Number Of CFU-F Research Articles

Pre-proenkephalin 1 is Downregulated Under Unloading and is Involved in Osteoblast Biology.

Pre-proenkephalin 1 (Penk1) is a pro-neuropeptide that belongs to the typical opioid peptide's family, having analgesic properties. We previously found Penk1 to be the most downregulated gene in a whole gene profiling analysis performed in osteoblasts subjected to microgravity as a model of mechanical unloading. In this work, Penk1 downregulation was confirmed in the bones of two in vivo models of mechanical unloading: tail-suspended and botulinum toxin A (botox)-injected mice. Consistently, in the sera from healthy volunteers subjected to bed rest, we observed an inverse correlation between PENK1 and bed rest duration. These results prompted us to investigate a role for this factor in bone. Penk1 was highly expressed in mouse bone, but its global deletion failed to impact bone metabolism in vivo. Indeed, Penk1 knock out (Penk1-/-) mice did not show an overt bone phenotype compared to the WT littermates. Conversely, in vitro Penk1 gene expression progressively increased during osteoblast differentiation and its transient silencing in mature osteoblasts by siRNAs upregulated the transcription of the Sost1 gene encoding sclerostin, and decreased Wnt3a and Col1a1 mRNAs, suggesting an altered osteoblast activity due to an impairment of the Wnt pathway. In line with this, osteoblasts treated with the Penk1 encoded peptide, Met-enkephalin, showed an increase of Osx and Col1a1 mRNAs and enhanced nodule mineralization. Interestingly, primary osteoblasts isolated from Penk1-/- mice showed lower metabolic activity, ALP activity, and nodule mineralization, as well as a lower number of CFU-F compared to osteoblasts isolated from WT mice, suggesting that, unlike the transient inhibition, the chronic Penk1 deletion affects both osteoblast differentiation and activity. Taken together, these results highlight a role for Penk1 in the regulation of the response of the bone to mechanical unloading, potentially acting on osteoblast differentiation and activity in a cell-autonomous manner.

Crosstalk of Endothelial and Mesenchymal Stromal Cells under Tissue-Related O2

Mesenchymal stromal cells (MSCs) are considered a valuable tool for cell therapy. After systemic administration, the outcome of MSCs and endothelial cells (ECs) interactions strongly depend on the local microenvironment and tissue O2 levels in particular. In vitro analysis of EC effects on MSC regenerative potential in co-culture was performed after short-term interaction at “physiological” hypoxia (5% O2) and acute hypoxic stress (0.1% O2). At 5% O2, MSCs retained stromal phenotype and CFU-f numbers, osteogenic RUNX2 was upregulated. A shift in the expression of adhesion molecules, and an increase in transcription/synthesis of IL-6, IL-8 contributed to facilitation of directed migration of MSCs. In the presence of MSCs, manifestations of oxidative stress in ECs were attenuated, and a decrease in adhesion of PBMCs to TNF-α-activated ECs was observed. Under 0.1% O2, reciprocal effects of ECs and MSCs were similar to those at 5% O2. Meanwhile, upregulation of RUNX2 was canceled, IL-6 decreased, and IL-8 significantly increased. “Protective” effects of MSCs on TNF-α-ECs were less pronounced, manifested as NOS3 downregulation and intracellular NO elevation. Therefore, interaction with ECs at “physiological” hypoxia enhanced pro-regenerative capacities of MSCs including migration and anti-inflammatory modulation of ECs. Under acute hypoxic stress, the stimulating effects of ECs on MSCs and the “protective” potential of MSCs towards TNF-α-ECs were attenuated.

Hematopoietic stem cell and mesenchymal stem cell population size in bone marrow samples depends on patient's age and harvesting technique.

Mesenchymal stem cells (MSCs) are heterogeneous population of cells with great potential for regenerative medicine. MSCs are relatively easy to expand in a cell culture, however determination of their concentration in harvested tissue is more complex and is not implemented as routine procedure. To identify MSCs collected from bone marrow we have used two combinations of cell markers (CD45-/CD73+/CD90+/CD105+ and CD45-/CD271+) and fibroblast colony-forming unit (CFU-F) assay. Further, in donors of various ages, mesenchymal stem cell concentration was compared with the result of CFU-F assay and with hematopoietic stem cell concentration, determined by a standardized flow cytometric assay. A positive correlation of MSC populations to the CFU-F numbers is observed, the population of the CD45-/CD271+ cells correlates better with CFU-F numbers than the population of the CD45-/CD73+/CD90+/CD105+ cells. The relationship between the hematopoietic CD45dim/CD34+ cell concentration and mesenchymal CFU-Fs or CD45-/CD271+ cells shows a positive linear regression. An age-related quantitative reduction of hematopoietic CD45dim/CD34+, mesenchymal CD45-/CD73+/CD90+/CD105+ and CD45-/CD271+ stem cells, and CFU-F numbers were noted. Additionally, statistically significant higher CFU-F numbers were observed when bone marrow samples were harvested from three different sites from the anterior iliac crest instead of harvesting the same sample amount only from one site.

The Effects of Radiation and Hindlimb Unloading on Rat Bone Marrow Progenitor Cells

Bone marrow cells of mesenchymal origin play an important role in adaptation of physiological systems to space flight. Hematopoiesis, immunity, and homeostasis of bone tissue depend on their functional activity. An investigation that was carried out in the framework of the Bion and Bion-M programs showed a decrease of the number of rat bone marrow hematopoietic progenitors and the inhibition of lympho- and erythropoiesis when the granulocyte-macrophage linage was activated. A negative influence on nonhematopoietic bone marrow cells was also revealed. The pathogenetic influence of radiation and microgravity on the bone marrow progenitor cells has remained unclear so far. The goal of this research was to study the effect of a 30-day unloading and 6 days of γ-irradiation on rat bone marrow progenitor cells. The study was conducted on male rats of four groups: vivarium control (VC), hindlimb unloading (HU), irradiation (IR), and combined action (HU + IR). The following parameters have been examined: the number of bone marrow mononuclear cells, proliferative activity of marrow mononuclear cells, immunophenotype, number of hematopoietic CFU and CFU-f, and differentiation potency of hematopoietic and stromal bone marrow precursors. It was found that the cellularity and proliferation activity of rat bone marrow cells did not change under simulation of space flight. The number of CFU-f was decreased. Irradiation was accompanied by an increase in the hematopoietic cell share among total bone marrow mononuclear cells, while their activity was attenuated. The osteopotential of the stromal precursors was unchanged. Adipogenic differentiation was stimulated with irradiation. The functional activity of bone marrow progenitor cells was restored after 2 weeks of recovery. Thus, 30-day simulation of space flight factors negatively affected the morphofunctional properties of rat bone marrow progenitor cells. These effects were reversible upon 2 weeks of recovery.

Comparative characteristics cultural properties of adherent cells derived from bone marrow of multiorgan donors and tissues donors

Purpose of work - compare cultural properties of adherent cells derived from bone marrow of multiorgan donors and tissues donors, to assess the possibility of using these cells in clinical practice. Materials and methods. Adherent culture cells from cadavers bone marrow were obtained using standart methods of isolation, scaling, and passaging cells. In culture, counted the number of CFU-F, proliferating activity of cell , their complies with the minimal criteria for defining multipotent mesenchymal stromal cells (hMSC) and compatibility with allogeneic tissue grafts. The Results. In cultures of cells derived from multiorgan donors and tissues donors the number of CFU-F was 0,0004% and 0,00024%, while the duration of the phase "rest proliferative" was 3-4 and 10-12 days respectively. All examined cell culture after the second passage have gained a high degree of homogeneity, and the cells were complies the minimum criteria hMSC. Conclusion: bone marrow of multiorgan donors and tissues donors can be used for obtain hMSC, suitable for creating a combined cellular-tissue grafts for clinical use.

Proliferation And Differentiation Potential Of Canine Synovial Fluid Cells

Abstract The aim of this study was to determine whether synovial fluid (SF) of dogs contains cells that have characteristics of MSCs and to describe their differentiation potential. SF adherent cells from 5 young German shepherd dogs (average 3.8 ± 0.9 years) were expanded (37°C, 5% CO2, humidified atmosphere) three weeks before their phenotype was characterized by flow-cytometry for the presence of CD90 and CD34. Population doubling time (PDT), number of CFU-F and adipogenic, osteogenic and chondrogenic potentials have been determined in vitro. In early passages PTD was 31 ± 10 hours and expansion fold after 3 sub cultivations (9 days) theoretically could be 372 ± 134. At P1, 0.55 ± 0.05% of SF cells had the ability to form CFU-F. Sixty-six percent of cells expressed CD90 and none of the cells expressed markers of hematopoietic cells. Oil Red O staining has shown accumulation of fat droplets in cells grown in adipogenic medium, while deposits of calcium in the osteogenic medium were evidenced with Alizarin red staining. SF cultured in hondrogenic and control medium in three-dimensional conditions formed a cartilage-like tissue. Alcian blue staining of pellets’ slides have shown a significant amount of glycosaminoglycans (GAGs) and immunohistochemistry analysis documented collagen type II expression. The amount of GAGs in pellets grown in both conditions showed no difference. SF cells in vitro exhibited osteogenic, adipogenic and chondrogenic differentiation potentials, suggesting the presence of different mesenchymal progenitors. These results also demonstrated that SF cells have a spontaneous chondrogenic potential that should be further explored for possible tissue engineering protocols.

Are MSCs Stem Cells? Demonstration of in Vitro and in Vivo Stem Cell Properties of Highly Enriched linneg/CD45neg/CD271pos/CD140alow/Neg Mesenchymal Stromal Cells from Adult Human Bone Marrow

Human bone marrow contains a rare population of non-hematopoietic mesenchymal stromal cells (BM-MSC) with multilineage differentiation capacity, which are essential constituents of the hematopoietic microenvironment. Self-renewal and differentiation are the two key properties of somatic stem cells, however, stem cell properties of human adult BM-MSC have not been demonstrated conclusively yet. We have previously shown that low/negative expression of PDGFRα on linneg/CD45neg/CD271pos cells identified a highly enriched population of primary BM-MSC in adult human bone marrow (Li et al. Blood, 2013, 122:3699). Based on this work, the current study aimed to investigate the in-vitro and in-vivo stem cell properties of this putative stromal stem cell population.The in-vitro clonogenic potential of freshly sorted human linneg/CD45neg/CD271pos/PDGFRlow/neg cells was evaluated by utilizing the CFU-F assay as well as the recently-developed mesensphere assay, which enables MSC amplification while preserving an immature phenotype (Isern et al, Cell Reports 2013, 30: 1714-24). Comparable colony frequencies were obtained with both assays (19.3 ± 2 and 17.5 ± 2.3 CFU-F and spheres per 100 plated cells, respectively, n=6, p=0.19). In order to test whether both assays identified the same population of clonogenic cells, colonies and spheres were replated under both conditions for up to three generations. The results showed comparable capacities of CFU-F and mesenspheres to form secondary and tertiary CFU-F and spheres. In-vitro self-renewal as indicated by increasing numbers of CFU-F and spheres (416.6 ± 431.7-fold and 49.5 ± 65.7-fold, respectively, n=3) was observed up to the third generation and decreased thereafter. The total number of generations was five (CFU-F) and six (spheres). In-vitro differentiation assays with both, CFU-F- and sphere-derived cells (tested until passage three) demonstrated tri-lineage differentiation potential (adipocytes, osteoblasts, chondrocytes). In addition, CFU-Fs and spheres had comparable surface marker profiles (CD73, CD90, CD105, and HLA-ABC positive; CD31, CD34 and HLA-DR negative), except for CD90, which was higher expressed on CFU-Fs.To investigate in-vivo self-renewal and differentiation potential of the putative stromal stem cells, linneg/CD45neg/CD271pos/PDGFRlow/neg -derived CFU-F and spheres were serially transplanted s.c into NSG mice. After 8 weeks, implants were harvested, human cells were FACS-isolated (CD90 and CD105 expression), and re-assayed under CFU-F and sphere conditions. Whereas in-vivo self-renewal of CFU-F could not be shown (111.5 ± 36 –fold decrease in total CFU-F numbers after primary transplantation, n=3), sphere self-renewal was clearly demonstrated by increased numbers of spheres after primary as well as secondary transplantation (1.13 ± 0.05 and 2.06 ± 0.26 –fold, respectively, n=3), which is remarkable given the fact that the number of recovered human cells is underestimated due to the isolation approach. Here, confirming GFP-marking experiments are ongoing. Finally, preliminary data indicate that linneg/CD45neg/CD271pos/PDGFRlow/neg –derived spheres display full in-vivo differentiation capacity in primary and secondary transplantations.Taken together, our data demonstrate - for the first time - that primary human linneg/CD45neg/CD271pos/PDGFRlow/neg cells meet stringent stem cell criteria, i.e. in-vitro and in-vivo self-renewal and differentiation. These findings answer the long-open question of the potential stem cell properties of adult human MSC and will enable to better understand the properties of native BM-MSC and their biological role in the bone marrow. DisclosuresNo relevant conflicts of interest to declare.

Comparative study of in vitro expansion of bone marrow-derived mesenchymal stem cells

Bone marrow-derived mesenchymal stem cells (BMD-MSCs) are of great interest for tissue engineering, but require expansion before they can be used for therapeutic applications. We compared three different culture techniques for their potential for large scale expansion of rat BMD-MSCs, i.e. monolayer cultures, stirred suspension cultures and pour-off cultures, and found that pour-off cultures supported the biggest expansion in BMD-MSCs as measured by the fibroblastic-colony forming unit assay (CFU-f). BMD-MSCs expanded in stirred suspension cultures stopped proliferating altogether and, although monolayer cultures allowed for expansion of BMD-MSCs, they favoured a differentiated phenotype over uncommitted MSCs. Only BMD-MSCs expanded in pour-off cultures were able to differentiate into both osteoblastic and adipocytic lineages and maintain CFU-f numbers. These data suggest that pour-off cultures are a viable method of BMD-MSC expansion.

The influence of cardiovascular risk factors on bone marrow mesenchymal stromal cell fitness

In the past, cell transplantation strategies for the treatment of heart failure have shown promising results in experimental and clinical studies. Bone marrow (BM)-derived stem cells represent the most frequently used cell population. Within this heterogeneous cell population, mesenchymal stromal cells (MSC) have been identified to induce therapeutic effects, mainly through paracrine mechanisms. Because of their low frequency in native tissues, in vitro cell culture expansion is mandatory prior to transplantation. We sought to identify patient-specific cardiovascular risk factors influencing the proliferative potential of MSC. BM aspirates from 51 patients undergoing elective cardiac surgery were analyzed for MSC frequency and cell culture expansion potential. Fibroblastic colony-forming units (CFU-F) were quantified for culture conditions applying autologous (AS) or fetal bovine serum (FBS) and different basic media. Univariate and multivariate analyzes were performed in order to determine the impact of patient-specific factors on CFU-F numbers. Expanded MSC showed a specific immune phenotype and displayed adipogenic, chondrogeneic and osteogeneic differentiation potential. CFU-F numbers did not differ under AS or FBS supplementation. Elevated numbers of mononuclear cells, diabetes mellitus, steroid treatment, chronic obstructive pulmonary disease, renal failure, high euroSCORE and impaired left ventricular function were significant determinants for higher CFU-F numbers. The impact of specific cardiovascular risk factors on MSC fitness could be determined. These results may help to establish patient profiling in order to identify patients suitable for autologous MSC transplantation, and might lead to the identification of disease-related mechanisms of stem cell activation.

Mesenchymal Stromal Cells Improve Salivary Function and Reduce Lymphocytic Infiltrates in Mice with Sjögren's-Like Disease

BackgroundNon-obese diabetic (NOD) mice develop Sjögren's-like disease (SS-like) with loss of saliva flow and increased lymphocytic infiltrates in salivary glands (SGs). There are recent reports using multipotent mesenchymal stromal cells (MSCs) as a therapeutic strategy for autoimmune diseases due to their anti-inflammatory and immunomodulatory capabilities. This paper proposed a combined immuno- and cell-based therapy consisting of: A) an injection of complete Freund's adjuvant (CFA) to eradicate autoreactive T lymphocytes, and B) transplantations of MSCs to reselect lymphocytes. The objective of this was to test the effectiveness of CD45−/TER119− cells (MSCs) in re-establishing salivary function and in reducing the number of lymphocytic infiltrates (foci) in SGs. The second objective was to study if the mechanisms underlying a decrease in inflammation (focus score) was due to CFA, MSCs, or CFA+MSCs combined.Methodology/Principal FindingsDonor MSCs were isolated from bones of male transgenic eGFP mice. Eight week-old female NOD mice received one of the following treatments: insulin, CFA, MSC, or CFA+MSC (combined therapy). Mice were followed for 14 weeks post-therapy. CD45−/TER119− cells demonstrated characteristics of MSCs as they were positive for Sca-1, CD106, CD105, CD73, CD29, CD44, negative for CD45, TER119, CD11b, had high number of CFU-F, and differentiated into osteocytes, chondrocytes and adipocytes. Both MSC and MSC+CFA groups prevented loss of saliva flow and reduced lymphocytic infiltrations in SGs. Moreover, the influx of T and B cells decreased in all foci in MSC and MSC+CFA groups, while the frequency of Foxp3+ (Treg) cell was increased. MSC-therapy alone reduced inflammation (TNF-α, TGF-β), but the combination of MSC+CFA reduced inflammation and increased the regenerative potential of SGs (FGF-2, EGF).Conclusions/SignificanceThe combined use of MSC+CFA was effective in both preventing saliva secretion loss and reducing lymphocytic influx in salivary glands.

The influence of pineal factors on the bone marrow cell profile in animals of different ages during changes of the thymus functional condition

The role of thymus on the effects of a pineal body on the number of stromal fibroblast colony-forming units (CFU-F), granulocyte and macrophage colony-forming units (CFU-GM), as well as CD3+-, CD4+-, and CD8+-cells, in the bone marrows of adult and old CBA/Ca mice was studied. Thymectomized and sham-operated mice received either melatonin in the evening (during spring) or epithalamin in the morning (during summer and autumn). After the administration of melatonin, the CFU-F/CFU-GM ratio decreased in adult sham-operated animals, and increased in adult animals after thymectomy. Melatonin induced an increase of the CD4+/CD8+ ratio in mice with an intact thymus and a decrease in mice after thymectomy due to the increase of number of CD8+-cells. Epithalamin caused the increase of the number of CFU-F in the summer but not in the autumn; this effect was less prominent in mice after thymectomy. The changes of the studied parameters under the influence of pineal factors in adult mice with an intact thymus correspond to their seasonal fluctuations. After administration of melatonin, the CFU-F/CFU-GM ratio slightly decreased in old sham-operated animals, and did not change in animals after thymectomy. The CD4+/CD8+ ratio in old mice slightly increased under the effect of melatonin, regardless of the presence or absence of thymus. Thus, in adult mice, the effect of pineal factors (melatonin, epithalamin) on the cell profile of bone marrow, and particularly on the cells of the microenvironment, is adaptive and involves thymus. In old mice, thymus is also involved in the effects of melatoninon on bone marrow, however, its influence is less pronounced and mostly appears as a change of the CFU-GM.

Effect of antigens on colony forming efficiency of stromal clonogenic cells and expression of cytokine genes in primary cultures of bone marrow and spleen of mice

Presence of microbial cells preparation (HCL – extract of group A streptococcus) in primary cell cultures of bone marrow (BMCC) and spleen (SCC) of CBA mice induced the expression mRNA of proinflammatory cytokines genes: IL-2, IL-6 and IL-8 in BMCC; in SCC cultures mRNA IL-1 β and IL-6 appeared and mRNA IL-4 disappeared. Injection of S. typhimurium antigen complex to mice CBA increased 5 times colony forming efficiency (CFE) and, respectively, content of stromal precursor cells (CFU-F) in femur bone marrow and 9 times in spleen of these animals with maximum at the first day. In both primary BMCC and SCC from immunized animals expression of proinflammatory cytokines genes IL-1 β, IL-6, IL-8 and TNF- α was revealed - in BMCC on 1 - 3 day after immunization, and in SCC – up to 15 days. In CBA mice injected with polyvinylpyrrolidone (PVP) CFE and number of CFU-F in BMCC and SCC increased 1, 9 and 1, 8 times only and expression of genes IL-6, IL-8 and TNF- was observed only on the first day. In СВА/N xid mice there was neither increase CFU-F in the corresponding organs, nor expression of proinflammatory cytokines genes in primary cultures. The data suggest the possibility of positive participation of stromal cells in the development on of immune response in organism. Degree of activation of stromal tissue in immunized mice, apparently, correlates with the degree of immune response, supposing a close relationship between stromal tissue and immune system.

Leukemia-Induced Alterations of Bone Marrow Microenvironment Contribute to Leukemogenesis.

AbstractAbstract 2597The impact of microenvironment on the regulation of normal hematopoietic process is being unveiled, but their role during leukemogenesis have been poorly understood. In this study, to characterize the leukemic microenvironment, we have analyzed mesenchymal stromal cells (MSC) of bone marrows from 55 cases of acute myelogenous leukemia (AML), and 17 of AML in the remission. When the colonogenic activity of MSCs were compared, 43% of AML-derived BM exhibited defect in colony (CFU-F) formation, where no colony formation or premature growth arrest of colonies were observed, whereas only 11% of normal BM exhibited growth arrest of colonies. In the AML subtypes, M3 type (FAB classification) exhibited most profound loss of CFU-Fs, where none of them (n=9) exhibited normal growth of colonies. In addition, numbers of colony (CFU-F) in AML was significantly lower than normal BM (52 vs. 155 per each ml of BM, respectively for AML and normal, p<0.05). In contrast, BMs of AML in remission did not exhibit significant difference in the number of CFU-F compared to numbers of normal BM (88 vs. 71.4 per 5×106 MNCs, respectively for normal and remission), indicating that the quantitative loss of CFU-F is correlated to disease activity of AML.We next examined the qualitative changes of MSCs in AML, i.e., 83% of MSCs in AML (5/6) exhibited poor osteogenic differentiation, and all examined AML MSCs (6/6) exhibited lower adipogenic differentiaton compared to normal MSCs. During in-vitro culture, MSCs from AML showed accelerated loss of colonogenic activity during passage cultures compared to normal MSCs and lower intensity levels of CD146 in the cell surface. Moreover, MSCs from AML showed significantly higher levels of senescence-associated beta-galactosidase without changes in telomere length, indicating that the MSC has undergone qualitative change during leukemogenesis. When co-cultured with umbilical cord blood-derived CD34+ cells, AML-derived MSCs inhibited proliferation of CD34+ cells and caused significantly lower levels of hematopoietic cell expansion compared to the levels from co-culture with normal BM-derived MSCs or stroma-free cultures, indicating that leukemic MSCs should contribute to the loss of normal hematopoietic cells in leukemia.Taken together, our study shows that microenvironment of BM undergoes quantitative and qualitative alteration during leukemogenesis and that such leukemic microenvironment contributes to the pathology of leukemia. Disclosures:No relevant conflicts of interest to declare.

G-CSF increases mesenchymal precursor cell numbers in the bone marrow via an indirect mechanism involving osteoclast-mediated bone resorption

During the course of studies to investigate whether MPC circulate in response to G-CSF, the agent most frequently used to induce mobilization of hematopoietic progenitors, we observed that while G-CSF failed to increase the number of MPC in circulation (assayed in vitro as fibroblast colony-forming cells, CFU-F), G-CSF administration nevertheless resulted in a time-dependent increase in the absolute number of CFU-F within the BM, peaking at Day 7. Treatment of BM cells from G-CSF-treated mice with hydroxyurea did not alter CFU-F numbers, suggesting that the increase in their numbers in response to G-CSF administration is not due to proliferation of existing CFU-F. Given previous studies demonstrating that G-CSF potently induces bone turnover in mice, we hypothesized that the increase in CFU-F may be triggered by the bone resorption that occurs following G-CSF administration. In accord with this hypothesis, administration of an inhibitor of osteoclast differentiation, osteoprotegerin (OPG), prevented the increase of CFU-F numbers induced by G-CSF. In conclusion, these data indicate that the cytokine treatment routinely used to mobilize hematopoietic stem cells could provide a readily applicable method to induce in vivo expansion of MPC for clinical applications.

MSC frequency correlates with blood vessel density in equine adipose tissue.

Mesenchymal stem cells (MSCs) are multipotent cells that have the capacity to develop into different mature mesenchymal cell types. They were originally isolated from bone marrow, but MSC-like cells have also been isolated from other tissues. The common feature of all of these tissues is that they all house blood vessels. It is, thus, possible that MSCs are associated with perivascular locations. The objective of this work was to test the hypothesis that MSCs are associated with blood vessels by verifying if MSC frequency positively correlates with blood vessel density. To this end, samples from highly and poorly vascularized adipose tissue sites of two equine donors were collected and processed for histology and cell isolation. MSC frequency in these samples was estimated by means of CFU-F assays, which were performed under MSC conditions. Culture-adherent cells from equine adipose tissue and bone marrow were culture expanded, tested for differentiation into mesenchymal cell types in vitro, and implanted in vivo in porous ceramic vehicles to assess their osteogenic capacity, using human MSCs and brain pericytes as controls. The differentiation assays showed a difference between adipose tissue-derived cells as compared to equine bone marrow MSCs. While differences in CFU-F frequencies between both donors were evident, the CFU-F numbers correlated directly with blood vessel densities (r(2) = 0.86). We consider these preliminary data as further evidence linking MSCs to blood vessels.

Bone Marrow Mesenchymal Stem Cells Are Altered in B-Cell Chronic Lymphocytic Leukemia.

In B-cell chronic lymphocytic leukemia (B-CLL), CD5+CD19+ malignant cells home into the bone marrow (BM) and circulate in the blood. While CLL tumor cells are not susceptible to apoptosis in vivo, they die rapidly in vitro in the absence of specialized non-hematopoietic feeder cells, such as mesenchymal stem cells (MSC). Recent observations have suggested that there is a functional relationship between B cell clone and the stroma. We have thus compared BM-MSC obtained from B-CLL patients and healthy subjects. We first evaluated the influence of in vitro culture conditions on the number of BM-derived CFU-F and the proliferation of MSC and, in parallel, we quantified in unmanipulated normal and malignant BM samples the CD45negCD14negCD73pos cell subset that was previously shown to contain CFU-F (Veyrat-Masson et al., BJH, 2007). Changes in the level of 42 cytokines/chemokines, were then evaluated in MSC-conditioned media (4 CLL vs 4 normal BM-MSCs) using protein-array (RayBio Human Cytokine Antibody Array IIITM, Tebu-bio SA,). In addition, total RNA was extracted (Rneasy MiniKit, Qiagen,) from 9 expanded MSC at passage 1 (P1) in the presence of bFGF (5 untreated B-CLL BM-MSC: 2 Binet stage A, 2 stage B and 1 stage C; 4 normal BM-MSC) and then reverse transcribed (High Capacity cDNA RT Kit, Applied BioSystems). Quantitative PCR reactions, using dedicated microfluid cards screening 384 selected genes, were then performed (TLDAs, Applied Biosystem Courtaboeuf, France). The expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used to normalize gene expression level. Despite a 16-fold increase in total cell numbers tested, we found that most BM-MSC cultures from B-CLL patients failed under standard culture conditions (IMDM/10%FCS), in contrast with our experience with normal BM (69 % n = 13 vs <0.05 % n = 205 ; p <0.005). In agreement, CD45negCD14negCD73pos cells were under the threshold of detection in most of B-CLL BM samples (11/16). In productive cultures, we found more CFU-F from B-CLL BM formed by large, polygonal mesenchymal cells (58.1 ± 12.7 % vs 11.4 ± 3.6 % ; p = 0.008 ). These cells proliferated poorly and in most cases could not be further amplified. The use of normal human AB serum, CLL serum, or bFGF enabled us to detect CFU-F in most malignant samples and to amplify mesenchymal cells (19/21 (90 %)), but their frequency remained lower than in control BM. By using protein-array, we observed that MSC tended to release lower amounts of IL-6, IL-7, and MCP-1 and sometimes higher amounts of IL-8. The concentrations of these cytokines/chemokines in the MSC culture supernatant are under validation by ELISA. Finally, among the 384 genes tested by RT-qPCR, we identified 16 statistically up-regulated genes and 41 down-regulated genes (Mann Whitney U test, P< .05; and SAM permutation analysis, FDR<5%). Up-regulated genes included several growth and angiogenic factors as well as key players of the stroma - tumor cell crosstalk. Most down-regulated genes were involved in differentiation pathways.Conclusions: These results show that the BM-MSC from B-CLL patients were quantitatively and functionally altered and are dependent for their in vitro growth on circulating soluble factors or on growth factors like bFGF. Interestingly, from this small series, we observed 57 differentially expressed genes which could be involved in the B-CLL specific stromal cell alterations previously reported (dysregulation of cytokine secretion, angiogenesis, host-tumor relationships). These findings suggest the possible permissive role of MSC on B-cell clone progression and raise the question as to whether we are dealing with selection of a mesenchymal subset or with alteration of mesenchymal cells induced by malignant B-cells

Effect of bone marrow grafting on the titanium porous-coated implant in bilateral total knee arthroplasty

Background and purpose Poor bone ingrowth into the porous coating of tibial components has been reported. We hypothesized that iliac marrow grafting might be useful to enhance bone ingrowth into a porous-coated implant. The first part of this study was to examine the presence of fibroblast colony-forming units (CFUF) containing osteogenic precursor cells in tibial bone marrow and iliac bone marrow. The second aim was to compare the clinical and radiographic results after bilateral total knee arthroplasty (TKA) with and without autologous bone marrow transplantation to the bone-implant interface.Methods Simultaneous bilateral TKA was performed in 21 patients with osteoarthritis. Aspirated iliac bone marrow was transplanted to the interface of one randomly selected porous-coated tibial component in each patient, and contralateral knees served as controls. All of the 21 patients were followed for 5 years.Results The average number of CFU-F was significantly lower in tibial marrow than in iliac marrow (p = 0.008). The final fluoroscopically-guided radiographs revealed a decrease in the number of knees with radiolucent lines after marrow grafting compared to those without grafting (p = 0.004).Interpretation Iliac bone marrow is useful as a bone grafting material to enhance the biological fixation in porous-coated implants.

Comparison of Different Strategies of MSC Isolation Revels Advantage To Expand MSC Directly from Purified CD105+ and CD271+ Cells.

The potential for multilineage differentiation together with the ability to expand in cultures are the reasons why Mesenchymal Stem Cells (MSC) are considered to be the population of stem cells for potential treatment for variety of disorders (e. g. Osteogenesis Imperfecta, Myocardium Infarction, GvHD). MSC are isolated from the bone marrow mononuclear cells (MNC) based on their adhesive properties. There have been few attempts to isolate MSC directly based on the expression of selected surface antigens, but these isolation strategies were not compared with “the gold standard” procedure which is still selection by plastic adherence. Nevertheless, it is obvious that a presence of different populations of cells “contaminating” MSC in adherent cell cultures (e.g., endothelial cells, macrophages, dendritic cells) may affect expansion of MSC.In this study we proposed new isolation strategies of bone marrow MSC based on RosetteSep Isolation Kit (Stem Cells Technologies Inc., Vancouver, Canada) and immunomagnetic isolation of CD105+ or CD271+ cell populations (Miltenyi Biotec, Germany). Four fractions of bone marrow mononuclear cells i) non-purified MNC, ii) MNC enriched in MSC by RosetteSep Isolation Kit, iii) sorted CD105+ and iv) sorted CD271+ cells were cultured for three passages. Subsequently, we evaluated i) number of CFU-F colonies, ii) expression of selected surface antigens (CD105, CD166, CD44, CD73, CD45, CD34), iii) in vitro osteogenic differentiation of expanded cells and iv) changes in the expression of genes related to osteogenesis (RQ-PCR). We found that the mean number of CFU-F colonies counted on the 9th day of culture was 26 (range 14,5–41,4), 49 (range 21,2–97,1), 105 (range 36,5–221) and 148 (range 55,3–211) per 107 MNC for non-purified MNC, MNC enriched in MSC by RosetteSep Isolation Kit, purified CD105+cells and purified CD271+cells, respectively. After 3rd passage the phenotype of cells was similar as we observed a comparable percentage of cells positive (over 90%) for CD105, CD166, CD44, CD73 and negative (below 5%) for CD45, CD34 surface antigens in all fractions. The RQ - PCR analysis of mRNA level of osteogenic (osteocalcin, PTHR, α1collagen), adipogenic (lipoprotein lipase, leptin, PPARγ2) and chondrogenic (aggrecan1) genes in all four populations revealed that MSC isolated by means of expression of 105 and CD271 antigens had higher level of mRNA for all assessed genes except for lipoprotein lipase and α1collagen prior to differentiation. After 30 days of osteogenic differentiation RQ - PCR analysis was repeated and compared with that before differentiation. We noticed an increased level of mRNA for osteocalcin and PTHR (markers of osteogenic differentiation) in all four populations, with the highest expression in MSC derived from non-purified MNC. However, this fraction had also the highest mRNA level of PPARγ2, lipoprotein lipase, and aggrecan genes (adipogenic and chondrogenic lineage respectively).Since the highest number of CFU-F was derived from purified CD105+ and CD271+ cells as well as these two populations seem to be the most homogenous based on RQ-PCR data, these cell fractions should be employed to expand most efficiently MSC for potential therapeutic purposes. Our data suggest that, non-MSC cells present in MNC and RosetteSep cultures may negatively affect both the expansion efficiency and differentiation along desired MSC lineage.

Effects of spinal cord injury on osteoblastogenesis, osteoclastogenesis and gene expression profiling in osteoblasts in young rats

Spinal cord injury (SCI) causes a significant amount of bone loss in the sublesional area in animals and humans, and this type of bone loss is different from other forms of osteoporosis such as disuse osteoporosis and postmenopausal osteoporosis. However, no data is available on the cellular and molecular changes of osteoblastogenesis and osteoclastogenesis during SCI-induced bone loss. SCI and SHAM rats were used in this study to investigate osteoblastogenesis and osteoclastogenesis in bone-marrow culture. We also measured bone mass and bone histomorphometry, as well as the expression of alkaline phosphatase (ALP), core binding factor alpha1 (Cbfa-1), osterix, receptor activator of NF-kappaB ligand (RANKL) and osteoprotegerin (OPG) in osteoblast-like cells in bone-marrow culture obtained from SCI and SHAM rats. Bone mineral density (BMD) measurement showed serious bone loss in the tibial ephiphyses and metaphyses of SCI rats compared with SHAM rats. In addition, bone histomorphometry analysis of the tibial metaphyses of SCI rats demonstrated that bone microarchitecture in SCI rats deteriorated further than in SHAM rats, and increased eroded surfaces and bone formation rates were observed in SCI rats. The number of osteoclasts that developed from bone marrow of SCI rats at equal density was significantly increased compared with SHAM rats, and the area of the resorption pits formed in the bone marrow culture from SCI rats was significantly greater than SHAM rats, whereas the number of CFU-F and CFU-OB was similar in both groups. RANKL mRNA and protein levels in osteoblast-like cells in culture obtained from SCI rats were significantly higher than those from the SHAM rats, whereas OPG levels decreased slightly. The ratios of RANKL to OPG expression in SCI rats were significantly higher than those in SHAM rats. However, osteogenic gene profiling of Cbfa-1, ALP and osterix in SCI rats remained similar with SHAM rats. These changes favor increased osteoclast activity over osteoblast activity, and may explain, in part, the imbalance in bone formation and resorption following SCI.

The Proliferative Ability of Adult Bone Marrow Native Mesenchymal Progenitors (NMP) Is Early Altered during In Vitro Passaging.

The rarity of adult bone marrow native mesenchymal stem/progenitor cells and the difficulty in characterizing them in fresh organs renders their amplification necessary before use. Little is known about NPM and the correlation between the properties of amplified cells and those of NPM remains unknown.To study the NPM, the sorted fresh CD45− CD14− CD73+ cell subsets (Boiret et al., Exp. Hematol. 2005) were seeded in a cloning assay. We studied the proliferation ability of mesenchymal cells (MC) from bone marrow (BM) grafts (n=35), filtered hematons (H) (n=10), and spongious bone from femoral head (FH) (n=131), until P2-3 (mean culture duration: 6 weeks in IMDM 10% FCS 1% L-Glutamin). The median age of patients was 38±3 years ranging from 21 to 75 years.Seeding of fresh FH CD45− CD14− CD73+ at very low densities showed that about 0.6% of these cells adhered with a fibroblast-like morphology. More than 60 % of them were NMP and capable of cloning, with a very heterogeneous proliferation amplitude. In the first 10 days, cell proliferation represented a mean of 3.8 doubling population (DP). Addition of EGF and PDGF accelerated this proliferation (6.4 DP) and increased the size of initial clones with no increase in the number of CFU-F. In BM, H and FH, the frequency of CD45− CD14− CD73+ cells in the mononuclear population was 0.02±0.004, 0.47±0.25 and 0.43±0.03%, respectively. That of mesenchymal progenitors (CFU-F) was 15.6±2.7 (BM), 85.18±17.3 (H) and 108.5±7.8 (FH)/106 initial cells, with a majority (60%) of large colonies (>50 cells). The frequency of CFU-F amongst native adherent cells (up to 60%) dramatically decreased since after P0 then appeared relatively stable (down to 7%) during passaging. In parallel, whatever the origin of the MC, the size of colonies decreased progressively, resulting in a majority of <50 cell-colonies at P3. Similarly, we observed that primary culture (P0) showed an average of 15 DP until confluence. After the first trypsinization, cell amplification clearly decreased and remained stable with a mean of 3 DP for each following passage (P1, P2, and P3). Cell proliferation was definitely reduced after about 25 DP, associated with a decrease in colony size.In conclusion, the majority of native BM adherent cells were mesenchymal progenitors. Their in vitro expansion was associated with a clearly decrease in cloning efficiency amongst their progeny from the earliest culture period and with a gradual decrease in their individual proliferative ability, revealing a marked reduction in NMP proliferative potential in standard culture medium. Expanded cells may not be biologically identical to NMP. Therefore, we confirmed here that most NMP from BM collections were in hematons, tissue aggregates usually eliminated by BM filtration. It is necessary now to define other conditions of culture for NMP amplification.