Stochastic branching model for hemopoietic progenitor cell differentiation

We present algebraic expressions describing the predictions of a stochastic branching model for differentiation of hemopoietic progenitor cells. The model assumes that there is a fixed probability, p (0 less than or equal to p less than or equal to 1), that commitment to a differentiative event occurs per progenitor cell division for each daughter cell. The model describes properties of in vitro hemopoietic cell differentiation including the population structure at the time the first progenitor cell becomes committed, the number of committed progenitor cells engendered by a single progenitor cell, and the probability of eventual commitment of all daughter cells derived from a single progenitor or stem cell. Application of the model to experimental data obtained from erythroid cultures suggests that the observed data can be explained by the stochastic branching model alone without making the deterministic assumption that there is a differentiative hierarchy in the lineage of the progenitors of erythropoiesis (BFU-E). The qualitative and quantitative aspects of the proposed stochastic model are discussed in conjunction with other analogous stochastic branching models.     

Effects of FGF2 and FGF9 on osteogenic differentiation of bone marrow-derived progenitors

Bone repair is a major concern in reconstructive surgery. Transplants containing osteogenically committed mesenchymal stem cells (MSCs) provide an alternative source to the currently used autologous bone transplants which have limited supply and require additional surgery to the patient. A major drawback, however is the lack of a critical mass of cells needed for successful transplantation. The purpose of the present study was to test the effects of FGF2 and FGF9 on expansion and differentiation of MSCs in order to establish an optimal culture protocol resulting in sufficient committed osteogenic cells required for successful in vivo transplantation. Bone marrow-derived MSCs cultured in αMEM medium supplemented with osteogenic supplements for up to three passages (control medium), were additionally treated with FGF2 and FGF9 in various combinations. Cultures were evaluated for viability, calcium deposition and in vivo osteogenic capacity by testing subcutaneous transplants in nude mice. FGF2 had a positive effect on the proliferative capacity of cultured MSCs compared to FGF9 and control medium treated cultures. Cultures treated with FGF2 followed by FGF9 showed an increased amount of extracted Alizarin red indicating greater osteogenic differentiation. Moreover, the osteogenic capacity of cultured cells transplanted in immunodeficient mice revealed that cells that were subjected to treatment with FGF2 in the first two passages and subsequently to FGF9 in the last passage only, were more successful in forming new bone. It is concluded that the protocol using FGF2 prior to FGF9 is beneficial to cell expansion and commitment, resulting in higher in vivo bone formation for successful bone tissue engineering.     

Isolation of human mesenchymal stem cells from amnion,chorion, placental decidua and umbilical cord: comparison of four enzymatic protocols

Objective

To compare four enzymatic protocols for mesenchymal stem cells (MSCs) isolation from amniotic (A-MSC) and chorionic (C-MSC) membranes, umbilical cord (UC-MSC) and placental decidua (D-MSC) in order to define a robust, practical and low-cost protocol for each tissue.

Results

A-MSCs and UC-MSCs could be isolated from all samples using trypsin/collagenase-based protocols; C-MSCs could be isolated from all samples with collagenase- and trypsin/collagenase-based protocols; D-MSCs were isolated from all samples exclusively with a collagenase-based protocol.

Conclusions

The trypsin-only protocol was least efficient; the collagenase-only protocol was best for C-MSCs and D-MSCs; the combination of trypsin and collagenase was best for UC-MSCs and none of tested protocols was adequate for A-MSCs isolation.

     

Growth kinetics,self-renewal,and the osteogenic potential of purified human mesenchymal stem cells during extensive subcultivation and following cryopreservation

Recent studies have demonstrated the existence of a subset of cells in human bone marrow capable of differentiating along multiple mesenchymal lineages. Not only do these mesenchymal stem cells (MSCs) possess multilineage developmental potential, but they may be cultured ex vivo for many passages without overt expression of a differentiated phenotype. The goals of the current study were to determine the growth kinetics, self-renewing capacity, and the osteogenic potential of purified MSCs during extensive subcultivation and following cryopreservation. Primary cultures of MSCs were established from normal iliac crest bone marrow aspirates, an aliquot was cryopreserved and thawed, and then both frozen and unfrozen populations were subcultivated in parallel for as many as 15 passages. Cells derived from each passage were assayed for their kinetics of growth and their osteogenic potential in response to an osteoinductive medium containing dexamethasone. Spindle-shaped human MSCs in primary culture exhibit a lag phase of growth, followed by a log phase, finally resulting in a growth plateau state. Passaged cultures proceed through the same stages, however, the rate of growth in log phase and the final number of cells after a fixed period in culture diminishes as a function of continued passaging. The average number of population doublings for marrow-derived adult human MSCs was determined to be 38 ± 4, at which time the cells finally became very broad and flattened before degenerating. The osteogenic potential of cells was conserved throughout every passage as evidenced by the significant increase in APase activity and formation of mineralized nodular aggregates. Furthermore, the process of cryopreserving and thawing the cells had no effect on either their growth or osteogenic differentiation. Importantly, these studies demonstrate that replicative senescence of MSCs is not a state of terminal differentiation since these cells remain capable of progressing through the osteogenic lineage. The use of population doubling potential as a measure of biological age suggests that MSCs are intermediately between embryonic and adult tissues, and as such, may provide an in situ source for mesenchymal progenitor cells throughout an adult's lifetime. J. Cell. Biochem. 64:278–294. © 1997 Wiley-Liss, Inc.     

Differential Expression of Surface Markers in Mouse Bone Marrow Mesenchymal Stromal Cell Subpopulations with Distinct Lineage Commitment

Bone marrow mesenchymal stromal cells (BM MSCs) represent a heterogeneous population of progenitors with potential for generation of skeletal tissues. However the identity of BM MSC subpopulations is poorly defined mainly due to the absence of specific markers allowing in situ localization of those cells and isolation of pure cell types. Here, we aimed at characterization of surface markers in mouse BM MSCs and in their subsets with distinct differentiation potential. Using conditionally immortalized BM MSCs we performed a screening with 176 antibodies and high-throughput flow cytometry, and found 33 markers expressed in MSCs, and among them 3 were novel for MSCs and 13 have not been reported for MSCs from mice. Furthermore, we obtained clonally derived MSC subpopulations and identified bipotential progenitors capable for osteo- and adipogenic differentiation, as well as monopotential osteogenic and adipogenic clones, and thus confirmed heterogeneity of MSCs. We found that expression of CD200 was characteristic for the clones with osteogenic potential, whereas SSEA4 marked adipogenic progenitors lacking osteogenic capacity, and CD140a was expressed in adipogenic cells independently of their efficiency for osteogenesis. We confirmed our observations in cell sorting experiments and further investigated the expression of those markers during the course of differentiation. Thus, our findings provide to our knowledge the most comprehensive characterization of surface antigens expression in mouse BM MSCs to date, and suggest CD200, SSEA4 and CD140a as markers differentially expressed in distinct types of MSC progenitors.     

Evaluation of chemokine and cytokine profiles in osteoblast progenitors from umbilical cord blood stem cells by BIO-PLEX technology

We have used cytokine protein array to analyze the secretion of cytokines from an osteoblastic clone derived from human umbilical cord blood mesenchymal stem cells (MSCs) cultured in an osteogenic differentiation medium. The analysis demonstrated the unexpected ability of osteoblast committed cells and their early progenitors to produce significant amounts of a range of soluble immune mediators without in vitro exposure to clinically relevant bacterial pathogens. The cells were expanded and their osteogenic potential analyzed over 45 days of culture was revealed by the expression of osteoblast-specific markers (alkaline phosphatase and Runx2), and by matrix mineralization. Over this culture period, the cells secreted particularly high levels of IL-8, MCP-1 and VEGF, but did not express IL-2, IL-7, IL-17, eotaxin, G-CSF and IFN-gamma. These findings should encourage the use of human umbilical cord blood as a potential stem cells source for bone regeneration.     

A multiplex PCR technique to characterize human bone marrow derived mesenchymal stem cells

Human mesenchymal stem cells (MSCs), with capacity to differentiate into adipocytes, osteoblasts and chondrocytes, offer potential for the development of novel treatments. A critical question in MSCs biology is whether this cell population possesses a relatively uniform differentiation capability or is comprised of distinct subsets of progenitors committed to differentiate in particular pathways. To quantify the changes during growth of MSCs, we analyzed the mesenchymal phenotype and differentiation ability using a multi-marker PCR with six primer sets specific for CD73, CD90, CD105, CD166, CD45 and β-actin allowing a gel-based differential detection of the PCR products. To determine degree of variability of MSCs populations in terms of proliferation, cell proliferation assays were performed on expanded MSCs up to the sixth passage. At each passage, the osteogenic and adipogenic differentiation potentials of MSCs were verified by culture in inductive media. RT-PCR and cytochemical analysis revealed that, despite the loss of multipotentiality during expansion, certain markers remain expressed, indicating that these markers are unlikely to be reflective of the MSC’s true ‘stem cell’ nature. Our results suggest that decrease in the expression of MSCs specific markers correlates with down-regulation of proliferation ability and differentiation efficiency of MSCs.     

Isolation, characterization, and mesodermic differentiation of stem cells from adipose tissue of camel (Camelus dromedarius)

Adipose-derived stem cells are an attractive alternative as a source of stem cells that can easily be extracted from adipose tissue. Isolation, characterization, and multi-lineage differentiation of adipose-derived stem cells have been described for human and a number of other species. Here we aimed to isolate and characterize camel adipose-derived stromal cell frequency and growth characteristics and assess their adipogenic, osteogenic, and chondrogenic differentiation potential. Samples were obtained from five adult dromedary camels. Fat from abdominal deposits were obtained from each camel and adipose-derived stem cells were isolated by enzymatic digestion as previously reported elsewhere for adipose tissue. Cultures were kept until confluency and subsequently were subjected to differentiation protocols to evaluate adipogenic, osteogenic, and chondrogenic potential. The morphology of resultant camel adipose-derived stem cells appeared to be spindle-shaped fibroblastic morphology, and these cells retained their biological properties during in vitro expansion with no sign of abnormality in karyotype. Under inductive conditions, primary adipose-derived stem cells maintained their lineage differentiation potential into adipogenic, osteogenic, and chondrogenic lineages during subsequent passages. Our observation showed that like human lipoaspirate, camel adipose tissue also contain multi-potent cells and may represent an important stem cell source both for veterinary cell therapy and preclinical studies as well.     

Comparative characterization of mesenchymal bone marrow stromal cells at early and late stages of culturing

The mesenchymal stromal cell is a multipotent precursor of osteoblasts, adipocytes, and some other cell types. In this study, a comparative analysis of cultured mesenchymal stromal cells from the rat bone marrow at the early and late stages of subculturing has been performed using molecular genetic and cytological methods. The culture has undergone 11 passages during 140 days. Upon long-term culturing, the mesenchymal stromal cells have proved to lose their potential for adipogenic differentiation but preserve the potential for osteogenesis. Morphological characters typical of osteogenic differentiation can be observed at the earlier stages of culturing (passages 1–4) but disappear at later stages (passages 9–11), despite mineralization of the extracellular matrix and the expression of osteogenic differentiation markers. A comparative analysis of the proliferation potential of stromal cells has shown that differences in the period of cell population doubling at the early and later stages of culturing are insignificant. An almost complete arrest of cell growth has been observed in the middle of the culture period (passages 5 and 6).     

Arsenic trioxide promotes senescence and regulates the balance of adipogenic and osteogenic differentiation in human mesenchymal stem cells

Arsenic trioxide (ATO) as an anti-tumor drug could induce differentiation and apoptosis in tumor cells. Mesenchymal stem cells (MSCs) play important roles in the hematogenesis of bone marrow. Many reports have shown that the disorder of MSC adipogenic and osteogenic differentiation occurs in some diseases. However, reports about the effects of ATO on MSCs are limited. In this study, we found that 1 μM ATO promoted MSC senescence mainly through p21, although it had no effect on apoptosis at this dose. Furthermore, ATO promoted adipogenic differentiation, but inhibited osteogenic differentiation in MSCs. Our study also showed that CCAAT/enhancer-binding protein alpha C/EBPα and peroxisome proliferator-activated receptor gamma PPARγ might be involved in the regulation of adipogenic and osteogenic differentiation induced by ATO. Our results indicated that ATO may exert an anti-tumor effect by influencing bone marrow micro-environment. Moreover, it may regulate the adipogenic and osteogenic differentiation of MSCs.     

Differentiating multipotent mesenchymal stromal cells generate factors that exert paracrine activities on exogenous MSCs: Implications for paracrine activities in bone regeneration

The mechanisms by which multipotent mesenchymal stromal cells (MSCs) contribute to tissue repair following transplantation into host tissues remains poorly understood. Current concepts suggest that, in addition to differentiation into cells of the host tissues, MSCs also generate trophic factors that modulate host tissue microenvironment to aid in the repair process. In this communication, we assessed whether factors secreted by MSCs undergoing osteogenic differentiation induce expression of osteoblast markers in exogenous MSCs as well as their migration. Murine MSCs were cultured in osteogenic medium, and at different time points, medium conditioned by the cells was collected and assessed for its effects on differentiation and migration of exogenous MSCs. In addition, we determined whether MSCs infused into mice femurs expressed genes encoding for factors predicted to play a role in paracrine activities. The results showed that MSCs maintained in osteogenic medium, secreted factors at specific time points that induced alkaline phosphatase activity (ALP) in exogenous MSCs as well as their migration. MSCs infused into mice femurs and retrieved at different days expressed genes that encoded predicted factors that play a role in cell differentiation and migration. Neutralizing antibodies to bone morphogenetic protein-2 (BMP-2) led to the decrease in ALP activity by exogenous MSCs. These data demonstrated that, as MSCs differentiate toward osteogenic lineage, they secrete factors that induce recruitment and differentiation of endogenous progenitors. These data reveal mechanisms by which donor MSCs may contribute to the bone reparative process and provide a platform for designing approaches for stem cell therapies of musculoskeletal disorders.     

Osteogenic differentiation of mesenchymal stem cells from osteopenic rats subjected to physical activity with and without nitric oxide synthase inhibition

Physical activity has potent and complex effects on bones. We hypothesized that physical activity has a positive effect upon osteopenic rat bones because it stimulates osteogenic differentiation of bone marrow mesenchymal stem cells (MSCs). We also postulated that local nitric oxide concentrations mediate the effects of physical activity on bones. The objective of this study was to investigate the osteogenic differentiation in vitro of MSCs from osteopenic female rats subjected to physical activity with and without nitric oxide synthase inhibition. We used MSCs from the femurs of Wistar female rats divided into six groups: Group 1, sham-operated (control); Group 2, sedentary osteopenic; Group 3, active osteopenic; Group 4, sham-operated with L-NAME; Group 5, sedentary osteopenic with L-NAME; and Group 6, active osteopenic with L-NAME. The cells were cultured at 37 °C and 5% CO₂. Cells were phenotypically characterized with anti-CD45, anti-CD90, anti-CD73, and anti-CD54 using a FACScan cytometer. MSCs were cultured in osteogenic medium for 7, 14 and 21 days. Alkaline phosphatase activity, the capacity of dimethylthiazol conversion in formazan crystals, collagen synthesis and the number of mineralized nodules were analyzed. The means of all of the variables were compared using the SNK test. MSCs did not express CD45 in 96.94% of the cells, but there was expression of CD73, CD54 and CD90 in 93.99%, 95.10% and 86.77% of the cells, respectively. MSCs from osteopenic rats showed less osteogenic differentiation. Surprisingly, physical activity increased the osteogenic differentiation of MSCs in osteopenic rats. Inhibition of nitric oxide synthase in vivo had a negative effect upon the osteogenic potential of MSCs from normal rats and from osteopenic rats subjected to physical activity. Our results suggest that nitric oxide stimulates MSCs osteogenic differentiation and that nitric oxide mediates the beneficial effects of physical activity upon MSCs osteogenic differentiation.     

Effect of TAK1 on osteogenic differentiation of mesenchymal stem cells by regulating BMP-2 via Wnt/β-catenin and MAPK pathway

Mesenchymal stem cells (MSCs) have the ability to differentiate into osteoblasts and chondrocytes. In vitro osteogenic differentiation is critical but the molecular mechanism has yet to be further clarified. The role of TGF-β activated kinase 1 (TAK1) in MSCs osteogenesis differentiation has not been reported. By adding si-TAK1 and rhTAK1, the osteogenic differentiation of MSCs was measured. Expression levels of the osteoblastic marker genes during osteogenic differentiation of MSCs were checked. As well as molecules involved in BMP and Wnt/β-catenin signaling pathways. The phosphorylation of p38 and JNK was also checked. TAK1 is essential for mineralization of MSCs at low concentration, but excessive rhTAK1 inhibits mineralization of MSCs. It up regulates the expression levels of bone sialoprotein (BSP), osteocalcin (OSC), Alkaline phosphatase (ALP), and RUNX2 during osteogenic differentiation of MSCs. It can also promote TGF-β/BMP-2 gene expression and β-catenin expression, and down regulate GSK-3β expression. Meanwhile, TAK1 promotes the phosphorylation of p38 and JNK. Additionally, TAK1 up regulates the expression of BMP-2 at all concentration under the inhibition of p38 and JNK. Our results suggested that TAK1 is essential in MSCs osteogenesis differentiation, and functions as a double-edged sword, probably through regulation of β-catenin and p38/JNK.     

Isolation and Osteogenic Differentiation of Rat Periosteum-derived Cells

Selection of appropriate cultures having an osteogenic potential is a necessity if cell/biomaterial interactions are studied in long-term cultures. Osteoblastic cells derived from rat long bones or calvaria have the disadvantage of being in an advanced differentiation stage which results in terminal differentiation within 21 days. In this regard, less differentiated periosteum-derived osteoprogenitors could be more suitable. Periosteum-derived cells were isolated from the tibiae of adult Wistar rats (n = 12). The osteogenic potential with regard to alkaline phosphatase activity, morphology, nodule formation and mineralization was studied by culturing them in an osteogenic medium for up to 4 months. Seventy-five percent of the cultures (n = 9) did not show any increase in alkaline phosphatase activity nor nodule formation during long-term culture for up to 4 months. Nevertheless, in 25% of the cultures, alkaline phosphatase activity started from negligible (<5 mM pNP/mg protein) and increased towards approximately 50 mM pNP/mg protein. Three-dimensional nodule formation was observed at passages 3–5. In further passages (P5–P7), nodule formation capacity decreased and a diffuse mineralization pattern was observed. Suitable cultures with osteogenic capacity, can be selected at early passages based on the presence of cuboidal cells. These cells have the advantage of retaining their osteogenic potential even after prolonged cultivation (6–7 passages) before final differentiation occurs. Although periosteal cells are suitable for long term in vitro evaluation of biomaterials, the isolation and selection is time consuming. Hence, a more appropriate source to study cell/biomaterial interactions should be more convenient.     

Phenotypic changes of adult porcine mesenchymal stem cells induced by prolonged passaging in culture

The in vitro culture of porcine bone marrow-derived mesenchymal stem cells (MSCs) was used for the investigation of adult stem cell biology. Isolated porcine MSCs possessed the ability to proliferate extensively in an antioxidants-rich medium containing 5% fetal bovine serum (FBS). Greater than 40 serial MSC passages and 100 cell population doublings have been recorded for some MSC batches. Early and late passage MSCs were defined here as those cultures receiving less than 5 trypsin passages and more than 15 trypsin passages, respectively. Consistent with their robust ability to proliferate, both the early and late passage MSCs expressed the cell-cycle promoting enzyme p34cdc2 kinase. Late MSCs, however, exhibited certain features reminiscent of cellular aging such as actin accumulation, reduced substrate adherence, and increased activity of lysosomal acid beta-galactosidase. Early MSCs retained the multipotentiality capable of chondrogenic, osteogenic, and adipogenic differentiation upon induction in vitro. In contrast, late MSCs were only capable of adipogenic differentiation, which was greatly enhanced at the expense of the osteochondrogenic potential. Along with these changes in multipotentiality, late MSCs expressed decreased levels of the bone morphogenic protein (BMP-7) and reduced activity of alkaline phosphatase. Late MSCs also exhibited attenuated synthesis of the hematopoietic cytokines granulocyte colony-stimulating factor (G-CSF), leukemia inhibitory factor (LIF), and stem cell factor (SCF). The long-term porcine MSC culture, thus, provides a model system to study the molecular interplay between multiple MSC differentiation cascades in the context of cellular aging.     

Bortezomib enhances the osteogenic differentiation capacity of human mesenchymal stromal cells derived from bone marrow and placental tissues

Bortezomib (BZB) is a chemotherapeutic agent approved for treating multiple myeloma (MM) patients. In addition, there are several reports showing that bortezomib can induce murine mesenchymal stem cells (MSCs) to undergo osteogenic differentiation and increase bone formation in vivo. MSCs are the multipotent stem cells that have capacity to differentiate into several mesodermal derivatives including osteoblasts. Nowadays, MSCs mostly bone marrow derived have been considered as a valuable source of cell for tissue replacement therapy. In this study, the effect of bortezomib on the osteogenic differentiation of human MSCs derived from both bone marrow (BM-MSCs) and postnatal sources such as placenta (PL-MSCs) were investigated. The degree of osteogenic differentiation of BM-MSCs and PL-MSCs after bortezomib treatment was assessed by alkaline phosphatase (ALP) activity, matrix mineralization by Alizarin Red S staining and the expression profiles of osteogenic differentiation marker genes, Osterix, RUNX2 and BSP. The results showed that 1 nM and 2 nM BZB can induce osteogenic differentiation of BM-MSCs and PL-MSCs as demonstrated by increased ALP activity, increased matrix mineralization and up-regulation of osteogenic differentiation marker genes, Osterix, RUNX2 and BSP as compared to controls. The enhancement of osteogenic differentiation of MSCs by bortezomib may lead to the potential therapeutic applications in human diseases especially patients with osteopenia.     

Differential activation of ERK1,2 MAP kinase signaling pathway in mesenchymal stem cell from control and osteoporotic postmenopausal women

Osteoblasts, the cells responsible for bone formation, derive from mesenchymal stem cells (MSCs) in bone marrow. To acquire a new cell phenotype, uncommitted MSCs must undergo several proliferation and differentiation changes. Although, it is known that extracellular signal-regulated protein kinases (ERKs) mitogen-activated protein (MAP) kinase pathway signaling is involved in the proliferation and differentiation processes, the role of ERKs in osteogenic differentiation it is controversial, at present. In addition, the function that ERK could play in MSCs derived from osteoporotic patients it is not well documented. In this study, we analyze whether previously observed differences in the dynamic response of MSCs from normal and osteoporotic postmenopausal women can be explained by changes in the activation of this signal transduction pathway. Levels of ERK phosphorylation and their correlation with osteogenic differentiation were evaluated in cultures of MSCs derived from osteoporotic postmenopausal women and "healthy" controls. The results show that, under basal conditions, MSCs derived from osteoporotic donors show a level of ERK phosphorylation 2.5 times higher than MSCs derived from control donors. The addition of the osteogenic stimulus only slightly increases the p-ERK level in cells derived from osteoporotic donors, and is higher in cells derived from control women. Important differences in the ability of PD98059 to inhibit phosphorylation of ERK in both types of cells were also observed, as well as the effect that this inhibition produced on calcium deposition. We conclude that the MAP kinase pathway signaling is differentially activated in MSCs derived from osteoporotic postmenopausal women. The high p-ERK levels in MSC derived from osteoporotic donors could determine the unresponsiveness of these cells to the osteogenic differentiation stimulus.     

Molecular-genetic and immunophenotypic analysis of antigen profile and osteogenic and adipogenic potentials of mesenchymal stromal cells from fetal liver and adult bone marrow in rats

Comparative characteristics of mesenchymal stromal cells (MSCs) from adult bone marrow and fetal liver are of great interest due to the similar functions performed by these organs on the organization of a hemopoietic microenvironment at various developmental periods. It is known that MSCs play a pivotal role in the formation of niches for hemopoietic stem cells. The histogenetic relation of MSCs from these two hemopoietic organs cannot be ruled out. An analysis of antigen profile using immunocytochemistry and RT-PCR has confirmed that the studied cell populations fit the MSC criteria and have no contaminations of hemopoietic, lymphoid, and endothelial cells beginning at the second passage. Comparative analysis of osteogenic and adipogenic marker expression revealed MSC from fetal liver to have a weaker potential for adipogenesis and the extremely low capability for terminal osteogenic differentiation, in contrast to pronounced osteo- and adipogenic potentials of adult bone marrow MSC. The similar cell phenotype but different differentiation potentials under identical conditions of cultivation in vitro seem to be due to different developmental programs of the pre- and postnatal histogenesis of these MSC.