Colony-forming Unit-fibroblast Frequency Research Articles

CYTOLOGICAL EFFECT OF SERUM ISOLATED FROM POLYTRAUMATIZED PATIENTS ON HUMAN BONE MARROW-DERIVED MESENCHYMAL STROMAL CELLS

Due to their immunomodulatory and regenerative capacity, human bone marrow-derived mesenchymal stromal cells (hBMSCs) are promising in the treatment of polytrauma patients. However, few studies evaluated the effects of sera from polytraumatized patients on hBMSCs. The aim of this study was to explore changes in hBMSCs exposed to serum from polytrauma patients from different time points after trauma.Sera from 84 patients on day 1 (D1), 5 (D5) and 10 (D10) after polytrauma (ISS ≥ 16) were pooled respectively to test the differential influence on hBMSC. As a control, sera from three healthy age- and gender-matched donors (HS) were collected. The pooled sera were analyzed by Multicytokine Array for pro-/anti-inflammatory cytokines. For the cell culture experiments, hBMSCs from four healthy donors were used. The influence of the different sera on hBMSC regarding cell proliferation, colony forming unit-fibroblast (CFU-F) assay, cell viability and toxicity, cell migration, as well as osteogenic and chondrogenic differentiation was analyzed. One-Way-ANOVA and LSD-test were used for the parametric, Kruskal-Wallis-test for non-parametric data. p≤0.05 was considered as statistically significant.The results showed that D5 serum reduced hBMSCs cell proliferation capacity by 41.26% (p=0.000) compared with HS and increased the proportion of dead cells by 3.19% (p=0.008) and 2.25% (p=0.020) compared with D1 and D10. The frequency of CFU-F was reduced by 49.08% (p=0.041) in D5 and 53.99% (p=0.027) in D10 compared with HS, whereas the other parameters were not influenced.The serological effect of polytrauma on hBMSCs was related to the time after trauma. It is disadvantageous to use BMSCs in polytraumatized patients five days after the incidence as obvious cytological changes could be found at that time point. However, it is promising to use hBMSCs to treat polytrauma after 10 days, combined with the concept of “Damage Control Orthopaedics” (DCO).

Cytological Effects of Serum Isolated from Polytraumatized Patients on Human Bone Marrow-Derived Mesenchymal Stem Cells.

Due to their immunomodulatory and regenerative capacity, human bone marrow-derived mesenchymal stem cells (hBMSCs) are promising in the treatment of patients suffering from polytrauma. However, few studies look at the effects of sera from polytraumatized patients on hBMSCs. The aim of this study was to explore changes in hBMSC properties in response to serum from polytrauma patients taken at different time points after the trauma incident. For this, sera from 84 patients with polytrauma (collected between 2010 and 2020 in our department) were used. In order to test the differential influence on hBMSC, sera from the 1st (D1), 5th (D5), and 10th day (D10) after polytrauma were pooled, respectively. As a control, sera from three healthy donors (HS), matched with respect to age and gender to the polytrauma group, were collected. Furthermore, hBMSCs from four healthy donors were used in the experiments. The pooled sera of HS, D1, D5, and D10 were analyzed by multicytokine array for pro-/anti-inflammatory cytokines. Furthermore, the influence of the different sera on hBMSCs with respect to cell proliferation, colony forming unit-fibroblast (CFU-F) assay, cell viability, cytotoxicity, cell migration, and osteogenic and chondrogenic differentiation was analyzed. The results showed that D5 serum significantly reduced hBMSC cell proliferation capacity compared with HS and increased the proportion of dead cells compared with D1. However, the frequency of CFU-F was not reduced in polytrauma groups compared with HS, as well as the other parameters. The serological effect of polytrauma on hBMSCs was related to the time after trauma. It is disadvantageous to use BMSCs in polytraumatized patients at least until the fifth day after polytrauma as obvious cytological changes could be found at that time point. However, it is promising to use hBMSCs to treat polytrauma after five days, combined with the concept of “Damage Control Orthopedics” (DCO).

Oral Mesenchymal Stromal Cells in Systemic Sclerosis: Characterization and Response to a Hyaluronic-Acid-Based Biomaterial

Introduction. As oral mesenchymal stromal cells (MSCs) have not, to date, been isolated from systemic sclerosis (SSc) patients, the aim of this in vitro experiment was to characterize gingival MSCs (SScgMSCs) and granulation tissue MSCs (SScgtMSCs) from SSc and to evaluate their functionality in comparison to healthy MSCs (hMSCs), in normal or hyaluronic acid (HA) culture media. Materials and Methods. Isolated cells were described by immunophenotyping of surface antigen make-up and by trilineage mesenchymal differentiation capacity. Colony-Forming Unit-Fibroblast (CFU-F) test and migration potential evaluated MSC functionality. Results. All types of MSCs displayed positivity for the following surface markers: CD29, CD73, CD90, CD105, CD44, and CD79a. These cells did not express CD34, CD45, HL-DR, and CD14. Isolated MSCs differentiated into osteoblasts, adipocytes, and chondroblasts. The frequency of CFU-F for SScgtMSCs was significantly lower than that of hMSCs (p = 0.05) and SScgMSCs (p = 0.004) in normal medium, and also markedly lower than that of SScgMSCs (p = 0.09) in HA medium. Following HA exposure, both SScgMSCs and SScgtMSCs migrated significantly less (p = 0.033 and p = 0.005, respectively) than hMSCs. Conclusions. A reduced functionality of MSCs derived from SSc as compared to hMSCs was observed. HA in culture medium appeared to significantly stimulate the migration potential of hMSCs.

Pro-fibrotic phenotype of bone marrow stromal cells in Modic type 1 changes.

Modic type 1 changes (MC1) are painful vertebral bone marrow lesions frequently found in patients suffering from chronic low-back pain. Marrow fibrosis is a hallmark of MC1. Bone marrow stromal cells (BMSCs) are key players in other fibrotic bone marrow pathologies, yet their role in MC1 is unknown. The present study aimed to characterise MC1 BMSCs and hypothesised a pro-fibrotic role of BMSCs in MC1. BMSCs were isolated from patients undergoing lumbar spinal fusion from MC1 and adjacent control vertebrae. Frequency of colony-forming unit fibroblast (CFU-F), expression of stem cell surface markers, differentiation capacity, transcriptome, matrix adhesion, cell contractility as well as expression of pro-collagen type I alpha 1, α-smooth muscle actin, integrins and focal adhesion kinase (FAK) were compared. More CFU-F and increased expression of C-X-C-motif-chemokine 12 were found in MC1 BMSCs, possibly indicating overrepresentation of a perisinusoidal BMSC population. RNA sequencing analysis showed enrichment in extracellular matrix proteins and fibrosis-related signalling genes. Increases in pro-collagen type I alpha 1 expression, cell adhesion, cell contractility and phosphorylation of FAK provided further evidence for their pro-fibrotic phenotype. Moreover, a leptin receptor high expressing (LEPRhigh) BMSC population was identified that differentiated under transforming growth factor beta 1 stimulation into myofibroblasts in MC1 but not in control BMSCs. In conclusion, pro-fibrotic changes in MC1 BMSCs and a LEPRhigh MC1 BMSC subpopulation susceptible to myofibroblast differentiation were found. Fibrosis is a hallmark of MC1 and a potential therapeutic target. A causal link between the pro-fibrotic phenotype and clinical characteristics needs to be demonstrated.

Bone marrow stromal cells from MDS and AML patients show increased adipogenic potential with reduced Delta-like-1 expression

Myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) are clonal hematopoietic stem cell disorders with a poor prognosis, especially for elderly patients. Increasing evidence suggests that alterations in the non-hematopoietic microenvironment (bone marrow niche) can contribute to or initiate malignant transformation and promote disease progression. One of the key components of the bone marrow (BM) niche are BM stromal cells (BMSC) that give rise to osteoblasts and adipocytes. It has been shown that the balance between these two cell types plays an important role in the regulation of hematopoiesis. However, data on the number of BMSC and the regulation of their differentiation balance in the context of hematopoietic malignancies is scarce. We established a stringent flow cytometric protocol for the prospective isolation of a CD73+ CD105+ CD271+ BMSC subpopulation from uncultivated cryopreserved BM of MDS and AML patients as well as age-matched healthy donors. BMSC from MDS and AML patients showed a strongly reduced frequency of CFU-F (colony forming unit-fibroblast). Moreover, we found an altered phenotype and reduced replating efficiency upon passaging of BMSC from MDS and AML samples. Expression analysis of genes involved in adipo- and osteogenic differentiation as well as Wnt- and Notch-signalling pathways showed significantly reduced levels of DLK1, an early adipogenic cell fate inhibitor in MDS and AML BMSC. Matching this observation, functional analysis showed significantly increased in vitro adipogenic differentiation potential in BMSC from MDS and AML patients. Overall, our data show BMSC with a reduced CFU-F capacity, and an altered molecular and functional profile from MDS and AML patients in culture, indicating an increased adipogenic lineage potential that is likely to provide a disease-promoting microenvironment.

Development and Validation of a Fully GMP-Compliant Process for Manufacturing Stromal Vascular Fraction: A Cost-Effective Alternative to Automated Methods.

The therapeutic use of adipose-derived stromal vascular fraction (SVF) is expanding in multiple pathologies. Various processes have been proposed for manufacturing SVF but they must be revisited based on advanced therapy medicinal product (ATMP) regulations. We report here the development and validation of a fully good manufacturing practices (GMP)-compliant protocol for the isolation of SVF. Adipose tissue was collected from healthy volunteers undergoing lipoaspiration. The optimal conditions of collagenase digestion and washing were determined based on measurements of SVF cell viability, yield recovery, and cell subset distribution. Comparability of the SVF obtained using the newly developed manufacturing process (n = 6) and the Celution-based automated method (n = 33), used as a reference, was established using inter-donor analyses. Characteristics of SVF (n = 5) generated using both manufacturing protocols were analyzed for an intra-donor comparison. In addition, these comparisons also included the determination of colony-forming unit fibroblast frequency, in vitro angiogenic activity, and in vivo regenerative effects in a mouse ischemic cutaneous wound model. We successfully developed a process for the generation of SVF presenting higher cell viability and yield recovery compared to the Celution device-based protocol. Characteristics of the SVF including phenotype, capacity for angiogenesis, and wound-healing promotion attested to the comparability of the two manufacturing processes. We validated an optimized non-automated process that should allow for a GMP-compliant, more affordable, and reduced-cost strategy to exploit the potential of SVF-based regenerative therapies.

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.

Human Platelet Lysate as a Functional Substitute for Fetal Bovine Serum in the Culture of Human Adipose Derived Stromal/Stem Cells.

Introduction: Adipose derived stromal/stem cells (ASCs) hold potential as cell therapeutics for a wide range of disease states; however, many expansion protocols rely on the use of fetal bovine serum (FBS) as a cell culture nutrient supplement. The current study explores the substitution of lysates from expired human platelets (HPLs) as an FBS substitute. Methods: Expired human platelets from an authorized blood center were lysed by freeze/thawing and used to examine human ASCs with respect to proliferation using hematocytometer cell counts, colony forming unit-fibroblast (CFU-F) frequency, surface immunophenotype by flow cytometry, and tri-lineage (adipocyte, chondrocyte, osteoblast) differentiation potential by histochemical staining. Results: The proliferation assays demonstrated that HPLs supported ASC proliferation in a concentration dependent manner, reaching levels that exceeded that observed in the presence of 10% FBS. The concentration of 0.75% HPLs was equivalent to 10% FBS when utilized in cell culture media with respect to proliferation, immunophenotype, and CFU-F frequency. When added to osteogenic, adipogenic, and chondrogenic differentiation media, both supplements showed appropriate differentiation by staining. Conclusion: HPLs is an effective substitute for FBS in the culture, expansion and differentiation of human ASCs suitable for pre-clinical studies; however, additional assays and analyses will be necessary to validate HPLs for clinical applications and regulatory approval.

CXCR4 Antagonist AMD3100 Promotes Mesenchymal Stem Cell Mobilization in Rats Preconditioned with the Hypoxia-Mimicking Agent Cobalt Chloride.

Mobilization of mesenchymal stem cells (MSCs) is an attractive strategy for cell therapy. Our previous study demonstrated that MSCs can be mobilized in circulating blood by short-term hypoxia, and hypoxia-inducible factor-1α is essential for MSC mobilization. In the present study, the effect of the hypoxia-mimicking agent CoCl2 was examined on MSC mobilization. The results indicated that the frequency of circulating MSCs increased slightly by administration of CoCl2. However, the mobilization efficiency was low. Considering the critical role of stromal cell-derived factor-1α (SDF-1)/CXCR4 axis in the regulation of MSC migration, the effects of granulocyte colony-stimulating factor (G-CSF) and the CXCR4 antagonist AMD3100 were investigated on MSC mobilization. The experiments were notably demonstrated in animals preconditioned with CoCl2. The frequency of colony-forming unit fibroblast and the proportion of CD45-CD90+ cells did not significantly increase in the peripheral blood of rats treated with G-CSF and/or AMD3100 alone. The concomitant administration of G-CSF with CoCl2 could not stimulate the release of MSCs. However, AMD3100 dramatically increased MSC mobilization efficiency in rats pretreated with CoCl2. Furthermore, we identified and compared the multilineage differentiation capacities of MSCs derived from bone marrow (BM-MSCs) and mobilized peripheral blood (PB-MSCs). The results indicated that PB-MSCs exhibited higher osteogenic potential and lower adipogenic differentiation as compared with BM-MSCs. The findings may inform studies investigating mechanisms of the regulation of MSC mobilization and can aid in the development of clinically useful therapeutic agents.

Fetal bovine serum-free cryopreservation methods for clinical banking of human adipose-derived stem cells

The use of fetal bovine serum (FBS) as a cryopreservation supplement is not suitable for the banking of mesenchymal stem cells (MSCs) due to the risk of transmission of disease as well as xenogeneic immune reactions in the transplanted host. Here, we investigated if human serum albumin (HSA), human serum (HS), or knockout serum replacement (KSR) can replace FBS for the cryopreservation of MSCs. In addition, we examined the characteristics of MSCs after multiple rounds of cryopreservation. Human adipose-derived stem cells (ASCs) cryopreserved with three FBS replacements, 9% HSA, 90% HS, or 90% KSR, in combination with 10% dimethyl sulfoxide (Me2SO) maintained stem cell properties including growth, immunophenotypes, gene expression patterns, and the potential to differentiate into adipogenic, osteogenic, and chondrogenic lineages, similar to ASCs frozen with FBS. Moreover, the immunophenotype, gene expression, and differentiation capabilities of ASCs were not altered by up to four freeze-thaw cycles. However, the performance of three or four freeze-thaw cycles significantly reduced the proliferation ability of ASCs, as indicated by the longer population doubling time and reduced colony-forming unit-fibroblast frequency. Together, our results suggest that HSA, HS, or KSR can replace FBS for the cryopreservation of ASCs, without altering their stemness, and should be processed with no more than two freeze-thaw cycles for clinical approaches.

Umbilical cord blood-derived non-hematopoietic stem cells retrieved and expanded on bone marrow-derived extracellular matrix display pluripotent characteristics.

BackgroundUmbilical cord blood (UCB) not only contains hematopoietic stem cells (HSCs), but also non-hematopoietic stem cells (NHSCs) that are able to differentiate into a number of distinct cell types. Based on studies published to date, the frequency of NHSCs in UCB is believed to be very low. However, the isolation of these cells is primarily based on their adhesion to tissue culture plastic surfaces.Methods and resultsIn the current study, we demonstrate that this approach overlooks some of the extremely immature NHSCs because they lack the ability to adhere to plastic. Using a native extracellular matrix (ECM), produced by bone marrow (BM) stromal cells, the majority of the UCB-NHSCs attached within 4 h. The colony-forming unit fibroblast frequency of these cells was 1.5 × 104/108 mononuclear cells, which is at least 4000-fold greater than previously reported for UCB-NHSCs. The phenotype of these cells was fibroblast-like and different from those obtained by plastic adhesion; they formed embryonic body-like clusters that were OCT4-positive and expressed other human embryonic stem cell-related markers. Importantly, when implanted subcutaneously for 8 weeks into immunocompromised mice, these ECM-adherent and expanded NHSCs generated three germ layer-derived human tissues including muscle, fat, blood vessel, bone, gland, and nerve. Moreover, injection of these cells into muscle damaged by cryoinjury significantly accelerated muscle regeneration.ConclusionsThese results indicate that UCB may be a virtually unlimited source of NHSCs when combined with isolation and expansion on ECM. NHSCs may be a practical alternative to embryonic stem cells for a number of therapeutic applications.

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.

Cell Autonomous Defects of Mesenchymal Stem Progenitor Cells of Patients with CMML Contribute to Impaired Hematopoietic Supportive Activity

Objective: Increasing evidence indicates a critical role of the hematopoietic microenvironment in the pathophysiology of myeloid malignancies. Mesenchymal stem/progenitor cells (MSPCs) represent an indispensible cellular component of the bone marrow microenvironment and play an important role in supporting and regulating the proliferation and differentiation of hematopoietic stem/progenitor cells (HSPCs). Abnormal biological characteristics of MSPCs involved in the initiating and development of hematopoietic disorders, such as chronic myelogenous leukemia and myelodysplastic syndromes. However, the characteristics and the role of primary MSPC defects in the pathogenesis of chronic myelomonocytic leukemia (CMML) remain poorly understood.Methods: MSPCs were generated from bone marrow aspirates of patients with CMML and that of healthy controls. To investigate the frequency of the MSPCs in the bone marrow of CMML patients and healthy controls in vivo, a colony-forming unit fibroblast (CFU-F) assay was performed. The phenotypic evaluation of the MSPCs was performed with flow cytometric analysis after staining with a cocktail of cell surface markers, including CD34, CD45, CD29, CD44, CD73 and CD105. To determine the functional difference between the CMML- and control-MSPCs, senescence, cell proliferation, and cell cycle were assessed by EDU incorporation assays, β-galactosidase activity, and flow cytometric analysis (following BrdU/7AAD staining, respectively. To determine the multi-lineage potential of MSPCs, MSPCs were subjected osteoblast, chondrocyte and adipocyte differentiation cultures. The hematopoietic supportive activity was assessed using cobblestone-area forming cell (CAFC) assays by coculturing of CD34+ cord blood cells with CMML- or control- MSPCs. Furthermore, to evaluate the soluble factors of these MSPCs in HSPC supportive activity, the transwell assay was performed with a subsequent flow cytometric analysis further determine the multi-lineage differentiation alteration. Cytokine array was used to determine the cytokines/growth factors in the MSPC conditioned media.Results: The expression levels of the surface markers were identical between CMML-MSPCs and control MSPCs. The frequency of CFU-F is identical between healthy control and CMML patients. However, CMML-MSPCs exhibited a poor proliferative capacity compared to controls MSPCs. CMML MSPCs exhibited an increased senescence and a S phase retardation in the cell cycle as assessed microscopically and flow cytometrically, respectively. Differentiation assay revealed that CMML MSPCs had increased chondrocyte differentiation and reduced adipocyte differentiation, while no alteration of osteoblast differentiation was noticed. In vitro co-cultures of MSPCs and CD34+ umbilical cord blood cells demonstrated that CMML-MSPCs had an impaired hematopoietic supportive capability as compared to control MSPCs as evidenced by reduced numbers of cobble-stone and reduced numbers of CFU-C. Furthermore, a reduced CFU-C and a reduced CFU-GEMM were observed in CAFC and transwell cultures containing CMML-MSPCs compared to that of control MSPCs. In contrast, an increased myeloid cells (CD33+/CD14-/CD15+) and an increased CFU-GM were observed in CD34+ cells and CMML-MSPC co-cultures as compared to that in CD34+ cells and control-MSPC co-cultures. Cytokine array showed that the conditional media of CMML-MSPCs has lower levels of multiple cytokines (IL-6, IL-8, and GRO-β) compared to that of control MSPC conditional media.Conclusion: Collectively, our study indicated that MSPCs of CMML patients had reduced proliferation, increased senescence and reduced cytokine production, while differentiation was not markedly altered. CMML-MSPCs display less hematopoietic supportive activity. Furthermore, CMML-MSPCs induced CD34+ cell differentiation favoring granulomonocytic lineage over erythroid cells. Our study suggests that the cell autonomous defects of CMML-MSPCs contribute to the pathogenesis of CMML and the interaction between hematopoietic compartment and microenvironment could serve as targets for therapeutic interventions. DisclosuresNo relevant conflicts of interest to declare.

Phenotypic and Functional Alterations of Bone Marrow Mesenchymal Stem and Progenitor Cells in Chronic Myeloid Leukemia

Chronic myeloid leukemia (CML) is a myeloproliferative stem cell neoplasm characterized by the presence of the BCR-ABL1 fusion gene. Although current treatment with tyrosine kinase inhibitors (TKI) has dramatically improved the prognosis of CML, these inhibitors do not eradicate leukemic stem cells (LSC) in most patients with the risk of recurrence of leukemia if TKI are stopped. In vitro studies have suggested that this might be attributable to protection of bone marrow (BM) stromal cells, such as osteoblasts, adipocytes, endothelial and mesenchymal stem cells (MSCs). However, how different BM stromal cells contribute to the persistence of LSC remains largely unknown. To investigate this issue we have compared freshly isolated BM stromal cell subsets including MSCs from newly diagnosed CML patients (n=10) with that from age-matched healthy donors (n=12). Distinct from the previous studies on culture-selected BM stromal cells, the naive stromal cells isolated by multi-color fluorescence activated cell sorting (FACS) were phenotypically, molecularly and functionally characterized in the present study.We observed: 1) Similar to the immunophenotype of normal MSCs (CD45-CD235a-CD31-CD44-, most of which were CD271+CD146+CD106+) (Qian et al., JBC, 2012), the CML MSCs, estimated by colony forming unit-fibroblast (CFU-F), were also enriched in the CD45-CD235a-CD31-CD44- cell fraction. 2) The frequency of CFU-Fs was significantly increased in CML BM compared to that in the age-matched healthy controls (p=0.005). 3) A decreased osteogenic, but enhanced adipogenic differentiation potential of CML MSC was revealed in multilineage differentiation assay. This suggests a skewed differentiation potential of the CML MSCs towards adipocytes, possibly related to an altered stromal cell composition in the patients; 4) An increased proportion of CD31+ endothelial cells was seen in CML BM stroma compared to controls (p=0.023) by FACS. 5) An upregulation of the adhesion receptor integrin α4/CD49D was seen in the CD44- MSCs from CML patients (p=0.0087). Conversely, a downregulation of transcripts of Angiopoietin 1, CXCL12, KIT ligand and LAMA4 in the patient MSCs was detected by Quantitative-PCR, indicating an altered hematopoiesis-supportive function of CML MSCs. 6) Importantly, no BCR-ABL fusion were found in the freshly sorted MSCs and mature stromal cells using Fluorescence In Situ Hybridization analysis, suggesting that these MSCs were not part of the leukemic clone. Taken together, our data provide evidence for phenotypic and functional alterations of BM mesenchymal cells in CML patients. The functional relationship between the stromal cell alterations and the growth of LSC as well as the underlying molecular mechanisms are currently under investigation. DisclosuresMustjoki:Finnish Cancer Institute: Research Funding; Sigrid Juselius Foundation: Research Funding; Academy of Finland: Research Funding; the Finnish Cancer Societies: Research Funding; Pfizer: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Signe and Ane Gyllenberg Foundation: Research Funding.

Innovative Cell Therapy in the Treatment of Serious Adverse Events Related to Both Chemo-Radiotherapy Protocol and Acute Myeloid Leukemia Syndrome: The Infusion of Mesenchymal Stem Cells Post-Treatment Reduces Hematopoietic Toxicity and Promotes Hematopoietic Reconstitution

Bone marrow stroma is damaged by chemotherapy and irradiation protocol. Bone marrow microenvironment supports haematopoiesis and comprises Mesenchymal Stem Cells (MSCs). Coinfusion of MSCs with hematopoietic stem cells (HSC) improves engraftment and accelerates haematopoietic recovery. Stroma-derived factor-1 (SDF-1) is a chemotactic factor which plays a crucial role in stem cell transplantation by enhancing the ability of HSC to engraft. In this study expression of SDF-1 in bone marrow MSCs and the level of Colony Forming Unit Fibroblast (CFU-F) were evaluated in 8 patients with Acute Myeloid leukemia (AML). Evaluation was done at diagnosis and after induction/consolidation chemotherapy before the onset of haematopoietic stem cell transplantation (HSCT). CFU-F frequency increases from diagnosis to remission. Nevertheless level of stromal derived factor-1 (SDF-1) transcripts in bone marrow MSCs of patients with AML stays low. Considering the role of SDF-1 in the homing of HSC, the consequences of SDF-1 deficiency observed in this study might be deleterious on the engraftment after HSCT and haematopoietic recovery. The whole result of this clinical study is an argument for MSC infusion to restore a normal level of SDF1 in the bone marrow microenvironment that could reduce hematopoietic toxicity of chemotherapy and improve HSC engraftment after HSCT.

Phenotypic and functional properties of feline dedifferentiated fat cells and adipose-derived stem cells

It has been reported that mature adipocyte-derived dedifferentiated fat (DFAT) cells show multilineage differentiation potential similar to that observed in mesenchymal stem cells. Since DFAT cells can be prepared from a small quantity of adipose tissue, they could facilitate cell-based therapies in small companion animals such as cats. The present study examined whether multipotent DFAT cells can be generated from feline adipose tissue, and the properties of DFAT cells were compared with those of adipose-derived stem cells (ASCs). DFAT cells and ASCs were prepared from the floating mature adipocyte fraction and the stromal vascular fraction, respectively, of collagenase-digested feline omental adipose tissue. Both cell types were evaluated for growth kinetics, colony-forming unit fibroblast (CFU-F) frequency, immunophenotypic properties, and multilineage differentiation potential.DFAT cells and ASCs could be generated from approximately 1g of adipose tissue and were grown and subcultured on laminin-coated dishes. The frequency of CFU-Fs in DFAT cells (35.8%) was significantly higher than that in ASCs (20.8%) at passage 1 (P1). DFAT cells and ASCs displayed similar immunophenotypes (CD44+, CD90+, CD105+, CD14−, CD34− and CD45−). Alpha-smooth muscle actin-positive cells were readily detected in ASCs (15.2±7.2%) but were rare in DFAT cells (2.2±3.2%) at P1. Both cell types exhibited adipogenic, osteogenic, chondrogenic, and smooth muscle cell differentiation potential in vitro. In conclusion, feline DFAT cells exhibited similar properties to ASCs but displayed higher CFU-F frequency and greater homogeneity. DFAT cells, like ASCs, may be an attractive source for cell-based therapies in cats.

This Niche Is a Maze; An Amazing Niche

The cellular identity of niche cells that regulate hematopoietic stem cell (HSC) self-renewal and differentiation has been debated for several years. Two recent studies in Nature (Ding and Morrison, 2013; Greenbaum etal., 2013) have shed light into the bone marrow stromal subsets making CXCL12, a chemokine critical for HSC maintenance.

Isolation and characterization of mesenchymal stem cells from whole human umbilical cord applying a single enzyme approach

Mesenchymal stem cells (MSCs) are multipotent cells traditionally derived from bone marrow (BM). They have been demonstrated to be widely applied in tissue regeneration and cellular therapy. As an alternative to BM, an umbilical cord (UC) is considered as a potential source of MSCs. Here, we showed that human UC-MSCs were easily isolated by a single enzymatic digestion and characteristic of plastic adherence and fibroblast-like morphology. UC-MSCs isolation was successful in 15 of 15 samples. The colony-forming unit-fibroblast frequency was obtained 54 ± 1.33 from 10³ UC-MSCs at passage 3, and the doubling time was (24.15 ± 0.49) h. Almost 10¹⁰ UC-MSCs were largely produced in about 30 days. By flow cytometry analysis, the adherent cells displayed an abundant presence of CD73, CD90 and CD105 and absence of CD34, CD45 and HLA-DR. When cultured in differentiation media, they can be differentiated into adipocytes, osteocytes and chondrocytes. RT-PCR reactions confirmed that their multidifferentiation related genes were positive. Moreover, stem cell-related transcription factors Nanog, Oct-4 and Sox-2 were positively expressed in UC-MSCs. On the basis of these findings, the single enzyme method is a good method to obtain large-scale production of MSCs from whole human UC in a short time, and the UC can be considered as a novel and convenient source of adult MSCs displaying high expansion potential and primitive pluripotent stem cells.

Evaluating the Impact of Oxygen Concentration and Plating Density on Human Wharton's Jelly-Derived Mesenchymal Stromal Cells

Isolates of mesenchymal stromal cells (MSCs) contain a mixed cell population of stem cells, multipotent and unipotent progenitors, and differentiated cells. It is speculated that the useful subpopulation for tissue engineering and cell therapy will be the multipotent progenitor cells or the stem cells. The colony forming unit-fibroblast (CFU-F) assay is an in vitro assay for clonogenicity, which is one property of the stem/progenitor cell population of MSCs. Our goal was to generate standard protocols that would permit the expansion and maintenance of CFU-F. Previous work reported that low plating density and/or exposure to 5% oxygen vs. 21% oxygen increased proliferation rate and enhanced expansion of MSCs. Here, we characterized the effect of both plating density and oxygen concentration on MSCs derived from Wharton's jelly (WJCs). We found that reducing oxygen concentration from 21% (room air) to 5% during expansion increased cell yield and maintained CFU-F, without affecting the expression of surface markers or the differentiation capacity of WJCs. In addition, reducing plating density from 100 cells/cm 2 to 10 cells/cm 2 increased CFU-F frequency. Therefore, plating density and oxygen concentration are two important variables that affect the expansion rate and frequency of CFU-F of WJCs. These results suggest that these two variables might be used to produce different input populations for tissue engineering or cellular therapy.

Implantation of biomaterial as an efficient method to harvest mesenchymal stem cells

Autologous mesenchymal stem cell (MSC) transplants have been used successfully to treat a number of diseases, and patients undergoing cell transplantation must have stem cells collected before transplantation. In this study, we developed a new method to harvest MSCs. Biomaterials were implanted into the spatium intermuscular of mice hind limbs, and a large number of migrating cells (MCs) were isolated from the transplanted biomaterials. The adherent cells in MCs showed the characteristics of MSCs. Further comparative study demonstrated that the characteristics of MC-MSCs were similar to that of bone marrow (BM)-MSCs, including morphology, phenotype, proliferation potential, multilineage differentiation capacity and hematopoiesis-supportive function. The colony-forming unit-fibroblast frequency of the MCs was equivalent to approximately 20-fold of that of the BM. In addition, a BM transplantation experiment demonstrated that MC-MSCs were derived from the peripheral blood. In conclusion, we successfully establish an efficient method to harvest MSCs, and together with the distinct advantages of this method, such as accessibility and possibility for autologous cell therapy, we conclude that our efficient method may be a promising alternative for clinical application.