scholarly journals Impact of humanised isolation and culture conditions on stemness and osteogenic potential of bone marrow derived mesenchymal stromal cells

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Salwa Suliman ◽  
Hassan R. W. Ali ◽  
Tommy A. Karlsen ◽  
Jerome Amiaud ◽  
Samih Mohamed-Ahmed ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Replicative Senescence ◽  
Therapeutic Potential ◽  
Culture Conditions ◽  
Human Cells ◽  
Osteogenic Potential ◽  
Delayed Differentiation ◽  
Osteogenic Response

Abstract Therapeutic potential of human bone marrow stromal/stem cells (hBMSC) must be developed using well defined xenogenic-free conditions. hBMSC were isolated from healthy donors (n = 3) using different isolation and expansion methods. Donor I was isolated and expanded by either bone marrow directly seeded and cells expanded in 10% AB human serum (AB) +5 ng/ml fibroblast growth factor-2 (FGF2) [Direct(AB + FGFlow)] or Ammonium-Chloride-Potassium Lysing Buffer was used before the cells were expanded in 10% AB +5 ng/ml FGF-2 [ACK(AB + FGFlow)] or Lymphoprep density gradient medium was used before the cells were expanded in 10% AB +5 ng/ml FGF2 [Lympho(AB + FGFlow)] or bone marrow directly seeded and cells expanded in 10% pooled platelet lysate plasma (PL) + heparin (2 I/U/mL) [Direct(PL)]. Groups for donors II and III were: Direct(AB + FGFlow) or 10% AB +10 ng/ml FGF2 [Direct(AB + FGFhigh)] or Direct(PL). HBMSCs were assessed for viability, multi-potency, osteogenic, inflammatory response and replicative senescence in vitro after 1 and 3 weeks. Pre-selected culture conditions, Direct(AB + FGFhigh) or Direct(PL), were seeded on biphasic calcium phosphate granules and subcutaneously implanted in NOD/SCID mice. After 1 and 11 weeks, explants were analysed for inflammatory and osteogenic response at gene level and histologically. To identify implanted human cells, in situ hybridisation was performed. hBMSC from all conditions showed in vitro multi-lineage potency. hBMSCs expanded in PL expressed stemness markers in vitro at significantly higher levels. Generally, cells expanded in AB + FGF2 conditions expressed higher osteogenic markers after 1 week both in vitro and in vivo. After 11 weeks in vivo, Direct(AB + FGFhigh) formed mature ectopic bone, compared to immature mineralised tissues formed by Direct(PL) implants. Mouse responses showed a significant upregulation of IL-1α and IL-1β expression in Direct(PL). After 1 week, human cells were observed in both groups and after 11 weeks in Direct(AB + FGFhigh) only. To conclude, results showed a significant effect of the isolation methods and demonstrated a relatively consistent pattern of efficacy from all donors. A tendency of hBMSC expanded in PL to retain a more stem-like phenotype elucidates their delayed differentiation and different inflammatory expressions.

scholarly journals Late Adherent Human Bone Marrow Stromal Cells Form Bone and Restore the Hematopoietic MicroenvironmentIn Vivo

2013 ◽  
Vol 2013 ◽  
pp. 1-11 ◽  
Author(s):  
Verônica Fernandes Vianna ◽  
Danielle Cabral Bonfim ◽  
Amanda dos Santos Cavalcanti ◽  
Marco Cury Fernandes ◽  
Suzana Assad Kahn ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stromal Cells ◽  
Bone Marrow Stromal Cells ◽  
Therapeutic Potential ◽  
Marrow Stromal Cells ◽  
Osteogenic Potential ◽  
Phenotypic Properties ◽  
L Cells

Bone marrow stromal cells (BMSCs) are a valuable resource for skeletal regenerative medicine because of their osteogenic potential. In spite of the very general term “stem cell,” this population of cells is far from homogeneous, and different BMSCs clones have greatly different phenotypic properties and, therefore, potentially different therapeutic potential. Adherence to a culture flask surface is a primary defining characteristic of BMSCs. We hypothesized that based on the adherence time we could obtain an enriched population of cells with a greater therapeutic potential. We characterized two populations of bone marrow-derived cells, those that adhered by three days (R-cells) and those that did not adhere by three days but did by six days (L-cells). Clones derived from L-cells could be induced into adipogenic, chondrogenic, and osteogenic differentiationin vitro. L-cells appeared to have greater proliferative capacity, as manifested by larger colony diameter and clones with higher CD146 expression. Only clones from L-cells developed bone marrow stromain vivo. We conclude that the use of late adherence of BMSCs is one parameter that can be used to enrich for cells that will constitute a superior final product for cell therapy in orthopedics.

scholarly journals Hypoxia Preconditioning of Human MSCs: a Direct Evidence of HIF-1α and Collagen Type XV Correlation

2018 ◽  
Vol 51 (5) ◽  
pp. 2237-2249 ◽  
Author(s):  
Elisabetta Lambertini ◽  
Letizia Penolazzi ◽  
Marco Angelozzi ◽  
Leticia Scussel Bergamin ◽  
Cristina Manferdini ◽  
...  
Keyword(s):  
Collagen Type ◽  
Therapeutic Potential ◽  
Hypoxia Inducible Factor ◽  
Culture Conditions ◽  
Luciferase Reporter ◽  
Osteogenic Potential ◽  
Human Mscs ◽  
Short Period ◽  
Hypoxia Preconditioning

Background/Aims: Mesenchymal stromal cells (MSCs) hold considerable promise in bone tissue engineering, but their poor survival and potency when in vivo implanted limits their therapeutic potential. For this reason, the study on culture conditions and cellular signals that can influence the potential therapeutic outcomes of MSCs have received considerable attention in recent years. Cell maintenance under hypoxic conditions, in particular for a short period, is beneficial for MSCs, as low O2 tension is similar to that present in the physiologic niche, however the precise mechanism through which hypoxia preconditioning affects these cells remains unclear. Methods: In order to explore what happens during the first 48 h of hypoxia preconditioning in human MSCs (hMSCs) from bone marrow, the cells were exposed to 1.5% O2 tension in the X3 Hypoxia Hood and Culture Combo – Xvivo System device. The expression modulation of critical genes which could be good markers of increased osteopotency has been investigated by Western blot, immunufluorescence and ELISA. Luciferase reporter assay and Chromatin immunoprecipitation was used to investigate the regulation of the expression of Collagen type XV (ColXV) gene. Results: We identified ColXV as a new low O2 tension sensitive gene, and provided a novel mechanistic evidence that directly HIF-1α (hypoxia-inducible factor-1 alpha) mediates ColXV expression in response to hypoxia, since it was found specifically in vivo recruited at ColXV promoter, in hypoxia-preconditioned hMSCs. This finding, together the evidence that also Runx2, VEGF and FGF-2 expression increased in hypoxia preconditioned hMSCs, is consistent with the possibility that increased ColXV expression in response to hypoxia is mediated by an early network that supports the osteogenic potential of the cells. Conclusion: These results add useful information to understand the role of a still little investigated collagen such as ColXV, and identify ColXV as a marker of successful hypoxia preconditioning. As a whole, our data give further evidence that hypoxia preconditioned hMSCs have greater osteopotency than normal hMSCs, and that the effects of hypoxic regulation of hMSCs activities should be considered before they are clinically applied.

scholarly journals Alendronate Can Improve Bone Alterations in Experimental Diabetes by Preventing Antiosteogenic, Antichondrogenic, and Proadipocytic Effects of AGEs on Bone Marrow Progenitor Cells

2016 ◽  
Vol 2016 ◽  
pp. 1-13 ◽  
Author(s):  
Sara Rocío Chuguransky ◽  
Ana María Cortizo ◽  
Antonio Desmond McCarthy
Keyword(s):  
Bone Marrow ◽  
Experimental Diabetes ◽  
Bone Microarchitecture ◽  
Bone Matrix ◽  
Diabetic Rats ◽  
Long Bone ◽  
Osteogenic Potential ◽  
Bone Marrow Progenitor

Bisphosphonates such as alendronate are antiosteoporotic drugs that inhibit the activity of bone-resorbing osteoclasts and secondarily promote osteoblastic function. Diabetes increases bone-matrix-associated advanced glycation end products (AGEs) that impair bone marrow progenitor cell (BMPC) osteogenic potential and decrease bone quality. Here we investigated the in vitro effect of alendronate and/or AGEs on the osteoblastogenic, adipogenic, and chondrogenic potential of BMPC isolated from nondiabetic untreated rats. We also evaluated the in vivo effect of alendronate (administered orally to rats with insulin-deficient Diabetes) on long-bone microarchitecture and BMPC multilineage potential. In vitro, the osteogenesis (Runx2, alkaline phosphatase, type 1 collagen, and mineralization) and chondrogenesis (glycosaminoglycan production) of BMPC were both decreased by AGEs, while coincubation with alendronate prevented these effects. The adipogenesis of BMPC (PPARγ, intracellular triglycerides, and lipase) was increased by AGEs, and this was prevented by coincubation with alendronate. In vivo, experimental Diabetes (a) decreased femoral trabecular bone area, osteocyte density, and osteoclastic TRAP activity; (b) increased bone marrow adiposity; and (c) deregulated BMPC phenotypic potential (increasing adipogenesis and decreasing osteogenesis and chondrogenesis). Orally administered alendronate prevented all these Diabetes-induced effects on bone. Thus, alendronate could improve bone alterations in diabetic rats by preventing the antiosteogenic, antichondrogenic, and proadipocytic effects of AGEs on BMPC.

scholarly journals Analysis of megakaryocyte ploidy in rat bone marrow cultures

Blood ◽  
1989 ◽  
Vol 74 (6) ◽  
pp. 1952-1962
Author(s):  
DJ Kuter ◽  
SM Greenberg ◽  
RD Rosenberg
Keyword(s):  
Bone Marrow ◽  
Culture Conditions ◽  
Recombinant Erythropoietin ◽  
Bone Marrow Cultures ◽  
Ploidy Changes ◽  
Macrophage Colony ◽  
Vitro Model ◽  
Rat Bone

Megakaryocytes undergo changes in ploidy in vivo in response to varying demands for platelets. Attempts to study the putative factor(s) regulating these ploidy changes have been frustrated by the lack of an appropriate in vitro model of megakaryocyte endomitosis. This report describes a culture system in which rat bone marrow is depleted of identifiable megakaryocytes and enriched in their precursor cells. Morphologically identifiable megakaryocytes appear when the depleted marrow is cultured in vitro. The total number of nucleated cells, as well as the number of megakaryocytes and their ploidy distribution, are quantitated very precisely by flow cytometry. Although the total number of nucleated cells declines by 35% to 40% over 3 days in culture, the number of megakaryocytes rises 10-fold. The number of nucleated cells, the number of megakaryocytes, and the extent of megakaryocyte ploidization behave as independent variables in culture and are dependent on the culture conditions. The addition of recombinant erythropoietin promotes a rise in the number of megakaryocytes and a shift in ploidy to higher values while recombinant murine granulocyte- macrophage colony stimulating factor is without effect on the cultured megakaryocytes. This in vitro system may provide a means to study those factors that affect megakaryocyte growth and ploidization.

scholarly journals Senolytics improve bone forming potential of bone marrow mesenchymal stem cells from aged mice

2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Yueying Zhou ◽  
Xiaonan Xin ◽  
Lichao Wang ◽  
Binsheng Wang ◽  
Li Chen ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Osteogenic Potential ◽  
Calvarial Defect ◽  
Defect Model ◽  
Aged Mice ◽  
Marrow Mesenchymal Stem Cells

AbstractThe osteogenic potential of bone marrow mesenchymal stem cells (BMSCs) declines dramatically with aging. By using a calvarial defect model, we showed that a senolytic cocktail (dasatinib+quercetin; D + Q) improved osteogenic capacity of aged BMSC both in vitro and in vivo. The study presented a model to assess strategies to improve bone-forming potential on aged BMSCs. D + Q might hold promise for improving BMSC function in aged populations.

An In Vitro and In Vivo Evidence That Downregulation of Leukemia Inhibitory Factor (LIF) Receptor (LIF-R) Decreases the Metastatic Potential of Human Rhabdomyosarcoma (RMS) Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2561-2561
Author(s):  
Marcin Wysoczynski ◽  
Katarzyna Miekus ◽  
Anna Marcinkowska ◽  
Anna Janowska-Wieczorek ◽  
Mariusz Z. Ratajczak
Keyword(s):  
Skeletal Muscle ◽  
Bone Marrow ◽  
Cell Line ◽  
Cell Lines ◽  
Leukemia Inhibitory Factor ◽  
Biological Significance ◽  
Human Cells ◽  
Inhibitory Factor

Abstract Rhabdomyosarcoma (RMS) and skeletal muscle-derived tumors frequently infiltrate bone marrow (BM). We have demonstrated that the stromal-derived factor (SDF)-1-CXCR4 receptor (Blood2002;100:2597) and hepatocyte growth factor (HGF)-c-Met receptor (Cancer Res. 2003;63:7926) play an important role in RMS metastasis to BM. Leukemia inhibitory factor (LIF) is a well known factor that plays an important role in skeletal muscle development/regeneration and similarly as SDF-1 and HGF is secreted by BM stroma. This prompted us to examine whether the LIF-LIF receptor (LIF-R) axis affects the biology/metastasis of RMS cells. We employed in our studies, human established RMS cell lines, as well as RMS samples isolated from patients and noticed that LIF-R was expressed not only on established human RMS cell lines (7/7) but more importantly, it was also detectable in patient samples (23/23). We also found that in RMS cells LIF stimulatesphosphorylation of MAPKp42/44, AKT and STAT3,chemotaxis and adhesion andincreases resistance to cytostatics (e.g., etoposide). These LIF-mediated effects were inhibited after downregulating the LIF-R by siRNA. To learn more on the biological significance of the LIF-LIF-R axis in vivo we employed two models. First, human RMS cells (RH-30) were exposed or not exposed to LIF-R siRNA and subsequently injected into SCID™-Beige immunodeficient mice. To estimate the number of RMS cells that seed to BM and liver in these animals, we isolated DNA and using real- time RT-PCR, amplified human a-satellite sequences and murine b-actin. The number of human cells present in murine organs was subsequently calculated from a standard curve derived from mixing varying numbers of human cells with a constant number of murine cells. We noticed that downregulation of LIF-R by siRNA significantly decreased the number of human RMS cells in murine BM and liver (x4 and x2 respectively). In a second model, the RH30 cell line was selected by repetitive chemotaxis for cells that are highly responsive to LIF (RH-30 L) and subsequently the cells from parental RH-30 cell line and RH-30 L cells were injected intramuscularly. Six weeks after tumour inoculation, we detected more metastasis in bone marrow and lungs in mice injected with RH-30L cells as compared to parental RH-30 clone (x6 and x3 respectively). In conclusion, we present evidence for the first time that the inhibition of LIF-LIF-R axis may decrease the invasive potential of human RMS both in vitro and in vivo. Hence, molecular targeting of LIF-LIF-R axis could possibly become a more effective new strategy to control the progression and metastasis of RMS.

A 3 Dimensional in Vitro Model to Test HL-60 Cell Line Sensitivity to Chemotherapy

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4882-4882
Author(s):  
Omar S. Aljitawi ◽  
Dandan Li ◽  
Da Zhang ◽  
Jonathan Mahnken ◽  
Suman Kambhampati ◽  
...  
Keyword(s):  
Cell Culture ◽  
Bone Marrow ◽  
3D Culture ◽  
Culture Conditions ◽  
Cytotoxic Chemotherapy ◽  
3 Dimensional ◽  
3D Microenvironment ◽  
3D Culture System

Abstract Abstract 4882 Introduction: Current in vitro drug testing models are based on 2-dimensional (2D) cell culture systems and therefore do not always predict in vivo responses. This lack of predictability of the 2D assays is believed to be related to the 3-dimensional (3D) microenvironment present in tissues or tumors. This 3D microenvironment, were cell-cell and cell-extracellular matrix (ECM) interactions occur, is fundamental for cell biologic activities. This is especially true for acute myeloid leukemia, were current 2-D cell culture models do not always predict clinical responses. This discrepancy in leukemia cell responses to chemotherapy in vivo, in comparison to in vitro, is at least partly related to leukemia cells interaction with the bone marrow microenvironment and their ability to establish niches. These niches offer partial protection from the effects of cytotoxic chemotherapy, otherwise termed cell adhesion-mediated drug resistance. In these experiments, we investigate the apoptotic effects of cytotoxic chemotherapy on HL-60 cell line cultured in a designed 3D AML cell culture model. In this 3D microenvironment, HL-60 cells were co-cultured with ex vivo expanded bone marrow mesenchyaml stem cells in a 3D synthetic scaffold. Aim: To examine the apoptotic effect of cytotoxic chemotherapy on HL-60 co-cultured with human bone marrow mesenchymal stem cells (huBM-MSCs) in 3D conditions. Methods: After several passages, expanded huBM-MSCs were seeded into PGA/PLLA 90/10 copolymer discs, 5-mm in diameter and 2-mm in thickness and allowed to attach to scaffold fibers and to expand over 2 weeks. Then, HL-60 were added and allowed to grow in the 3D culture system for another 10 days. HL-60 cells in 3D culture system were then exposed to doxorubicin given in two concentrations (25 and 50 μM) and incubated for 24 hours. HL-60 were then retrieved applying a combination of mechanical forces and using cell dissociation solution. FITC Annexin V Apoptosis Detection Kit was used to determine apoptosis. Apoptosis was confirmed by TUNEL assay. Proliferation of HL-60 cells in the 3D scaffold was assessed using Ki-67 stain of scaffold's cryosections. All tests were done in triplicates, and untreated HL-60 served as controls for treatment. Comparison was made with HL-60 cells alone and with HL-60 cells growing on a hu-BM-MSC monolayer. SAS version 9.2 (SAS Institute, Inc., 2002–2008) was used for statistical analysis Results: Virtually, all HL-60 cells treated with 25 or 50 μM underwent late apoptosis. Around.03% of HL-60 cells survived 25 μM concentration, none, however, survived 50 μM concentration. In 2D, most of HL-60 cells underwent necrosis, and to lesser extent late apoptosis. In sharp contrast, 17.8% of HL-60 cells survived 25μM concentration, nevertheless, only.27% of HL-60 cells treated with 50 μM concentration survived. The differences in apoptosis patterns between the three groups was statistically significant (P<.0001). Conclusion: compared to traditional cell culture conditions, the designed 3D culture conditions protected a higher percentage of HL-60 cells from undergoing apoptosis and necrosis. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Nestin+ Mesenchymal Precursors Generate Distinct Spleen Stromal Cell Subsets and Have Immunomodulatory Function

2021 ◽  
Author(s):  
Jing Huang ◽  
Minrong Li ◽  
Ronghai Deng ◽  
Weiqiang Li ◽  
Meihua Jiang ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stromal Cells ◽  
Therapeutic Potential ◽  
Inflammatory Diseases ◽  
Cell Phenotype ◽  
Therapeutic Effects ◽  
Inflammatory Bowel ◽  
Inflammatory Milieu

Abstract Background Mesenchymal stromal cells (MSCs) are known to be widespread in many tissues and possess a broad spectrum of immunoregulatory properties. They have been used in the treatment of a variety of inflammatory diseases; however, the therapeutic effects are still inconsistent owing to their heterogeneity. Spleen stromal cells have evolved to regulate the immune response at many levels as they are bathed in a complex inflammatory milieu during infection. Therefore, it is unknown whether they have stronger immunomodulatory effects than their counterparts derived from other tissues. Methods Here, using a transgenic mouse model expressing GFP driven by the Nestin (Nes) promoter, Nes-GFP+ cells from bone marrow and spleen were collected. Artificial lymphoid reconstruction in vivo was performed. Cell phenotype, inhibition of T cell inflammatory cytokines, and in vivo therapeutic effects were assessed. Results We observed Nes-GFP+ cells colocalized with splenic stromal cells and further demonstrated that these Nes-GFP+ cells had the ability to establish ectopic lymphoid-like structures in vivo. Moreover, we showed that the Nes-GFP+ cells possessed the characteristics of MSCs. Spleen-derived Nes-GFP+ cells exhibited greater immunomodulatory ability in vitro, and more remarkable therapeutic efficacy in inflammatory diseases, especially inflammatory bowel disease (IBD) than bone marrow-derived Nes-GFP+ cells. Conclusions Overall, our data showed that Nes-GFP+ cells contributed to subsets of spleen stromal populations and possessed the biological characteristics of MSCs with a stronger immunoregulatory function and therapeutic potential than bone marrow-derived Nes-GFP+ cells.

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