after therapy, also showed that AZA treatment improves significantly the proliferation rate of MDS-MSCs (P<0.05). Similarly, a cumulative and paired analysis showed that the defective (compared to healthy individuals) clonogenic potential of patient MSCs at P2, increased significantly following AZA treatment (P<0.05 and P<0.05, respectively), although not at the level of the healthy individuals. Karyotypic analysis did not reveal any chromosomal abnormalities in MSC samples, before or after AZA treatment. An aCGH analysis of P2 MSCs has - thus far - been performed in three MDS patients before and after AZA therapy. Results have indicated genetic abnormalities (duplications/deletions) in all three samples before treatment that remained unchanged after AZA therapy whereas some new abnormalities also appeared.The data of this ongoing study on the biological properties of MSCs from MDS patients receiving AZA, show an improvement of the clonogenic and proliferative potential of MSCs following treatment. According to aCGH analysis, the genetic abnormalities of MSCs remain unaffected. The findings of the functional and cytogenetic characteristics of patient MSCs before and after therapy will be individually correlated with the MSC methylation status and with patients' outcome and the results are anticipated to provide novel insights in the role of AZA therapy in the BM microenvironment of MDS patients. DisclosuresNo relevant conflicts of interest to declare.

Long-Term Trypsin Treatment Promotes Stem Cell Potency of Canine Adipose-Derived Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) isolated from adipose tissue (adipose-derived stem cells [ADSCs]) are considered one of the most promising cell types for applications in regenerative medicine. However, the regenerative potency of ADSCs may vary because of heterogeneity. Long-term trypsin treatment (LTT) is known to significantly concentrate multilineage-differentiating stress-enduring (Muse) cells from human MSCs. In this study, we aimed to generate cells with high stem cell potency from canine ADSCs using LTT. After 16 h of treatment with trypsin, surviving ADSCs (LTT-tolerant cells) had significantly enhanced expression of stage-specific embryonic antigen (SSEA)-1, a mouse embryonic stem cell marker, and fucosyltransferase 9, one of several fucosyltransferases for SSEA-1 biosynthesis. However, LTT-tolerant cells did not enhance the expression of SSEA-3, a known human Muse cell marker. LTT-tolerant cells, however, showed significantly higher self-renewal capacity in the colony-forming unit fibroblast assay than ADSCs. In addition, the LTT-tolerant cells formed cell clusters similar to embryoid bodies and expressed undifferentiated markers. Moreover, these cells differentiated into cells of all three germ layers and showed significantly higher levels of α 2-6 sialic acid (Sia)-specific lectins, known as differentiation potential markers of human MSCs, than ADSCs. LTT-tolerant cells had a normal karyotype and had low telomerase activity, showing little carcinogenetic potency. LTT-tolerant cells also showed significantly increased activity of transmigration in the presence of chemoattractants and had increased expression of migration-related genes compared with ADSCs. In addition, LTT-tolerant cells had stronger suppressive activity against mitogen-stimulated lymphocyte proliferation than ADSCs. Overall, these results indicated that the LTT-tolerant cells in canine ADSCs have similar properties as human Muse cells (although one of the undifferentiated markers is different) and are expected to be a promising tool for regenerative therapy in dogs.

Radial extracorporeal shockwave promotes subchondral bone stem/progenitor cell self-renewal by activating YAP/TAZ and facilitates cartilage repair in vivo

BackgroundRadial extracorporeal shockwave (r-ESW), an innovative and noninvasive technique, is gaining increasing attention in regenerative medicine due to its mechanobiological effects. Subchondral bone stem/progenitor cells (SCB-SPCs), originating from the pivotal zone of the osteochondral unit, have been shown to have multipotency and self-renewal properties. However, thus far, little information is available regarding the influences of r-ESW on the biological properties of SCB-SPCs and their therapeutic effects in tissue regeneration.MethodsSCB-SPCs were isolated from human knee plateau osteochondral specimens and treated with gradient doses of r-ESW in a suspension stimulation system. The optimized parameters for SCB-SPC self-renewal were screened out by colony-forming unit fibroblast assay (CFU-F). Then, the effects of r-ESW on the proliferation, apoptosis, and multipotency of SCB-SPCs were evaluated. Moreover, the repair efficiency of radial shockwave-preconditioned SCB-SPCs was evaluated in vivo via an osteochondral defect model. Potential mechanisms were explored by western blotting, confocal laser scanning, and high-throughput sequencing.ResultsThe CFU-F data indicate that r-ESW could augment the self-renewal of SCB-SPCs in a dose-dependent manner. The CCK-8 and flow cytometry results showed that the optimized shockwave markedly promoted SCB-SPC proliferation but had no significant influence on cell apoptosis. Radial shockwave exerted no significant influence on osteogenic capacity but strongly suppressed adipogenic ability in the current study. For chondrogenic potentiality, the treated SCB-SPCs were mildly enhanced, while the change was not significant. Importantly, the macroscopic scores and further histological analysis strongly demonstrated that the in vivo therapeutic effects of SCB-SPCs were markedly improved post r-ESW treatment. Further analysis showed that the cartilage-related markers collagen II and proteoglycan were expressed at higher levels compared to their counterpart group. Mechanistic studies suggested that r-ESW treatment strongly increased the expression of YAP and promoted YAP nuclear translocation in SCB-SPCs. More importantly, self-renewal was partially blocked by the YAP-specific inhibitor verteporfin. Moreover, the high-throughput sequencing data indicated that other self-renewal-associated pathways may also be involved in this process.ConclusionWe found that r-ESW is capable of promoting the self-renewal of SCB-SPCs in vitro by targeting YAP activity and strengthening its repair efficiency in vivo, indicating promising application prospects.

Cryopreserved bone marrow aspirate concentrate as a cell source for the colony-forming unit fibroblast assay

PurposeThe prevalence of connective tissue progenitor cells within a cell-based therapy is often quantified using the colony-forming unit fibroblast (CFU-F) assay. The present study investigates the feasibility of using cryopreserved bone marrow aspirate concentrate (BMAC) as an alternative cell source to fresh BMAC for CFU-F quantification. MethodsFreshly prepared and corresponding cryopreserved BMAC samples from patients receiving autologous cell therapy (n = 98) were analyzed using the CFU-F assay for comparison. Cultures were established by directly plating BMAC at low cell densities and maintained for a 2-week growth period. Colonies were enumerated to determine CFU-F frequency, and a subset of cultures was imaged and analyzed to quantify colony area and density. ResultsA nonlinear relationship was observed between plating density and CFU-F frequency over a wide range in plating densities (~30-fold). Cryopreserved BMAC yielded recoverable (77 ± 23%) and viable (73 ± 9%) nucleated cells upon thawing. After cryopreservation, CFU-F frequencies were found to be significantly lower (56.6 ± 34.8 vs. 50.3 ± 31.7 colonies per million nucleated cells). Yet the number of CFU-F in fresh and cryopreserved BMAC were strongly correlated (r = 0.87) and had similar area and densities. Further, moderate correlations were observed between the number of CFU-F and nucleated cells, and both the mean colony area and density were negatively correlated with patient age. Notably, no relationship was found between CFU-F frequency and age, regardless of whether fresh or cryopreserved BMAC was used. ConclusionsFreshly prepared and cryopreserved BMAC yielded correlated results when analyzed using the CFU-F assay. Our findings support the cryogenic storage of patient BMAC samples for retrospective CFU-F analyses, offering a potential alternative for characterizing BMAC and furthering our understanding of progenitor cells in relation to clinical outcome.

Comprehensive analysis of skeletal muscle- and bone-derived mesenchymal stem/stromal cells in patients with osteoarthritis and femoral neck fracture

BackgroundMesenchymal stem/stromal cells (MSCs) can replenish the aged cells of the musculoskeletal system in adult life. Stem cell exhaustion and decrease in their regenerative potential have been suggested to be hallmarks of aging. Here, we investigated whether muscle- and bone-derived MSCs of patients with osteoarthritis and osteoporosis are affected by this exhaustion, compared to healthy donors.MethodsPatients with primary osteoarthritis, femoral neck fractures due to osteoporosis, and healthy donors (controls) were included. MSCs were isolated from the skeletal muscle and subchondral bone from each patient and compared using ex vivo and in vitro analyses, including immunophenotyping, colony-forming unit fibroblast assays, growth kinetics, cell senescence, multilineage potential, and MSC marker gene expression profiling.ResultsFreshly isolated cells from muscle from patients with osteoarthritis showed a lower proportion of CD45/CD19/CD14/CD34-negative cells compared to patients with osteoporosis and healthy donors. Freshly isolated muscle cells from patients with osteoarthritis and osteoporosis also showed higher clonogenicity compared to healthy donors. MSCs from both tissues of osteoarthritis patients showed significantly reduced osteogenesis and MSCs from the bone also reduced adipogenesis. Chondrogenic pellet diameter was reduced in bone-derived MSCs from both patient groups compared to healthy donors. A significant positive correlation was observed between adipogenesis and CD271 expression in muscle-derived MSCs. CD73 was significantly lower in bone-derived MSCs from osteoarthritis patients, compared to osteoporosis patients. Gene expression profiling showed significantly lower expression of MSC marker gene leptin receptor, LEPR, previously identified as the major source of the bone and adipocytes in the adult bone marrow, in bone-derived MSCs from patients with osteoarthritis in comparison with osteoporotic patients and healthy donors.ConclusionsOur results show deficient ex vivo and in vitro properties of both skeletal muscle- and bone-derived MSCs in osteoarthritis and osteoporosis patients, compared to healthy donors. In bone-derived MSCs from patients with osteoarthritis, we also identified a lower expression of the leptin receptor, a marker of MSCs that present a major source of MSCs in the adult bone marrow. This suggests that exhaustion of skeletal muscle- and bone-derived MSCs is a hallmark of osteoarthritis and osteoporosis, which defines the need for further clinical trials of stem cell transplantation in these patients.

Human Adipose-Derived Hydrogel Characterization Based on In Vitro ASC Biocompatibility and Differentiation.

Hydrogels serve as three-dimensional scaffolds whose composition can be customized to allow attachment and proliferation of several different cell types. Extracellular matrix-derived hydrogels are considered close replicates of the tissue microenvironment. They can serve as scaffolds for in vitro tissue engineering and are a useful tool to study cell-scaffold interaction. The aim of the present study was to analyze the effect of adipose-derived stromal/stem cells (ASCs) and decellularized adipose tissue-derived (DAT) hydrogel interaction on ASC morphology, proliferation, differentiation, and DAT hydrogel microstructure. First, the ASCs were characterized using flow cytometry, adipogenic/osteogenic differentiation, colony-forming unit fibroblast assay and doubling time. The viability and proliferation assays showed that ASCs seeded in DAT hydrogel at different concentrations and cultured for 21 days remained viable and displayed proliferation. ASCs were seeded on DAT hydrogel and cultured in stromal, adipogenic, or osteogenic media for 14 or 28 days. The analysis of adipogenic differentiation demonstrated the upregulation of adipogenic marker genes and accumulation of oil droplets in the cells. Osteogenic differentiation demonstrated the upregulation of osteogenic marker genes and mineral deposition in the DAT hydrogel. The analysis of DAT hydrogel fiber metrics revealed that ASC seeding, and differentiation altered both the diameter and arrangement of fibers in the matrix. Matrix metalloproteinase-2 (MMP-2) activity was assessed to determine the possible mechanism for DAT hydrogel remodeling. MMP-2 activity was observed in all ASC seeded samples, with the osteogenic samples displaying the highest MMP-2 activity. These findings indicate that DAT hydrogel is a cytocompatible scaffold that supports the adipogenic and osteogenic differentiation of ASCs. Furthermore, the attachment of ASCs and differentiation along adipogenic and osteogenic lineages remodels the microstructure of DAT hydrogel.

Evaluation of an Autologous Bone Mesenchymal Stem Cell-Derived Extracellular Matrix Scaffold in a Rabbit and Minipig Model of Cartilage Repair.

BackgroundThis study aimed to evaluate an autologous bone mesenchymal stem cell (MSC)-derived extracellular matrix (ECM) scaffold in two animal models of cartilage repair.Material/MethodsA rabbit model (n=16) and a minipig model (n=8) of cartilage repair were created with cartilage defects of the knee joints treated with bone marrow stimulation (BMS). In the ECM group, autologous bone MSC-derived ECM scaffolds were implanted into the cartilage defects after bone marrow stimulation. In the BMS group, the cartilage defects were treated by bone marrow stimulation only. The renewal capacity of bone MSCs was measured with a colony-forming unit fibroblast (CFU-F) in vitro assay. The extent of cartilage repair was as-sessed at 6 months after surgery.ResultsIn the rabbit model, the macroscopic appearance of the exudate of the healing wounds in the ECM group showed less fibrosis, and the histology showed more evenly distributed chondrocytes compared with the BMS group. The CFU-F assay showed that the number of bone MSCs in the ECM group was approximately was twice that of the BMS group. In the minipig model, the macroscopic appearance and magnetic resonance imaging (MRI) findings of the ECM group were improved when compared with the BMS group. The repaired tissue in ECM group had similar histological characteristics and biochemical content to normal hyaline cartilage.ConclusionsIn two animal models of knee joint cartilage repair, the use of an ECM scaffold increased the number of bone MSCs and improved the extent of cartilage repair.

The Analysis of In Vivo Aging in Human Bone Marrow Mesenchymal Stromal Cells Using Colony-Forming Unit-Fibroblast Assay and the CD45lowCD271+ Phenotype

Uncultured mesenchymal stromal cells (MSCs) are increasingly used in therapies; however, the effects of donor age on their biological characteristics and gene expression remain unclear. The aim of this study was to investigate age-related changes in bone marrow (BM) MSCs following minimal or no culture manipulation. Iliac crest BM was aspirated from 67 healthy donors (19-89 years old) and directly used for the colony-forming unit-fibroblast (CFU-F) assay or CD45lowCD271+ cell enumeration. The colonies were analysed for colony area and integrated density (ID) when grown in standard MSC media or media supplemented with human serum from young (YS) or old (OS) donors. There was a notable age-related decline in the number of MSCs per millilitre of BM aspirate revealed by the CFU-F assay (r = −0.527, p < 0.0001) or flow cytometry (r = −0.307, p = 0.0116). Compared to young donors (19-40 years old), colony IDs were significantly lower in older donors (61-89 years old), particularly for smaller-sized colonies (42% lower, p < 0.01). When cultured in media supplemented with OS, young and old donor MSCs formed colonies with lower IDs, by 21%, p < 0.0001, and 27%, p < 0.05, respectively, indicating the formation of smaller sparser colonies. No significant differences in the expression of selected adipogenic, osteogenic, stromal, and bone remodelling genes as well as CD295, CD146, CD106, and connexin 43 surface molecules were found in sorted CD45lowCD271+ MSCs from young and old donors (n = 8 donors each). Altogether, these results show similar trends for age-related decline in BM MSC numbers measured by the CFU-F assay and flow cytometry and reveal age-related effects of human serum on MSC colony formation. No significant differences in selected gene expression in uncultured CD45lowCD271+ MSCs suggest that old donor MSCs may not be inferior in regard to their multipotential functions. Due to large donor-to-donor variation in all donor groups, our data indicate that an individual's chronological age is not a reliable predictor of their MSC number or potency.

Skeletal-muscle-derived mesenchymal stem/stromal cells from patients with osteoarthritis show superior biological properties compared to bone-derived cells

Mesenchymal stem/stromal cells (MSCs) are being exploited for patient-derived stem-cell therapies. As the biological properties of MSCs derived from skeletal muscle of osteoarthritis patients are poorly understood, the aim of this study was to compare muscle MSCs with well-recognized bone and bone marrow-derived MSCs from these patients.Paired samples of skeletal muscle and trabecular bone tissue were obtained from 21 patients with osteoarthritis. Isolated cells were compared using ex vivo immunophenotyping and detailed in vitro analyses. These included the colony forming unit fibroblast assay, growth kinetics, senescence, multilineage potential, immunophenotyping, and MSC marker gene expression profiling.Freshly isolated MSCs from muscle showed improved viability over bone-derived MSCs, with similar mesenchymal fraction. Muscle-derived MSCs showed superior clonogenicity, higher growth rates, and lower doubling times. Muscle-derived MSCs also showed superior osteogenic and myogenic properties and a positive correlation between CD271 expression and adipogenesis. Senescence rate as well as adipogenic and chondrogenic potentials were similar.Skeletal muscle-derived MSCs of osteoarthritis patients have superior clonogenicity and growth kinetics compared to bone-derived MSCs, making them a good candidate for autologous stem-cell therapies. Moreover, the positive correlation between CD271 and adipogenesis suggest that CD271 expressing muscle MSCs might contribute to muscle steatosis observed in osteoarthritis.

Enhanced expansion of human bone marrow- and adipose tissue derived- mesenchymal stromal cells in an improved animal component-free culture medium

Background & Aim MesenCult™-ACF-Plus (MACF-P) is an improved animal component-free (ACF) culture medium for the derivation and expansion of mesenchymal stromal cells (MSCs) from primary tissues. We characterized MSCs derived from human bone marrow (BM) and adipose (AD) tissues in MACF-P and in medium containing fetal bovine serum (FBS). Methods, Results & Conclusion Clonogenic growth was evaluated by plating BM mononuclear cells (MNCs) or AD-derived stromal cells (SCs) at low density in the Colony-Forming Unit-Fibroblast (CFU-F) assay. The proliferative potential of BM and AD-derived MSCs was measured by determining cell number at each passage (P) up to P6 (BM-MSCs) or P9 (AD-MSCs), and paired t-test was used for statistical analysis. MSCs from both tissue sources were plated at 1.5–3 × 103 cells/cm2 in each medium for long-term cell expansion. Immunosuppression of CD4+ T cells by BM-MSCs cultured in MACF-P was evaluated by co-culture of BM-MSCs with PBMCs. The PBMCs were labelled with eFluor450, activated with human CD3/CD28 T Cell Activator and then analyzed by flow cytometry for T cell proliferation after 5 days. Total CFU-F per 106 AD-SCs and per 106 BM-MNCs was comparable in MACF-P and FBS containing medium (AD-SCs: 236 ± 127 vs. 268 ± 120 [mean ± SEM; n=3]; BM-MNCs: 37 ± 7 vs. 32 ± 5 [mean ± SEM; n=6] in MACF-P and FBS-containing media, respectively). However, the average fold-expansion of AD-MSCs at each subculture over 9 passages was significantly higher in MACF-P medium (12.2 ± 0.9; mean ± SEM; n=5) than in FBS-containing medium (3.8 ± 0.3; mean ± SEM; n=5; p Co-culture of BM-MSCs with PBMCs in MACF-P indicated that proliferation of CD4+ T cells is suppressed in a cell concentration-dependent manner when BM-MSCs were added in vitro. These data demonstrate that improved MACF-P medium supports derivation and efficient expansion of both BM-MSCs and AD-MSCs under complete ACF conditions and that BM-MSCs exhibit immunosuppressive activity in vitro.

Tendon contains more stem cells than bone at the rotator cuff repair site

The rotator cuff (RC) repair failure rate is high. Tendon and bone represent sources of mesenchymal stem cells (MSCs), but the number of MSCs from each has not been compared. Bone channeling may increase bone-derived MSC numbers participating in enthesis re-formation at the "footprint" repair site. The effect of preoperative channeling on increasing bone MSC numbers has never been reported. We asked (1) whether bone contains more MSCs than tendon at the time of arthroscopic repair and (2) whether bone preoperative channeling at the RC repair site increases the number of bone-derived MSCs at the time of surgery. In 23 participants undergoing arthroscopic RC repair, bone was sampled from the footprint and tendon was sampled from the distal supraspinatus. We randomized participants to the channeling or no-channeling group5 to 7 days before surgery. We enumerated MSCs from both tissues using the colony-forming unit-fibroblast (CFU-F) assay (10 per group). We identified MSC identity using flow cytometry and MSC tri-differentiation capacity (n = 3). Tendon CFU-F per gram exceeded bone CFU-F per gram for both groups (479 ± 173 CFU-F/g vs. 162 ± 54 CFU-F/g for channeling [P = .036] and 1334 ± 393 CFU-F/g vs. 284 ± 88 CFU-F/g for no channeling [P = .009]). Ninety-nine percent of cultured cells satisfied the MSC definition criteria. The distal supraspinatus tendon contained more MSCs per gram than the humeral footprint. Tendon may represent an important and overlooked MSC source for postoperative enthesis re-formation. Further studies are needed to evaluate the repair role of tendon MSCs and to recommend bone channeling in RC repair.