Number Of Colony-forming Unit Fibroblasts Research Articles

Osteogenic differentiation from mouse adipose-derived stem cells and bone marrow stem cells.

Mesenchymal stem cells (MSCs) have been successfully cultured and proliferated in vitro and can differentiate into a variety of specific cell types, such as adipocytes or osteocytes, through chemical stimulation. One of the major applications of MSCs is in regenerative medicine research. MSCs can be collected from many adult tissues. In this experiment, an 8-week-old expresses green fluorescent protein (EGFP) transgenic mouse, FVB/NCrl-Tg(Pgk1-EGFP)01Narl, was used to obtain adipose-derived stem cells (ADSCs) from abdominal adipose tissue and bone marrow stem cells (BMSCs) from femur bone marrow. We compared the differences in the growth rate and differentiation ability of ADSCs and BMSCs. The growth curves of different generations (P1 and P3) of the stem cells showed that the proliferation rate of ADSCs was significantly higher than that of BMSCs. The purity of stem cells was measured by the number of colony-forming unit fibroblast. The results show that the number of colonies of ADSCs at different generations (P1 and P3) was significantly higher than that of BMSCs and that the purity of ADSCs was greater than that of BMSCs. Comparing the ability of ADSCs and BMSCs to induce osteogenic differentiation and the expression of Runx2 and Opn genes, the results show that ADSCs had a higher rate of osteogenic differentiation than BMSCs. In summary, mouse ADSCs display similar osteogenic differentiation ability to BMSCs but have a better capacity than BMSCs in terms of stem cell purity and cell proliferation in vitro.

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.

Isolation of Amniotic Fluid Mesenchymal Stem Cells Obtained from Cesarean Sections

BACKGROUND: The term amniotic fluid (AF) can be an ideal alternative as a source of mesenchymal stem cells (MSCs), originating from the neonate. Preclinical studies of the second- and third-trimester amnion fluid cells confirmed the number of potential donors from this wasted material.
 AIM: This study aims to look at the forming time of stem cells derived from AF-MSCs.
 MATERIALS AND METHODS: AF samples from healthy human donors were collected during full-term C-sections and kept at 4°C until processed. The number of colony-forming unit-fibroblast was assessed microscopically by calculating spindle-shaped colonies that clearly resembled of fibroblasts and did not include colonies with rounded epithelioid morphology. The immunophenotyping of their independent AF preparations was done using the human MSC phenotyping kit which was done according to the manufacturer’s instruction by flow cytometry.
 RESULTS: The result showed that it succeeded in getting 8 million cells which will be used for research on pelvic organ prolapse therapy using AF-MSCs. The stem cell isolation totally takes 6 weeks. We got 2 million stem cells in one flask.
 CONCLUSION: This study concludes that the time needed for differentiation of AF-MSCs is 6 weeks and AF-MSCs express mesenchymal markers such as CD90, CD73 (SH3, SH), and CD105 (SH2) and these cells also express HLA antigens – ABC, CD 34, and CD 45 which are hematopoietic markers and endothelial CD31 markers.

Cryopreservation of Stromal Vascular Fraction Cells Reduces Their Counts but Not Their Stem Cell Potency.

Background:Adipose-derived stem cells are derived from the nonfat component of adipose tissue termed the stromal vascular fraction (SVF). The use of freshly isolated autologous SVF cells as an alternative to adult stem cells is becoming more common. Repeated SVF administration for improved clinical outcomes is complicated by the need for repeated liposuction. This can be overcome by cryopreservation of SVF cells. The current study aimed to assess whether SVF cells retain their stem cell potency during cryopreservation.Methods:SVF cells isolated from lipoaspirates (donor age: 46.1 ± 11.7 y; body mass index: 29.3 ± 4.8 kg/m2) were analyzed either immediately after isolation or following cryopreservation at −196°C. Analyses included assessment of nucleated cell counts by methylene blue staining, colony-forming unit fibroblast counts, surface marker expression using a flow cytometric panel (CD45, CD34, CD31, CD73, CD29, and CD105), expansion in culture, and differentiation to fat and bone.Results:While cryopreservation reduced the number of viable SVF cells, stem cell potency was preserved, as demonstrated by no significant difference in the proliferation, surface marker expression in culture, bone and fat differentiation capacity, and the number of colony-forming unit fibroblasts in culture, in cryopreserved versus fresh SVF cells. Importantly, reduced cell counts of cryopreserved cells were due, mainly, to a reduction in hematopoietic CD45+ cells, which was accompanied by increased proportions of CD45−CD34+CD31− stem cell progenitor cells compared to fresh SVF cells.Conclusions:Cryopreservation of SVF cells did not affect their in vitro stem cell potency and may therefore enable repeated SVF cell administrations, without the need for repeated liposuction.

Mesenchymal stem cells from sternum: the type of heart disease, ischemic or valvular, does not influence the cell culture establishment and growth kinetics

BackgroundIn an attempt to increase the therapeutic potential for myocardial regeneration, there is a quest for new cell sources and types for cell therapy protocols. The pathophysiology of heart diseases may affect cellular characteristics and therapeutic results.MethodsTo study the proliferative and differentiation potential of mesenchymal stem cells (MSC), isolated from bone marrow (BM) of sternum, we made a comparative analysis between samples of patients with ischemic (IHD) or non-ischemic valvular (VHD) heart diseases. We included patients with IHD (n = 42) or VHD (n = 20), with average age of 60 years and no differences in cardiovascular risk factors. BM samples were collected (16.4 ± 6 mL) and submitted to centrifugation with Ficoll-Paque, yielding 4.5 ± 1.5 × 107 cells/mL.ResultsMorphology, immunophenotype and differentiation ability had proven that the cultivated sternal BM cells had MSC features. The colony forming unit-fibroblast (CFU-F) frequency was similar between groups (p = 0.510), but VHD samples showed positive correlation to plated cells vs. CFU-F number (r = 0.499, p = 0.049). The MSC culture was established in 29% of collected samples, achieved passage 9, without significant difference in expansion kinetics between groups (p > 0.05). Dyslipidemia and the use of statins was associated with culture establishment for IHD patients (p = 0.049 and p = 0.006, respectively).ConclusionsTogether, these results show that the sternum bone can be used as a source for MSC isolation, and that ischemic or valvular diseases do not influence the cellular yield, culture establishment or in vitro growth kinetics.

Mobilisation of Joint-resident Mesenchymal Stromal Cells for Articular Cartilage Regeneration

Background and Objectives:Microfracture is a recognized procedure used to treat isolated cartilage injuries or defects, in which bone marrow mesenchymal stromal cells (BM-MSCs) are thought to migrate into the resulting blood clot, leading to subsequent cartilage repair via fibrocartilage formation. The discovery of MSCs in the synovium and synovial fluid (SF) provides a potential mechanism for repairing cartilage from the top down via their migration and homing to the microfracture site, however SF-MSCs low in number and usually lost with joint irrigation. The purpose of this work was threefold; first to test the hypothesis that SF-MSCs can be replaced, and also their numbers further increased by synovial agitation, second that these cells were capable of rapid adhesion to clots and third that the clot composition improve MSC migration.Materials-Methods:Ex-vivo mechanical agitation of human superficial synovium and in vivo intra-operative agitation of synovium in patients undergoing arthroscopy were performed using a Cytology brush and custom designed Synovial brush. Colony-forming unit-fibroblast (CFU-F) assay was performed to quantify released MSCs. Adhesion to clots was studied by comparing Platelet Rich Plasma (PRP), Whole Blood (WB) and Fibrin Glue (FG). Migration studies were performed using passage 2-4 synovial MSCs in trans-well migration assay. MSC migration, over a five hour period, was compared between PRP and pooled human Platelet Lysate (hPL).Results:Ex-vivo mechanical agitating of the synovium with the cytology brush compared to irrigation alone increased MSC number 2.7-fold (n=10, p=0.002). Irrigation during arthroscopy was seen to effectively remove the majority of CFU-Fs for the synovial fluid. Use of a custom designed synovial brush, compared to the cytology brush resulted in a median 65-fold increase in the number of CFU-Fs (n=8, p=0.0148). Trilineage differentiation of released synovial MSCs was at least comparable to donor match synovial fluid MSCs. These MSCs adhered to clots within 30 minutes with no difference seen between clot compositions. Released synovial MSCs demonstrated a trend for a better migration towards hPL compared to PRP.Conclusions:Existing surgical procedures wash away SF-MSCs. Using a novel brushing technique and a custom designed synovial brush, synovial MSCs can be mechanically released in vivo, and these cells were capable of migration and rapid adhesion to clots. Collectively these findings aid in the rapid replenishment of endogenous minimally manipulated MSCs, and provide a one-stage procedure combining synovial brushing with microfracture, as a strategy for cost effective joint repair.

Electro-Acupuncture Promotes Endogenous Multipotential Mesenchymal Stem Cell Mobilization into the Peripheral Blood

Background/Aims: Mobilization of endogenous stem cells is an appealing strategy for cell therapy However, there is little evidence for reproducible, effective methods of mesenchymal stem cell (MSC) mobilization. In the present study, we investigated the mobilizing effect of electro-acupuncture (EA) on endogenous MSCs. Methods: Normal adult rats were randomly divided into six groups, namely, EA for 14 days (EA14d), sham EA14d, EA21d, sham EA21d and matched control groups. MSC mobilization efficiency was determined by colony-forming unit fibroblast (CFU-F) assays. Mobilized peripheral blood (PB)-derived MSCs were identified by immunophenotype and multi-lineage differentiation potential. Results: CFU-F frequency was significantly increased in the PB of EA14d rats compared with the sham EA and control groups. Moreover, the number of CFU-Fs was increased further in the EA21d group. MSCs derived from EA-mobilized PB were positive for CD90 and CD44, but negative for CD45. Additionally, these cells could differentiate into adipocytes, osteoblasts, chondrocytes and neural-like cells in vitro. Finally, stromal cell-derived factor-1α (SDF-1α) was increased in the PB of rats subjected to EA, and the migration of MSCs was improved in response to SDF-1α. Conclusions: MSCs with multi-lineage differentiation potential can be mobilized by EA. Our data provide a promising strategy for MSC mobilization.

Ruxolitinib Does Not Affect Bone Marrow Stromal Cell (BMSC) Clonogenicity and Growth but Interferes with JAK Signaling Under Inflammatory Conditions - Implications for Fibrosis in Myeloproliferative Diseases?

The JAK2 V617F gain-of-function mutation is found in hematopoietic cells of nearly all patients with myeloproliferative diseases (MPDs). Ruxolitinib (Ruxo), a potent oral JAK1/JAK2 inhibitor, attenuates cytokine signaling by inhibition of JAK signaling, leading to anti-proliferative and pro-apoptotic effects. In patients with myelofibrosis (MF), effects of Ruxolitinib on splenomegaly, symptom improvement and possibly survival were successfully demonstrated in the two COMFORT studies. Furthermore, resolution of bone marrow fibrosis after long-term treatment with Ruxo was recently reported (Wilkins et al., Haematologica 2013). However, bone marrow stromal cells (BMSC), which contribute to fibrosis, do not harbor the JAK2 mutation or other chromosomal abnormalities that can be found in hematopoietic stem/progenitor cells. Therefore, the current study aimed to investigate possible effects of Ruxo on these non-hematopoietic cells.Bone marrow mononuclear cells were harvested from consenting donors and Ruxo effects on stroma progenitor cells were investigated using the standard CFU-F (colony-forming unit, fibroblast) assay. Our data showed that CFU-F numbers were not decreased by Ruxo at doses ranging from 0.2 to 10 μM (n = 9, p = 0.41, one-way ANOVA), but were even found to be increased at the 5 μΜ dose as indicated by a Ruxo/DMSO ratio of over one (p = 0.04, multiple comparisons test, control-vs-5 μΜ) (Figure 1). In contrast, statistically significant differences were observed in CFU-F colony size with a tendency to decreased sizes with higher doses of Ruxo (p < 0.0001, one-way ANOVA) (Figure 1). Next, we tested if Ruxo affected standard culture-derived bone marrow stromal cell (BMSC) growth, both, in short-term (6 h, 12 h, 24 h and 48 h, n = 3) and in long-term exposure experiments (up to a total of 21 days, n = 3). Neither short-term nor long-term exposure with Ruxo at 0.2 μΜ, 0.5 μΜ, 1 μΜ, 5 μΜ and 10 μΜ caused significant changes of BMSC numbers when compared to their corresponding DMSO controls (p > 0.05, two-way ANOVA), however, a trend to lower BMSC counts with higher Ruxo doses was observed. BMSC were reduced by maximum 25.3±5.7 % (mean ± SD) after 24-hour exposure with 10 μΜ Ruxo. After 21 days drug exposure, ratios of Ruxo-treated BMSC relative to their corresponding DMSO controls were 1.1±0.2, 1.1±0.3, 0.9±0.2, 0.7±0.4%, and 0.6±0.2 for 0.2 μΜ, 0.5 μΜ, 1 μΜ, 5 μΜ and 10 μΜ Ruxo, respectively.These data indicated that Ruxo might have a cytotoxic effect on BMSC, however, only at very high concentrations. We therefore went on to study Ruxo effects on JAK signaling in BMSC after stimulation with IL-6, in order to mimic the inflammatory environment in MPDs. Western blot assays revealed that IL-6 treatment (100 ng/ml) induced pJAK2 in BMSC, which was not detectable in unstimulated BMSC. Furthermore, exposure of IL-6 stimulated BMSC to Ruxo (1 μΜ) diminished pSTAT3 and reduced downstream STAT targets such as pAKT and pMAPK. Furthermore, preliminary results on the cytokine expression profile of supernatants from IL-6-activated BMSC treated with Ruxo showed significant differences compared to controls. Specifically, IL-23 and CCL4 were elevated in Ruxo-treated/IL-6 activated BMSC whereas MCP-1 was strongly reduced compared to the non-Ruxo treated controls.Taken together, these data show that clinically-relevant doses of Ruxo did not affect the clonogenic potential and proliferation of primary marrow stroma cells, indicating that Ruxo most likely has no or little direct effect on the fibrosis-causing cells in MPD. However, Ruxo considerably affects JAK-STAT signaling in activated BMSCs, leading to an altered cytokine expression profile which potentially contributes to the amelioration of fibrosis, and, accordingly, ongoing experiments address this question. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Standardized quantification of human bone marrow mesenchymal stromal cells based on a flow cytometric assay

Conclusion The mesenchymal stromal cell (MSC) content is a major quality determinant of human bone marrow aspirates (BMA). It depends on the aspiration site, contamination with peripheral blood, as well as the aspiration procedure, and it negatively correlates with donor age. The common way of quantifying MSCs is based on the cultivation of cells using the colony forming unit– fibroblast (CFU-F) assay, which is time consuming and highly variable. Cuthbert et al. described a close linear relationship between the number of CFU-F colonies counted manually after 14 days of culture and the number of CD271bright cells per mL of BMA1. The aim of this work was the optimization of a fast and reproducible flow cytometry–based MSC quantification method enabling prospective quality assessment of BMA. In previous experiments we have shown that only CD271bright cells, which also express MSCA-1, give rise to CFU-F. MSCs were isolated from 2×107 bone marrow mononuclear cells (BM MNC) using CD271 (LNGFR)-APC and Anti-APC MicroBeads. Samples of all fractions were labeled with CD45-FITC. PI fluorescence and light scatter signals were used for gating live cells. The positive fraction contained CD271+ cells with a purity of about 86% (Mean: 83±4.1%; n = 4). This population contained about 73% CD271CD45 cells (A, orange fraction) and about 13% CD271CD45 cells (A, red fraction). Further separation of CD271 and CD271 cells was achieved by flow sorting. The different fractions were cultivated and CFU-F numbers were counted after 14 days. The numbers of CFU-F were about 40-fold higher when MSCs were cultivated from the CD271/ CD45 fraction compared to MSCs obtained by plastic adherence (PA). No CFU-Fs were detected in the CD271– and CD271CD45 cell fractions (A). Counterstaining of isolated CD271+ cells revealed a 100% co-expression of MSCA-1 in CD271CD45 cells (B). CD271CD45 MSCs met ISCT criteria2 after culture expansion (3 passages) with respect to MSCspecific marker expression (C) and their differentiation potential towards osteocytes, adipocytes, and chondrocytes (D, left to right)

Easily-handled method to isolate mesenchymal stem cells from coagulated human bone marrow samples.

To establish an easily-handled method to isolate mesenchymal stem cells (MSCs) from coagulated human bone marrow samples. Thrombin was added to aliquots of seven heparinized human bone marrow samples to mimic marrow coagulation. The clots were untreated, treated with urokinase or mechanically cut into pieces before culture for MSCs. The un-coagulated samples and the clots were also stored at 4 °C for 8 or 16 h before the treatment. The numbers of colony-forming unit-fibroblast (CFU-F) in the different samples were determined. The adherent cells from different groups were passaged and their surface profile was analyzed with flow cytometry. Their capacities of in vitro osteogenesis and adipogenesis were observed after the cells were exposed to specific inductive agents. The average CFU-F number of urokinase-treated samples (16.85 ± 11.77/10(6)) was comparable to that of un-coagulated control samples (20.22 ± 10.65/10(6), P = 0.293), which was significantly higher than those of mechanically-cut clots (6.5 ± 5.32/10(6), P < 0.01) and untreated clots (1.95 ± 1.86/10(6), P < 0.01). The CFU-F numbers decreased after samples were stored, but those of control and urokinase-treated clots remained higher than the other two groups. Consistently, the numbers of the attached cells at passage 0 were higher in control and urokinase-treated clots than those of mechanically-cut clots and untreated clots. The attached cells were fibroblast-like in morphology and homogenously positive for CD44, CD73 and CD90, and negative for CD31 and CD45. Also, they could be induced to differentiate into osteoblasts and adipocytes in vitro. Urokinase pretreatment is an optimal strategy to isolate MSCs from human bone marrow samples that are poorly aspirated and clotted.

MSCs Isolated From Patients With Ischemic Vascular Disease Have Normal Angiogenic Potential

MSCs Isolated From Patients With Ischemic Vascular Disease Have Normal Angiogenic Potential

Cancer Risk Is Not Increased in Patients Treated for Orthopaedic Diseases with Autologous Bone Marrow Cell Concentrate

There is concern that regenerative cell-based therapies could result in increased risk of tumor formation. We investigated the long-term risks for systemic and site-specific cancers in patients who had received autologous bone marrow-derived stromal progenitor cells to treat orthopaedic lesions. A total of 1873 patients were treated from 1990 to 2006 with bone marrow-derived concentrated cells. Patients were monitored for cancer incidence from the date of the first operation (1990) until death, or until December 31, 2011. The mean follow-up time was 12.5 years (range, five to twenty-two years). The average number of colony-forming unit fibroblasts returned to the patients was 483,000 fibroblasts (range, 62,000 to 2,095,000 fibroblasts). The primary outcome was to evaluate with radiographs and/or magnetic resonance imaging the risk of tumorigenesis at the cell therapy treatment sites. The secondary outcome was to evaluate the risk of cancer diagnosed in areas other than the treatment site during the follow-up period. The relative risk of cancer was expressed as the ratio of observed and expected number of cases, that is, the standardized incidence ratio, according to the cancer incidence in the French population. No tumor formation was found at the treatment sites on the 7306 magnetic resonance images and 52,430 radiographs among the 1873 patients. Fifty-three cancers were diagnosed in areas other than the treatment site. On the basis of cancer incidence in the general population during the same period, the expected number of cancers was between ninety-seven and 108 for the same age and sex distribution. The range of the standardized incidence ratio for the follow-up period was between 0.49 and 0.54 (95% confidence interval, 0.30 to 0.80). This study found no increased cancer risk in patients after application of autologous cell-based therapy using bone marrow-derived stromal progenitor cells either at the treatment site or elsewhere in the patients after an average follow-up period of 12.5 years.

CD146 expression on mesenchymal stem cells is associated with their vascular smooth muscle commitment

Bone marrow mesenchymal stem cells (MSCs) are plastic adherent cells that can differentiate into various tissue lineages, including osteoblasts, adipocytes and chondrocytes. However, this progenitor property is not shared by all cells within the MSC population. In addition, MSCs vary in their proliferation capacity and expression of markers. Because of heterogeneity of CD146 expression in the MSC population, we compared CD146−/Low and CD146High cells under clonal conditions and after sorting of the non-clonal cell population to determine whether this expression is associated with specific functions. CD146−/Low and CD146High bone marrow MSCs did not differ in colony-forming unit-fibroblast number, osteogenic, adipogenic and chondrogenic differentiation or in vitro haematopoietic-supportive activity. However, CD146−/Low clones proliferated slightly but significantly faster than did CD146High clones. In addition, a strong expression of CD146 molecule was associated with a commitment to a vascular smooth muscle cell (VSMC) lineage characterized by a strong up-regulation of calponin-1 and SM22α expression and an ability to contract collagen matrix. Thus, within a bone marrow MSC population, certain subpopulations characterized by high expression of CD146, are committed towards a VSMC lineage.

Expression of C/EBPβ in Bone Marrow Mesenchymal Stem Cells Is Mandatory for Early-Stage B Cell Lymphopoiesis

AbstractAbstract 3457The interplay between hematopoietic cells and bone marrow microenvironment organized by mesenchymal stem cells is important for the maintenance of hematopoiesis. With respect to B cell lymphopoiesis, several constituents of bone marrow microenvironment specific for B cells (“B cell niche”) have been identified, including CXCL12/stromal cell-derived factor-1 (SDF-1)-abundant reticular cells as cellular factors, and CXCL12/SDF-1, interleukin (IL)-7, stem cell factor (SCF), fms-related tyrosine kinase 3 ligand (Flt3-L), and nuclear factor kappa-B ligand (RANKL), as essential humoral factors. However, the precise mechanism through which mesenchymal stem cells in the bone marrow microenvironment support B cell lymphopoiesis, especially the role of transcription factors, remains unknown. We show that the mesenchymal stem cells lacking a transcription factor, CCAAT enhancer binding protein (C/EBP) b, are functionally abnormal, which contribute to the impairment of B cell lymphopoiesis in C/EBPb knockout mice.In C/EBPb knockout mice, the number of B cells, in particular, B220+CD43+ precursor B cells, was significantly decreased in bone marrow compared with that in wild-type littermates (Figure 1A and 1B). As shown in Fig. 1A, the percentage of total B220+ B cells was decreased at 19.1 ± 7.1% in the bone marrow of C/EBPb knockout mice (KO, n = 13) compared to wild-type mice (WT, n = 14, 26.5 ± 7.3%: *P<0.05). The percentage of B220+CD43+ precursor B cells was also decreased at 5.2 ± 1.5% in the bone marrow of C/EBPb knockout mice (KO, n = 13) compared to wild-type mice (WT, n = 14, 7.4 ± 1.6%: **P<0.01). Intriguingly, in vivo bone marrow transplantation experiments demonstrated that the bone marrow cells derived from C/EBPb knockout mice were engrafted in lethally-irradiated (10 Gy) wild-type mice with equivalently B cell recovery compared to the bone marrow cells from normal wild-type mice. Conversely, when normal wild-type c-kit+ Sca-1+ lineages− hematopoietic stem cells (KSL cells) were co-cultured with C/EBPb deficient mesenchymal stem cells in vitro (KO), they showed impaired B cell differentiation compared to the co-culture with normal wild-type mesenchymal stem cells (WT, Figure 1C). Mechanistically, the CXCL12/SDF-1 production by C/EBPb deficient mesenchymal stem cells was reduced compared with that by wild-type mesenchymal stem cells (KO, n = 5, 4.47 ± 1.16 ng/mL; WT, n = 5, 9.90 ± 1.93 ng/mL; **P < 0.01). These results suggest a possibility that abnormal C/EBPƒÀ deficient mesenchymal stem cells in bone marrow microenvironment contribute to impaired B cell lymphopoiesis in C/EBPb knockout mice.We further found that C/EBPb deficient mesenchymal stem cells displayed several functional abnormalities. First, calcium accumulation was significantly reduced in 4 week osteogenesis-inducing cultures of C/EBPb-deficient mesenchymal stem cells compared to cultures of wild-type mesenchymal stem cells. This occurred along with the down-regulated expression of the principal osteogenic master molecule runt-related transcription factor 2 (Runx2). Second, lipid deposition was significantly reduced in 1 week adipogenesis-inducing cultures of C/EBPb-deficient mesenchymal stem cells. The expression of adipogenic markers, including peroxisome proliferator-activated receptor b (PPARb) was significantly reduced in adipogenic cultures of C/EBPb-deficient mesenchymal stem cells compared with cultures of wild-type mesenchymal stem cells Finally, the number of colony-forming unit fibroblast (CFU-F) was higher in the bone marrow of C/EBPb knockout mice than in that of wild-type mice. Collectively, C/EBPb-deficient mesenchymal stem cells have aberrant multi-differentiation capability and increased proliferation activity compared with wild-type mesenchymal stem cells, further supporting that C/EBPb-deficient mesenchymal stem cells were functionally abnormal. [Display omitted] Altogether, this work demonstrates that impaired B cell lymphopoiesis in C/EBPb knockout mice is attributed to abnormal mesenchymal stem cells in bone marrow microenvironment, at least in a steady-state, an effect that is due in part to the impaired CXCL12/SDF-1 production. Disclosures:No relevant conflicts of interest to declare.

Recapitulating Aspects of the Oxygen and Substrate Environment of the Damaged Joint Milieu for Stem Cell-Based Cartilage Tissue Engineering

Human infrapatellar fat pad contains a source of mesenchymal stem cells (FPSCs) that potentially offer a novel population for the treatment of damaged or diseased articular cartilage. Existing cartilage repair strategies such as microfracture harness the presence of a low-oxygen microenvironment, fibrin clot formation at sites of microfracture, and elevations in growth factors in the damaged joint milieu. Bearing this in mind, the objective of this study was to determine the chondrogenic potential of diseased human FPSCs in a model system that recapitulates some of these features. In the first phase of the study, the role of transforming growth factor beta-3 (TGF-β3) and fibroblast growth factor-2 (FGF-2), in addition to an altered oxygen-tension environment, on the colony-forming unit-fibroblast (CFU-F) capacity and growth kinetics of human FPSCs during monolayer expansion was evaluated. The subsequent chondrogenic capacity of these cells was quantified in both normoxic (20%) and low- (5%) oxygen conditions. Expansion in FGF-2 was shown to reduce CFU-F numbers, but simultaneously increase both the colony size and the cell yield compared to standard expansion conditions. Supplementation with both FGF-2 and TGF-β3 significantly reduced cell-doubling time. Expansion in FGF-2, followed by differentiation at 5% oxygen tension, was observed to synergistically enhance subsequent sulfated glycosaminoglycan (sGAG) accumulation after chondrogenic induction. FPSCs expanded in FGF-2 were then encapsulated in either agarose or fibrin hydrogels in an attempt to engineer cartilaginous grafts. sGAG synthesis was higher in fibrin constructs, and was further enhanced by differentiation at 5% oxygen tension, accumulating 2.7% (ww) sGAG after 42 days in culture. These results indicate that FPSCs, a readily accessible cell population, form cartilage in an in vitro environment that recapitulates several key biological features of cartilage repair during microfracture and also point toward the potential utility of such cells when combined with fibrin hydrogel scaffolds.

Comparison between Stem Cells Harvested from Wet and Dry Lipoaspirates

Adipose-derived stem cells (ASC) are usually isolated from lipoaspirates, but it is not known if the anesthetic solution injected into adipose tissue affects cell yield and functions. Two different samples were drawn from the abdominal region of female subjects. In the first, a physiological solution containing lidocaine/adrenaline was injected (wet liposuction, WL), while in the contralateral area, the sample was collected without injecting any solution (dry liposuction, DL). The aspirates were processed to investigate the yield of the stromal-vascular fraction (SVF) cells and ASC frequency, growth rate, apoptosis, and differentiation potential. The solid dried mass of fresh WL isolates was lower than that of DL isolates (p < 0.01) due to the presence, in the former, of a liquid solution. As a consequence, the amount of WL-SVF cells was 18.7% lower than those obtained from DL (p < 0.01); this difference was also observed under culture conditions. In addition, the number of colony-forming unit-fibroblasts (CFU-Fs) obtained from 1 × 103 SVF cells was 25.5% lower in WL-aspirates than DL-aspirates (p < 0.05) owing, at least in part, to the observed presence of apparent diffusion coefficient in the liquid solution of the WL isolates. After WL and DL, no differences were observed in ASC growth rate, apoptosis, or differentiation potential toward adipogenic, osteogenic, and endothelial cell lineages. In conclusion, WL yields about 40% fewer ASC than DL due to the combined effect of tissue dilution and the reduced frequency of ASC in the SVF. The main biological features of ASC are suitable for cell-based therapies.

MRI of the proximal femur predicts marrow cellularity and the number of mesenchymal stem cells

To determine whether the marrow conversion index (MCI) in MRI is related to the total number of mononuclear and mesenchymal stem cells (MSCs) in proximal femoral metaphysis of patients with hip osteoarthritis. Thirty-two hips of 32 consecutive patients who underwent total hip arthroplasty (THA) for hip osteoarthritis were included in this study. MRI of the hip was performed preoperatively and MCI was subsequently calculated. Three-milliliter bone marrow samples were obtained from the proximal femur during THA and the number of total mononuclear cells was determined using a hemocytometer. Colony forming unit-fibroblasts (CFU-Fs) assays of MSCs were performed by transferring a total of 2 × 10(4) mononuclear cells to each of five 60-mm plates. One week later, the numbers of colonies were counted. The total number of mononuclear cells decreased with increasing MCI. Likewise, the prevalence and total number of CFU-Fs increased with increasing number of total mononuclear cells, and decreased with increasing MCI. Our results suggest that measurement of MCI in MRI can be an objective and noninvasive method to predict marrow cellularity and the number of MSCs in patients with hip osteoarthritis.

Mesenchymal Stem Cells Can Be Mobilized Into Peripheral Blood by Short-Term Hypoxia: a Possible Role of HIF-1α.

AbstractAbstract 1450 Background:Mesenchymal stem cells (MSCs) constitute a population of multipotential cells giving rise to adipocytes, osteoblasts and chondrocytes. Combining with their engraftment promoting capacity and immunosuppressive property, MSCs may be therapeutically useful for haematopoietic stem cell transplantation. There is growing evidence that these cells can, under the right circumstances, enter peripheral circulation. Previous study revealed that MSCs are mobilized into peripheral blood (PB) by 3 weeks of chronic hypoxia, but the mobilization effect of short-term hypoxia and the underlying mechanisms are currently unknown. In this study, we used rat model to determine whether short-term hypoxia can mobilize MSCs into PB and investigated the related factors which may regulate the mobilization process. Design and Methods:Rats were housed in a hypoxic chamber (FiO2=10%) for 1, 2, 3, 5, 7 and 14 days, respectively, while control ones were housed in normoxic environment for equal periods. To quantify the number of MSCs and evaluate mobilization efficiency, PB and bone barrow (BM) samples of each group were collected and colony-forming unit fibroblast (CFU-F) assays were performed. Mobilized PB derived MSCs were identified by immunophenotype and trilineage differentiation. Since BM is the main reservoir and typical microenvironment of MSCs, we investigated the response of BM environment exposed to hypoxia which may potentially facilitate MSCs mobilization. Hypoxia-inducible factor 1α (HIF-1α) expression of BM cells was detected by Western blot; vascular endothelial growth factor (VEGF) in BM was qualified by ELISA and Immunohistochemistry (IHC). To evaluate the change of BM sinusoid vessels (BMSVs), VEGFR3 was stained by IHC and positive vessels were counted. The levels of stromal cell-derived factor-1α (SDF-1α) and VEGF in PB were tested by ELISA. Moreover, we compared migration capacity of MSCs in hypoxic condition (PO2=1% or 8%) with normal condition (PO2=21%) in vitro using Transwell assay. Results:We found that MSCs were mobilized into PB by exposing to short-term hypoxia (2d) and the CFU-F frequency was 5.80±0.58 vs. 1.40±0.24 CFU-Fs per 3×106 cells (p<0.05, n=5). From 2d to 14d of hypoxic exposure, the number of CFU-Fs mobilized in PB of hypoxic group was gradually increased in a time dependent manner. However, no significant differences were observed in bone marrow CFU-Fs among varies groups (P>0.05). Mobilized PB derived adherent cells were positive for CD90, CD29 and CD44, but negative for CD34 and CD45 and they could differentiate into adipocyte, osteoblast, and chondrocyte, which indicated that mobilized PB-derived cells are bona fide MSCs. What's more, we showed here that during hypoxic exposure, HIF-1α was stabilized and expressed continuously in BM of rats which is a main niche of MSCs. Stabilization and up-regulation of HIF-1α suggested that BM is hypoxia-sensitive and during hypoxic exposure it became a lower oxygen environment (PO2<1%). Previous studies have proved that VEGF and SDF-1α are directly regulated by the transcription factor HIF-1α. Our results showed that, induced by HIF-1α, VEGF was elevated from 2d to 7d in the BM of hypoxic rats which may increase BM vascular permeability and induce vasodilatation; VEGFR3(+) BMSVs increased in 7d and 14d hypoxic BM which may further facilitate the egress of MSCs. SDF-1α in PB increased from 2d to 14d, especially 7d of hypoxia (1976.7±148.1 vs. 663.6±56.7pg/ml, P<0.01). In addition, exposure of MSCs to low oxygen (8% PO2) significantly promoted their in vitro migration and a further increase was observed under lower oxygen condition (1% PO2). MSCs migrated more rapidly in response to SDF-1α exposed to hypoxia. Conclusion:Taken together, we show here that MSCs can be mobilized into PB by short-term hypoxia and the mobilization efficacy increased in a time dependent manner. Our results suggest the mechanisms of hypoxia inducing MSCs mobilization relate to the lower oxygen milieu of BM and stabilization of HIF-1α may play a pivotal role in MSCs mobilization. Our data provide meaningful clues to clarify the mechanisms of MSCs mobilization and important evidence for further exploring the exact agents that of clinical use. Disclosures:No relevant conflicts of interest to declare.

Red-Light Light-Emitting Diode Irradiation Increases the Proliferation and Osteogenic Differentiation of Rat Bone Marrow Mesenchymal Stem Cells

The objective of this study was to investigate the effects on the proliferation and osteogenic differentiation of rat mesenchymal stem cells (MSCs) by using red-light light-emitting diode (LED) irradiation. Low-level light irradiation (LLLI) has been shown to enhance proliferation and cytokine secretion of a number of cells. MSCs are capable of regenerating various mesenchymal tissues and are essential in supporting the growth and differentiation of hematopoietic stem cells within the bone marrow. Rat bone marrow MSCs were treated with single or multiple doses of LLLI from an LED array (630 nm) at the irradiances of 5 and 15 mW/cm(2), and radiant exposures of 2 and 4 J/cm(2). The proliferation, clonogenic potential, and osteogenic differentiation of MSCs were evaluated after illumination. The growth of MSCs was enhanced by red-light LLLI, and the effect became more obvious at low cell density. A single dose of LLLI led only to a short-term increase in MSCs proliferation. A maximal increase in cell proliferation was observed with multiple exposures of LLLI at 15 mW/cm(2) and 4 J/cm(2). The number of colony-forming unit fibroblasts increased when cells were illuminated under the optimal parameter. During osteogenesis, significant increases (p < 0.01) in both alkaline phosphatase and osteocalcin expressions were found in the MSCs that received light irradiation. Our data demonstrated that MSCs proliferation was enhanced by multiple exposures to LLLI from 630-nm LEDs, and cell growth depended on the plating density. Furthermore, multiple dose of LLLI could enhance the osteogenic potential of rat MSCs.

Mesenchymal Stromal Cells, Colony-Forming Unit Fibroblasts, From Bone Marrow of Untreated Advanced Breast and Lung Cancer Patients Suppress Fibroblast Colony Formation From Healthy Marrow

We have shown that bone marrow (BM) from untreated advanced lung and breast cancer patients (LCP and BCP) have a reduced number of colony-forming unit fibroblasts (CFU-Fs) or mesenchymal stem cells (MSCs). Factors that regulate the proliferation and differentiation of CFU-F are produced by the patients' BM microenvironment. We have now examined whether conditioned media (CM) from patients' CFU-F-derived stromal cells also inhibits the colony-forming efficiency (CFE) of CFU-F in primary cultures from healthy volunteers (HV)-BM. Thus the number and proliferation potential of HV-CFU-F were also found to be decreased and similar to colony numbers and colony size of patients' CFU-F. Stromal cells from both of these types of colonies appeared relatively larger and lacked the characteristic spindle morphology typically seen in healthy stromal cells. We developed an arbitrary mesenchymal stromal cell maturational index by taking three measures consisting of stromal cell surface area, longitudinal and horizontal axis. All stromal indices derived from HV-CFU-F grown in patients' CM were similar to those from stromal elements derived from patients' CFU-F. These indices were markedly higher than stromal indices typical of HV-CFU-F cultured in healthy CM or standard medium [alpha-medium plus 20% heat-inactivated fetal bovine serum (FBS)]. Patients' CM had increased concentrations of the CFU-F inhibitor, GM-CSF, and low levels of bFGF and Dkk-1, strong promoters of self-renewal of MSCs, compared to the levels quantified in CM from HV-CFU-F. Moreover, the majority of patients' MSCs were unresponsive in standard medium and healthy CM to give CFU-F, indicating that the majority of mesenchymal stromal cells from patients' CFU-F are locked in maturational arrest. These results show that alterations of GM-CSF, bFGF, and Dkk-1 are associated with deficient cloning and maturation arrest of CFU-F. Defective autocrine and paracrine mechanisms may be involved in the BM microenvironments of LCP and BCP.