A non-contact suspension culture approach to the culture of osteogenic cells derived from a CD49elow subpopulation of human bone marrow-derived cells

We demonstrate that adult human bone marrow (BM) contains a population of mesenchymal stromal cells (MSCs) that can be expanded in non-adherent, cytokine-dependent, suspension culture conditions for at least 42 days. The cells generated during suspension culture lacked detectable levels of gene expression associated with differentiated mesenchymal cell types, including bone, muscle and fat, suggesting that suspension culture maintains MSCs in an uncommitted state. However, when these undifferentiated cells were taken out of suspension culture and placed in adherent osteogenic conditions, osteogenic genes were upregulated and morphologically identifiable bone matrix was elaborated. Flow cytometric analysis of uncultured, density gradient-separated human BM revealed that colony forming unit-fibroblast (CFU-F) and CFU-osteoblast (CFU-O) activity was associated with a CD45(-) CD49e(low) phenotype. Importantly, suspension-grown MSCs, capable of CFU-F and CFU-O development, maintained the CD45(-)CD49e(low) phenotype whereas MSCs directly cultured under adherent conditions rapidly upregulated CD49e expression and were associated with a CD45(-)CD49e(high) phenotype. Tracking the CD49e(low) expression under suspension culture conditions provides a mechanism to isolate an expanding suspension-grown MSC population with osteogenic potential. This could provide a potential strategy to isolate populations of MSCs, with functional osteogenic capacity, in a scalable and controllable culture system for therapeutic applications.     

Pre CFU-F in bone marrow cultures from children with hematopoietic disease including acute leukemia

Stromal precursor cells from bone marrow aspirates of children have been studied in culture. In 7 day liquid cultures normal individuals and patients with acute leukemia in remission grew 110 +/- 50 CFU-F and 100 +/- 40 CFU-F (colony forming unit--fibroblasts) respectively, per 6 X 10(5) buffy coat mononuclear cells. Staining with monoclonal antibodies suggests that stromal cells from CFU-F colonies are fibroblasts. CFU-F colony growth from the bone marrow of patients with active leukemia was low. After cultivation periods of more than 21 days, we observed, in addition, still more immature, clonogenic fibroblast precursor cells, "pre CFU-F", and round cells attached to stromal cells from pre CFU-F colonies. From the round cells, we have passaged pre CFU-F and CFU-GM (colony forming unit--granulocytic, monocytic) in secondary cultures. Our observations are in agreement with the concept that the bone marrow stromal cell matrix serves as a sanctuary for reversibly attached clonogenic cells of both the hematopoietic and fibroblast lineages.     

Canonical and non-canonical Wnts differentially affect the development potential of primary isolate of human bone marrow mesenchymal stem cells

This study examines the role of Wnt signaling events in regulating the differential potential of mesenchymal stem cells (MSCs) from adult bone marrow (BM). Immunohistochemical analysis of BM revealed co-localization of Wnt5a protein, a non-canonical Wnt, with CD45(+) cells and CD45(-) STRO-1(+) cells, while Wnt3a expression, a canonical Wnt, was associated with the underlying stroma matrix, suggesting that Wnts may regulate MSCs in their niche in BM. To elucidate the role of Wnts in MSC development, adult human BM-derived mononuclear cells were maintained as suspension cultures to recapitulate the marrow cellular environment, in serum-free, with the addition of Wnt3a and Wnt5a protein. Results showed that Wnt3a increased cell numbers and expanded the pool of MSCs capable of colony forming unit -- fibroblast (CFU-F) and CFU -- osteoblast (O), while Wnt5a maintained cell numbers and CFU-F and CFU-O numbers. However, when cells were cultured directly onto tissue culture plastic, Wnt5a increased the number of CFU-O relative to control conditions. These findings suggest the potential dual role of Wnt5a in the maintenance of MSCs in BM and enhancing osteogenesis ex vivo. Our work provides evidence that Wnts can function as mesenchymal regulatory factors by providing instructive cues for the recruitment, maintenance, and differentiation of MSCs.     

In vivo osteoinductive effect and in vitro isolation and cultivation bone marrow mesenchymal stem cells

Bone marrow contains cell type termed mesenchymal stem cells (MSC), first recognized in bone marrow by a German pathologist, Julius Cohnheim in 1867. That MSCs have potential to differentiate in vitro in to the various cells lines as osteoblast, chondroblast, myoblast and adipoblast cells lines. Aims of our study were to show in vivo capacity of bone marrow MSC to produce bone in surgically created non critical size mandible defects New Zeland Rabbits, and then in second part of study to isolate in vitro MSC from bone marrow, as potential cell transplantation model in bone regeneration. In vivo study showed new bone detected on 3D CT reconstruction day 30, on all 3 animals non critical size defects, treated with bone marrow MSC exposed to the human Bone Morphogenetic Protein 7 (rhBMP-7). Average values of bone mineral density (BMD), was 530 mg/cm3, on MSC treated animals, and 553 mg/cm3 on control group of 3 animals where non critical size defects were treated with iliac crest autologue bone graft. Activity of the Alkaline Phosphatase enzyme were measurement on 0.5, 14, 21, 30 day and increased activity were detected day 14 on animals treated with bone marrow MSCs compared with day 30 on iliac crest treated animals. That results indicates strong osteoinduction activity of the experimental bone marrow MSCs models exposed to the rhBMP-7 factor Comparing ALP activity, that model showed superiorly results than control group. That result initiates us in opinion that MSCs alone should be alternative for the autolologue bone transplantation and in vitro study we isolated singles MSCs from the bone marrow of rat's tibia and femora and cultivated according to the method of Maniatopoulos et all. The small initial colonies of fibroblast like cells were photo-documented after 2 days of primary culture. Such isolated and cultivated MSCs in future studies will be exposed to the growth factors to differentiate in osteoblast and indicate their clinically potential as alternative for conventional medicine and autologue bone transplantation. That new horizons have potential to minimize surgery and patient donor morbidity, with more success treatment in bone regenerative and metabolism diseases.     

Translation of a standardized manufacturing protocol for mesenchymal stromal cells: A systematic comparison of validation and manufacturing data

BackgroundMany data are available on expansion protocols for mesenchymal stromal cells (MSCs) for both experimental settings and manufacturing for clinical trials. However, there is a lack of information on translation of established protocols for Good Manufacturing Practice (GMP) from validation to manufacturing for clinical application. We present the validation and translation of a standardized pre-clinical protocol for isolation and expansion of MSCs for a clinical trial for reconstitution of alveolar bone.MethodsKey parameters of 22 large-scale expansions of MSCs from bone marrow (BM) for validation were compared with 11 expansions manufactured for the clinical trial “Jaw bone reconstruction using a combination of autologous mesenchymal stromal cells and biomaterial prior to dental implant placement (MAXILLO1)” aimed at reconstruction of alveolar bone.ResultsDespite variations of the starting material, the robust protocol led to stable performance characteristics of expanded MSCs. Manufacturing of the autologous advanced therapy medicinal product MAXILLO-1-MSC was possible, requiring 21 days for each product. Transport of BM aspirates and MSCs within 24 h was guaranteed. MSCs fulfilled quality criteria requested by the national competent authority. In one case, the delivered MSCs developed a mosaic in chromosomal finding, showing no abnormality in differentiation capacity, growth behavior or surface marker expression during long-term culture. The proportion of cells with the mosaic decreased in long-term culture and cells stopped growth after 38.4 population doublings.ConclusionsClinical use of freshly prepared MSCs, manufactured according to a standardized and validated protocol, is feasible for bone regeneration, even if there was a long local distance between manufacturing center and clinical site. Several parameters, such as colony forming units fibroblasts (CFU-F), percentage of CD34+ cells, cell count of mononuclear cells (MNCs) and white blood cells (WBCs), of the BM may serve as a predictive tool for the yield of MSCs and may help to avoid unnecessary costs for MSC manufacturing due to insufficient cell expansion rates.     

Impact of Aging on the Regenerative Properties of Bone Marrow-, Muscle-, and Adipose-Derived Mesenchymal Stem/Stromal Cells

Mesenchymal stem/stromal cells (MSCs) are promising cell sources for regenerative therapies due to their multipotency and ready availability, but their application can be complicated by patient-specific factors like age or illness. MSCs have been investigated for the treatment of many musculoskeletal disorders, including osteoarthritis and osteoporosis. Due to the prevalence of these diseases in older populations, researchers have studied how aging affects MSC properties and have found that proliferation and differentiation potential are impaired. However, these effects have never been compared among MSCs isolated from multiple tissue sources in the same, healthy donor. Revealing differences in how MSCs are affected by age could help identify an optimal cell source for musculoskeletal therapies targeting older patients. MSCs were isolated from young and old rabbit bone marrow, muscle, and adipose tissue. Cell yield and viability were quantified after isolation procedures, and expansion properties were assessed using assays for proliferation, senescence, and colony formation. Multipotency was also examined using lineage-specific stains and spectrophotometry of metabolites. Results were compared between age groups and among MSC sources. Results showed that MSCs are differentially influenced by aging, with bone marrow-derived stem cells having impaired proliferation, senescence, and chondrogenic response, whereas muscle-derived stem cells and adipose-derived stem cells exhibited no negative effects. While age reduced overall cell yield and adipogenic potential of all MSC populations, osteogenesis and clonogenicity remained unchanged. These findings indicate the importance of age as a factor when designing cell-based therapies for older patients.     

Investigation of the mesenchymal stem cell compartment by means of a lentiviral barcode library

The hematopoietic bone marrow microenvironment is formed by proliferation and differentiation of mesenchymal stem cells (MSCs). The MSC compartment has been less studied than the hematopoietic stem cell compartment. To characterize the structure of the MSC compartment, it is necessary to trace the fate of distinct mesenchymal cells. To do so, mesenchymal progenitors need to be marked at the single-cell level. A method for individual marking of normal and cancer stem cells based on genetic “barcodes” has been developed for the last 10 years. Such approach has not yet been applied to MSCs. The aim of this study was to evaluate the possibility of using such barcoding strategy to mark MSCs and their descendants, colony-forming units of fibroblasts (CFU-Fs). Adherent cell layers (ACLs) of murine long-term bone marrow cultures (LTBMCs) were transduced with a lentiviral library with barcodes consisting of 32 + 3 degenerate nucleotides. Infected ACLs were suspended, and CFU-F-derived clones were obtained. DNA was isolated from each individual colony, and barcodes were analyzed in marked CFU-F-derived colonies by means of conventional polymerase chain reaction and Sanger sequencing. Barcodes were identified in 154 marked colonies. All barcodes appeared to be unique: there were no two distinct colonies bearing the same barcode. It was shown that ACLs included CFU-Fs with different proliferative potential. MSCs are located higher in the hierarchy of mesenchymal progenitors than CFU-Fs, so the presented data indicate that MSCs proliferate rarely in LTBMCs. A method of stable individual marking and comparing the markers in mesenchymal progenitor cells has been developed in this work. We show for the first time that a barcoded library of lentiviruses is an effective tool for studying stromal progenitor cells.     

AcSDKP抑制体外培养条件下人骨髓间充质干细胞的增殖

N-乙酰基-丝氨酰-天冬氨酰-赖氨酰-脯氨酸(N-acetyl-seryl-aspartyl-lysyl-proline,AcSDKP)是一种具有生理调控活性的四肽因子,对造血干/祖细胞增殖具有抑制作用。本研究采用集落形成实验、甲基偶氮唑盐(MTT)比色法、细胞分裂指数测定等方法,考察了AcSDKP对体外培养的人骨髓间充质干细胞(mesenchymal stem cell,MSC)增殖的影响。结果显示,在AcSDKP浓度为1×10-12mol／L-1×10-9mol／L的培养体系中,人骨髓MSC集落生成率和大小、活力细胞数和分裂指数均降低,最大效应浓度为1×10-11mol／L。以上实验结果表明,在体外培养条件下,一定浓度的AcSDKP对人骨髓MSC 的增殖具有抑制作用。     

Purified Human Synovium Mesenchymal Stem Cells as a Good Resource for Cartilage Regeneration

Mesenchymal stem cells (MSCs) have the ability to differentiate into a variety of lineages and to renew themselves without malignant changes, and thus hold potential for many clinical applications. However, it has not been well characterized how different the properties of MSCs are depending on the tissue source in which they resided. We previously reported a novel technique for the prospective MSC isolation from bone marrow, and revealed that a combination of cell surface markers (LNGFR and THY-1) allows the isolation of highly enriched MSC populations. In this study, we isolated LNGFR⁺ THY-1 ⁺ MSCs from synovium using flow cytometry. The results show that the synovium tissue contained a significantly larger percentage of LNGFR ⁺ THY-1 ⁺ MSCs. We examined the colony formation and differentiation abilities of bone marrow-derived MSCs (BM-MSCs) and synovium-derived MSCs (SYN-MSCs) isolated from the same patients. Both types of MSCs exhibited a marked propensity to differentiate into specific lineages. BM-MSCs were preferentially differentiated into bone, while in the SYN-MSC culture, enhanced adipogenic and chondrogenic differentiation was observed. These data suggest that the tissue from which MSCs are isolated should be tailored according to their intended clinical therapeutic application.     

The clonogenic potential of hematopoietic stem cells and mesenchymal stromal cells in various hematologic diseases: a pilot study

Background aimsMesenchymal stromal cells (MSC) are the most popular cells used in regenerative medicine and biotechnology. The clonogenic potential of these cells is defined by colony-forming unit-fibroblasts (CFU-F). It is well known that there is an interaction between hematopoietic cells and stromal cells in disease formation pathogenesis. Therefore we hypothesized that there should be a quantitative and qualitative relationship between MSC colonies (CFU-F) and hematopoietic stem cell colonies (colony-forming unit-granulocyte-macrophages; CFU-GM) among patients with and without hematologic diseases.MethodsForty-two patients were included in this study. Patients were divided into three groups: group A, patients with hematologic malignancies (n = 20); group B, patients with bone marrow (BM) failure (n = 11); group C, patients without hematologic diseases (n = 11). BM aspirates were plated in different densities for CFU-F culture. The plating density was the same for CFU-GM culture.ResultsCFU-GM colonies grew in 90% of group A cells and all of group B and C cells (P = 0.0001). CFU-F colonies became visible on the ninth day of plating in group A and on the eight day in groups B and C. There was no statistically significant difference between the groups for the duration of CFU-F colony formation (P = 0.12). There were differences in the morphology of the colonies among the groups.ConclusionsThis is the first study that has compared the clonogenic potential of stromal cells and hematopoietic stem cells in the same subjects with and without hematologic diseases. No correlation was shown between the clonogenic potential of stromal cells and hematopoietic cells.     

Improved proliferation and differentiation capacity of human mesenchymal stromal cells cultured with basement-membrane extracellular matrix proteins

Background aimsIn vitro cultured mesenchymal stromal cells (MSC) are characterized by a short proliferative lifespan, an increasing loss of proliferation capacity and progressive reduction of differentiation potential. Laminin-1, laminin-5, collagen IV and fibronectin are important constituents of the basement membrane extracellular matrix (ECM) that are involved in a variety of cellular activities, including cell attachment and motility.Methods and resultsThe in vitro proliferation capacity of MSC was significantly improved when the cells were incubated in the presence of basement membrane ECM proteins. For example, a mixture of proteins improved proliferation capacity 250-fold in comparison with standard conditions after five passages. Furthermore, in colony-forming unit–fibroblast (CFU-F) assays colony numbers and size were significantly extended. Blocking specific integrin cell-surface receptors, positive effects on the proliferation capacity of MSC were inhibited. Additionally, when MSC were co-cultivated with ECM proteins, cells maintained their multipotential differentiation capacity throughout many culture passages in comparison with cells cultivated on plastic. However, expansion of MSC on laminin-5 suppressed any subsequent chondrogenic differentiation.ConclusionsOur results suggest that expansion of bone marrow-derived MSC in the presence of ECM proteins is a powerful approach for generating large numbers of MSC, showing a prolonged capacity to differentiate into mesodermal cell lineages, with the exception of the lack of chondrogenesis by using laminin-5 coating.     

Back Cover Image,Volume 117, Number 1, January 2020

In vivo mesenchymal stem cell (MSC) survival is relevant to therapeutic applications requiring engraftment and potentially to nonengraftment applications as well. MSCs are a mixture of progenitors at different stages of cellular aging, but the contribution of this heterogeneity to the survival of MSC implants is unknown. Here, we employ a biomarker of cellular aging, the decoy TRAIL receptor CD264, to compare the survival kinetics of two cell populations in human bone marrow MSC (hBM-MSC) cultures. Sorted CD264⁺ hBM-MSCs from two age-matched donors have elevated β-galactosidase activity, decreased differentiation potential and form in vitro colonies inefficiently relative to CD264⁻ hBM-MSCs. Counterintuitive to their aging phenotype, CD264⁺ hBM-MSCs exhibited comparable survival to matched CD264⁻ hBM-MSCs from the same culture during in vitro colony formation and in vivo when implanted ectopically in immunodeficient NIH III mice. In vitro and in vivo survival of these two cell populations were independent of colony-forming efficiency. These findings have ramifications for the preparation of hBM-MSC therapies given the prevalence of aging CD264⁺ cells in hBM-MSC cultures and the popularity of colony-forming efficiency as a quality control metric in preclinical and clinical studies with MSCs.     

Reduced reactivation from dormancy but maintained lineage choice of human mesenchymal stem cells with donor age

Mesenchymal stem cells (MSC) are promising for cell-based regeneration therapies but up to date it is still controversial whether their function is maintained throughout ageing. Aim of this study was to address whether frequency, activation in vitro, replicative function, and in vitro lineage choice of MSC is maintained throughout ageing to answer the question whether MSC-based regeneration strategies should be restricted to younger individuals. MSC from bone marrow aspirates of 28 donors (5-80 years) were characterized regarding colony-forming unit-fibroblast (CFU-F) numbers, single cell cloning efficiency (SSCE), osteogenic, adipogenic and chondrogenic differentiation capacity in vitro. Alkaline phosphatase (ALP) activity, mineralization, Oil Red O content, proteoglycan- and collagen type II deposition were quantified. While CFU-F frequency was maintained, SSCE and early proliferation rate decreased significantly with advanced donor age. MSC with higher proliferation rate before start of induction showed stronger osteogenic, adipogenic and chondrogenic differentiation. MSC with high osteogenic capacity underwent better chondrogenesis and showed a trend to better adipogenesis. Lineage choice was, however, unaltered with age. CONCLUSION: Ageing influenced activation from dormancy and replicative function of MSC in a way that it may be more demanding to mobilize MSC to fast cell growth at advanced age. Since fast proliferation came along with high multilineage capacity, the proliferation status of expanded MSC rather than donor age may provide an argument to restrict MSC-based therapies to certain individuals.     

CFU-F circulating in cord blood

CFU-F (colony forming units-fibroblast) were studied from cord blood and, as controls, from normal bone marrow of older children and adults. Numbers of CFU-F in cord blood buffy coat cells are lower by a factor of 10 in comparison to bone marrow CFU-F. Cytomorphology and staining with monoclonal antibody identify the progeny cells of CFU-F as fibroblasts. Cord blood CFU-F derived fibroblasts have properties supporting hematopoiesis: They produce CSF (colony stimulating factor) to which fresh cord blood CFU-GM (colony forming units-granulocytic, monocytic) react by colony formation in a dose-response manner. In addition, fibroblast colonies discharge clonogenic round cells into the medium forming CFU-GM and CFU-F colonies in secondary methyl cellulose cultures. We conclude that fetal blood contains clonogenic stromal cells (CFU-F) that give rise to fibroblasts with properties of hematopoietic support.     

Mesenchymal stromal cells from infants with simple polydactyly modulate immune responses more efficiently than adult mesenchymal stromal cells

Bone marrow–derived stromal cells or mesenchymal stromal cells (BMSCs or MSCs, as we will call them in this work) are multipotent progenitor cells that can differentiate into osteoblasts, adipocytes and chondrocytes. In addition, MSCs have been shown to modulate the function of a variety of immune cells. Donor age has been shown to affect the regenerative potential, differentiation, proliferation and anti-inflammatory potency of MSCs; however, the impact of donor age on their immunosuppressive activity is unknown. In this study, we evaluated the ability of MSCs derived from very young children and adults on T-cell suppression and cytokine secretion by monocytes/macrophages. MSCs were obtained from extra digits of children between 10 and 21 months and adults between 28 and 64 years of age. We studied cell surface marker expression, doubling time, lineage differentiation potential and immunosuppressive function of the MSCs. Young MSCs double more quickly and differentiate into bone and fat cells more efficiently than those from older donors. They also form more and dense colonies of fibroblasts (colony forming unit–fibroblast [CFU-F]). MSCs from both young and adult subjects suppressed T-cell proliferation in a mitogen-induced assay at 1:3 and 1:30 ratios. At a 1:30 ratio, however, MSCs from adults did not, but MSCs from infants did suppress T-cell proliferation. In the mixed lymphocyte reaction assay, MSCs from infants produced similar levels of suppression at all three MSC/T-cell ratios, but adult MSCs only inhibited T-cell proliferation at a 1:3 ratio. Cytokine analyses of co-cultures of MSCs and macrophages showed that both adult and young MSCs suppress tumor necrosis factor alpha (TNF-α) and induce interleukin-10 (IL-10) production in macrophage co-culture assay in a similar manner. Overall, this work shows that developing MSCs display a higher level of immunosuppression than mature MSCs.     

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 CD45^dim/CD34⁺ cell concentration and mesenchymal CFU-Fs or CD45^?/CD271⁺ cells shows a positive linear regression. An age-related quantitative reduction of hematopoietic CD45^dim/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.     

Effects of recombinant human interferon alpha on human megakaryocyte and fibroblast colony formation

Effects of recombinant human interferon alpha (HuIFN-alpha) on human megakaryocyte (CFU-MK) and fibroblast (CFU-F) colony-forming cell growth were studied. Concentration-dependent inhibition of both CFU-MK and CFU-F by HuIFN-alpha was demonstrated. Statistically significant suppression of both CFU-MK and CFU-F was seen at a HuIFN-alpha concentration of 1000 U/ml or greater. No significant difference was found between HuIFN-alpha treated cultures and controls for the distribution of CFU-MK types and for the size and cell morphology of CFU-F. When a concentration of 1000 u/ml HuIFN-alpha was added at varying time points during the marrow cultures, decreased numbers of megakaryocyte and fibroblast colonies only appeared at the early days of cultures. When bone marrow cells were incubated with HuIFN-alpha for different periods of time prior to initiation of cultures, a reduction of megakaryocyte colony formation also occurred. These studies demonstrate a suppressive effect of HuIFN-alpha on human CFU-MK and CFU-F growth. This effect seems to occur at the initial stages of CFU-MK and CFU-F development.     

Impaired differentiation potential of human trabecular bone mesenchymal stromal cells from elderly patients

Background aimsAdvances in bone tissue engineering with mesenchymal stromal cells (MSC) as an alternative to conventional orthopedic procedures has opened new horizons for the treatment of large bone defects. Bone marrow (BM) and trabecular bone are both sources of MSC. Regarding clinical use, we tested the potency of MSC from different sources.MethodsWe obtained MSC from 17 donors (mean age 64.6 years) by extensive washing of trabecular bone from the femoral head and trochanter, as well as BM aspirates of the iliac crest and trochanter. The starting material was evaluated by histologic analysis and assessment of colony-forming unit–fibroblasts (CFU-F). The MSC populations were compared for proliferation and differentiation potential, at RNA and morphologic levels.ResultsMSC proliferation potential and immunophenotype (expression of CD49a, CD73, CD90, CD105, CD146 and Stro-1) were similar whatever the starting material. However, the differentiation potential of MSC obtained by bone washing was impaired compared with aspiration; culture-amplified cells showed few Oil Red O-positive adipocytes and few mineralized areas and formed inconsistent Alcian blue-positive high-density micropellets after growth under adipogenic, osteogenic and chondrogenic conditions, respectively. MSC cultured with 1 ng/mL fibroblast growth factor 2 (FGF-2) showed better differentiation potential.ConclusionsTrabecular bone MSC from elderly patients is not good starting material for use in cell therapy for bone repair and regeneration, unless cultured in the presence of FGF-2.     

Dental mesenchymal stromal/stem cells in different microenvironments— implications in regenerative therapy

Current research data reveal microenvironment as a significant modifier of physical functions, pathologic changes, as well as the therapeutic effects of stem cells. When comparing regeneration potential of various stem cell types used for cytotherapy and tissue engineering, mesenchymal stem cells (MSCs) are currently the most attractive cell source for bone and tooth regeneration due to their differentiation and immunomodulatory potential and lack of ethical issues associated with their use. The microenvironment of donors and recipients selected in cytotherapy plays a crucial role in regenerative potential of transplanted MSCs, indicating interactions of cells with their microenvironment indispensable in MSC-mediated bone and dental regeneration. Since a variety of MSC populations have been procured from different parts of the tooth and tooth-supporting tissues, MSCs of dental origin and their achievements in capacity to reconstitute various dental tissues have gained attention of many research groups over the years. This review discusses recent advances in comparative analyses of dental MSC regeneration potential with regards to their tissue origin and specific microenvironmental conditions, giving additional insight into the current clinical application of these cells.