Sequential exposure to fibroblast growth factors (FGF) 2, 9 and 18 enhances hMSC chondrogenic differentiation.

OBJECTIVE: To test the effects of sequential exposure to FGF2, 9 and 18 on human Mesenchymal Stem Cells (hMSC) differentiation during in vitro chondrogenesis. DESIGN: Control and FGF2-expanded hMSC were cultured in aggregates in the presence of rhFGF9, rhFGF18 or rhFGFR3-specific signaling FGF variants, starting at different times during the chondroinductive program. Quantitative real time polymerase chain reaction (qRT-PCR) and immunocytochemistry were performed at different stages. The aggregate cultures were switched to a hypertrophy-inducing medium along with rhFGFs and neutralizing antibodies against FGFR1 and FGFR3. Histological/immunohistochemical/biochemical analyses were performed. RESULTS: FGF2-exposed hMSC during expansion up-regulated Sox9 suggesting an early activation of the chondrogenic machinery. FGF2, FGF9 and 18 modulated the expression profile of FGFR1 and FGFR3 in hMSC during expansion and chondrogenesis. In combination with transforming growth factor-beta (TGF-ß), FGF9 and FGF18 inhibited chondrogenesis when added at the beginning of the program (≤ d7), while exhibiting an anabolic effect when added later (≥d14), an effect mediated by FGFR3. Finally, FGFR3 signaling induced by either FGF9 or FGF18 delayed the appearance of spontaneous and induced hypertrophy-related changes. CONCLUSIONS: The stage of hMSC-dependent chondrogenesis at which the growth factors are added impacts the progression of the differentiation program: increased cell proliferation and priming (FGF2); stimulated early chondrogenic differentiation (TGF-ß, FGF9/FGF18) by shifting the chondrogenic program earlier; augmented extracellular matrix (ECM) production (FGF9/FGF18); and delayed terminal hypertrophy (FGF9/FGF18). Collectively, these factors could be used to optimize pre-implantation conditions of hMSC when used to engineer cartilage grafts.

Human bone marrow-derived mesenchymal stem cells rescue neonatal CPAP-induced airway hyperreactivity.

Continuous positive airway pressure (CPAP) is a primary non-invasive mode of respiratory support for preterm infants. However, emerging evidence suggests CPAP could be an underlying contributor to the unintended pathophysiology of wheezing and associated airway hyperreactivity (AHR) in former preterm infants. The therapeutic benefits of mesenchymal stem cells (MSCs) have been demonstrated in a variety of animal models and several clinical trials are currently underway to assess their safety profiles in the setting of prematurity and bronchopulmonary dysplasia (BPD). In the present study, using a mouse model of neonatal CPAP, we investigated whether conditioned medium harvested from cultures of human bone-marrow derived mesenchymal stem cells (hMSC) could rescue the CPAP-induced AHR, based upon previous observations of their anti-AHR properties. Newborn mice (male and female) were fitted with a custom-made mask for delivery of daily CPAP 3 h/day for the first 7 postnatal days. At postnatal day 21 (two weeks after CPAP ended), lungs were removed, precision-cut lung slices were sectioned and incubated for 48 h in vitro in conditioned medium collected from cultures of three different hMSC donors. As expected, CPAP resulted in AHR to methacholine compared to untreated control mice. hMSC conditioned medium from the cultures of all three donors completely reversed AHR. These data reveal potential therapeutic benefits of hMSC therapy, which may be capable of rescuing the long-term adverse effects of neonatal CPAP on human airway function.

Why are MSCs therapeutic? New data: new insight.

Adult marrow-derived mesenchymal stem cells (MSCs) are able to differentiate into bone, cartilage, muscle, marrow stroma, tendon-ligament, fat and other connective tissues. The questions can be asked, what do MSCs do naturally and where is the MSC niche? New insight and clinical experience suggest that MSCs are naturally found as perivascular cells, summarily referred to as pericytes, which are released at sites of injury, where they secrete large quantities of bioactive factors that are both immunomodulatory and trophic. The trophic activity inhibits ischaemia-caused apoptosis and scarring while stimulating angiogenesis and the mitosis of tissue intrinsic progenitor cells. The immunomodulation inhibits lymphocyte surveillance of the injured tissue, thus preventing autoimmunity, and allows allogeneic MSCs to be used in a variety of clinical situations. Thus, a new, enlightened era of experimentation and clinical trials has been initiated with xenogenic and allogeneic MSCs.

Umbilical Cord-derived Mesenchymal Stem Cells for COVID-19 Patients with Acute Respiratory Distress Syndrome (ARDS).

The coronavirus SARS-CoV-2 is cause of a global pandemic of a pneumonia-like disease termed Coronavirus Disease 2019 (COVID-19). COVID-19 presents a high mortality rate, estimated at 3.4%. More than 1 out of 4 hospitalized COVID-19 patients require admission to an Intensive Care Unit (ICU) for respiratory support, and a large proportion of these ICU-COVID-19 patients, between 17% and 46%, have died. In these patients COVID-19 infection causes an inflammatory response in the lungs that can progress to inflammation with cytokine storm, Acute Lung Injury (ALI), Acute Respiratory Distress Syndrome (ARDS), thromboembolic events, disseminated intravascular coagulation, organ failure, and death. Mesenchymal Stem Cells (MSCs) are potent immunomodulatory cells that recognize sites of injury, limit effector T cell reactions, and positively modulate regulatory cell populations. MSCs also stimulate local tissue regeneration via paracrine effects inducing angiogenic, anti-fibrotic and remodeling responses. MSCs can be derived in large number from the Umbilical Cord (UC). UC-MSCs, utilized in the allogeneic setting, have demonstrated safety and efficacy in clinical trials for a number of disease conditions including inflammatory and immune-based diseases. UC-MSCs have been shown to inhibit inflammation and fibrosis in the lungs and have been utilized to treat patients with severe COVID-19 in pilot, uncontrolled clinical trials, that reported promising results. UC-MSCs processed at our facility have been authorized by the FDA for clinical trials in patients with an Alzheimer's Disease, and in patients with Type 1 Diabetes (T1D). We hypothesize that UC-MSC will also exert beneficial therapeutic effects in COVID-19 patients with cytokine storm and ARDS. We propose an early phase controlled, randomized clinical trial in COVID-19 patients with ALI/ARDS. Subjects in the treatment group will be treated with two doses of UC-MSC (l00 × 106 cells). The first dose will be infused within 24 hours following study enrollment. A second dose will be administered 72 ± 6 hours after the first infusion. Subject in the control group will receive infusion of vehicle (DPBS supplemented with 1% HSA and 70 U/kg unfractionated Heparin, delivered IV) following the same timeline. Subjects will be evaluated daily during the first 6 days, then at 14, 28, 60, and 90 days following enrollment (see Schedule of Assessment for time window details). Safety will be determined by adverse events (AEs) and serious adverse events (SAEs) during the follow-up period. Efficacy will be defined by clinical outcomes, as well as a variety of pulmonary, biochemical and immunological tests. Success of the current study will provide a framework for larger controlled, randomized clinical trials and a means of accelerating a possible solution for this urgent but unmet medical need. The proposed early phase clinical trial will be performed at the University of Miami (UM), in the facilities of the Diabetes Research Institute (DRI), UHealth Intensive Care Unit (ICU) and the Clinical Translational Research Site (CTRS) at the University of Miami Miller School of Medicine and at the Jackson Memorial Hospital (JMH).

Biochemical and ultrastructural properties of osmotically lysed rat-liver mitochondria.

Isolated rat-liver mitochondria were osmotically lysed by suspension and washing 3 times in cold, distilled water. Pellets obtained by centrifugation at 105,000 g for 30 min were resuspended, fixed with glutaraldehyde and OsO(4), and embedded in Epon 812. Thin sections show the presence of two distinct membranous populations, each of which is relatively homogeneous in size and appearance. Swollen mitochondria ( approximately 1.5 micro in diameter), which have been stripped of their outer membranes, are largely devoid of matrix and normal matrix granules and are referred to as "ghosts." The smaller (0.2 to 0.4 micro in diameter), empty appearing, vesicular elements, derived primarily from the outer mitochondrial membrane, can be differentiated from the ghosts on the basis of their smaller size and complete absence of internal structures, especially cristae. Each membranous element is enclosed by a single, continuous membrane; the "double membrane" organization typical of intact mitochondria is not observed. These findings indicate that the outer membrane of rat-liver mitochondria is spatially dissociated from the inner mitochondrial membrane by osmotic lysis of the mitochondria in distilled water. Three parameters of structural and functional significance in freshly isolated rat-liver mitochondria have been correlated with the structural alterations observed: (a) chemical composition (total protein, lipid phosphate and total phosphate), (b) specific and total activities of marker enzymes for mitochondrial matrix and membranes (malate dehydrogenase (MDH), D-beta-hydroxybutyrate dehydrogenase (BDH) and cytochromes), and (c) integrated multienzyme functions (respiration, phosphorylation, and contraction). The data presented indicate that all mitochondrial membranes are completely conserved in the crude ghost preparation and that, in addition, about (1/3) of the matrix proteins (estimated by assays for MDH activity and protein) are retained. The study of integrated mitochondrial functions shows that a number of physiologically important multienzyme activities also are preserved in the water-washed preparation. The respiratory rate of ghosts per milligram of protein is 1.5 to 2.0 times that of intact mitochondria, which shows that the respiratory chain in the ghosts is functionally intact. The rate of phosphorylation is reduced, however, to about 25% of that measured in freshly isolated mitochondria and accounts for lowered P:O ratios using succinate as substrate (P:O ranges from 0.4 to 0.9). The phosphorylation of ADP to ATP is the only biochemical function, so far investigated, that is greatly affected by osmotic lysis. In addition, two lines of evidence suggest that the ghosts undergo an energy-dependent transformation resulting in contraction: (a) suspensions of the crude ghost preparation in 0.02 M Tris-0.125 M KCl medium show a marked increase in optical density upon the addition of ATP, and (b) ghost preparations incubated in ion-uptake medium in the absence of added calcium but in the presence of added ATP contain a large number of highly condensed ghosts (about 50% of the total profiles) when viewed as thin sections in the electron microscope. The correlated biochemical and morphological study presented here shows that the outer membrane of rat-liver mitochondria can be removed by controlled osmotic lysis without greatly impairing a number of integrated biochemical functions associated with the inner membrane.

The effects of osmotic lysis on the oxidative phosphorylation and compartmentation of rat liver mitochondria.

Rat liver mitochondria isolated in 0.25 M sucrose were osmotically lysed with distilled water. The effect of osmotic lysis on mitochondrial compartmentation was monitored by following the changes in the specific Mg(++)-ATPase and the stimulation of this activity by DNP. Each resuspension in distilled water caused a progressive increase in the specific Mg(++)-ATPase and a decrease in DNP-stimulation. Lysed mitochondria yielded P:O ratios of slightly less than 1.0 when each of the "site-specific" substrates, NADH, D-beta-hydroxybutyrate, succinate, and ascorbate, were oxidized. These data indicate that only site 3 phosphorylation remained undiminished. The crude, lysed mitochondria were subfractionated by centrifugation on linear sucrose density gradients. Assays for protein, malate dehydrogenase, D-beta-hydroxybutyrate dehydrogenase, and succinate dehydrogenase indicated that the inner compartment could be clearly separated from the outer membrane vesicles. The results also suggested that the small vesicle fraction contained a small proportion of vesiculated inner membranes. Inner mitochondrial compartments, "contracted" by preincubation in the presence of ATP, sedimented to a markedly lower density on the gradients than did the unincubated preparations and about 50% of the ghosts showed a highly condensed morphology. In the contracted preparations, relatively low malate dehydrogenase and D-beta-hydroxybutyrate dehydrogenase activities were found in the fractions comprised of the inner compartments. The specific activity and distribution of succinate dehydrogenase were about the same as were found with the unincubated, lysed mitochondria.

Ion-induced ultrastructural transformations in isolated mitochondria. The energized uptake of calcium.

The energized uptake of low levels of Ca(2+) in the presence and absence of phosphate by isolated rat liver mitochondria, and the perturbation effected by this activity on ultrastructural and metabolic parameters of mitochondria have been investigated. In the presence of phosphate, low levels of Ca(2+) are taken up by mitochondria and result in various degrees of ultrastructural expansion of the inner mitochondrial compartment. This indicates that low levels of Ca(2+) in the presence of phosphate, are accumulated in an osmotically active form into the water phase of the inner compartment. The first clearly observable quantitative increase in the volume of the inner compartment occurs after the accumulation of 100 nmoles Ca(2+)/mg protein. An accumulation of 150-200 nmoles Ca(2+)/mg protein, which is equivalent to the osmolar concentration of endogenous K(+), is required to effect a doubling of the volume of the inner compartment. This degree of osmotic perturbation occurs as mitochondria transform from a condensed to an orthodox conformation. The osmotically induced orthodox conformation differs from the mechanochemically induced orthodox conformation previously described, in that its development is concomitant with a marked decrease in acceptor control and oxidative phosphorylation efficiency and it fails to transform to a condensed conformation in response to addition of ADP. In the absence of added phosphate, a maximum of 190 nmoles Ca(2+)/mg protein was found to be taken up by mitochondria (state 6). Ca(2+) is apparently bound under state 6 conditions since the uptake does not effect an ultrastructural expansion of the inner compartment. Phosphate added after state 6 Ca(2+) binding, however, results in an immediate ultrastructural expansion of the inner compartment. The addition of phosphate to mitochondria in the absence of exogenous Ca(2-) fails to effect an osmotic ultrastructural transformation. Under state 6 conditions, the binding of between 40 and 190 nmoles Ca(2+)/mg protein results in the formation of dense matrix inclusions which appear to be composed of tightly packed, concentrically oriented membranes. Under conditions in which the bound Ca(2+) is subsequently released, there is a concomitant loss in the density of these matrix inclusions, leaving behind morphologically distinct membrane whorls in the mitochondrial matrix.

Electron microscopic characterization of chick embryonic skeletal muscle proteoglycans.

In this article, proteoglycans from embryonic chick leg muscle are quantitatively and qualitatively compared with day 8 high density cell culture cartilage proteoglycans by electron microscopy of proteoglycan-cytochrome c monolayers. The visualized proteoglycan profiles were separated into four categories according to shape, size, and complexity. The two major categories were further characterized by lengths of core proteins, lengths of side projections, and distance between side projections. Two large proteoglycans are identifiable in spread leg muscle preparations. One group has a core protein (mean length of 205 nm) from which extend long thin side projections that we interpret to be groups of chondroitin sulfate glycosaminoglycans with a mean length of 79 nm. This large chondroitin sulfate proteoglycan is the only type found in muscle cultures as determined both biochemically in the past and now by electron microscopy and is referred to as muscle proteoglycan. The second large proteoglycan has a mean core protein length of 250 nm and side projections that are visibly shorter (mean length of 38 nm) and thicker than those of the muscle proteoglycan. This group is referred to as the mesenchymal proteoglycan since its biosynthetic origin is still uncertain. We compare these two profiles with the chick cartilage chondroitin sulfate proteoglycan that has a mean core protein length of 202 nm and side projections with a mean length of 50 nm. The data presented here substantiate the earlier biochemical characterization of these noncartilage proteoglycans and establish the unique structural features of the muscle proteoglycan as compared with the similar profiles of the cartilage and mesenchymal proteoglycans.

3-acetylpyridine: effects in vitro related to teratogenic activity in chicken embryos.

Production of skeletal muscle hypoplasia by 3-acetylpyridine and its complete reversal by nicotinamide in developing chicken embryos have been confirmed. Cultures of developing embryonic chicken muscle show degenerative effects produced by 3-acetylpyridine; these effects are reversed by nicotinamide. Cartilage production in cultured chondrogenic cells is potentiated by 3-acetylpyridine; this potentiation is completely reversed by nicotinamide. It is suggested that nicotinamide-or pyridine-nucleotide-dependent reactions influence normal differentiation of limb mesoderm cells by inhibiting chondrogenic-cell and potentiating muscle-cell expression or proliferation.

Irreversible gene repression model for control of development.

As the pluripotent cells of early embryos differentiate, each progressively loses the potency to develop into several phenotypes. Ultimately, each cell becomes irreversibly restricted to the expression of a single phenotype. Although in many instances details regarding those restriction events are well known, there is little information concerning the nature of the gene transcription changes involved. A model that accounts for the diminution of developmental potential as resulting from progressive, irreversible repression of previously active genes is presented. A scheme of progressive gene repression, rather than selective gene activation, is most consistent with observations from experimental embryology as well as from more recent biochemical experimentation.

Molecular and cell isoforms during development.

Development proceeds by way of a discrete yet overlapping series of biosynthetic and restructuring events that result in the continued molding of tissues and organs into highly restricted and specialized states required for adult function. Individual molecules and cells are replaced by molecular and cellular variants, called isoforms; these arise and function during embryonic development or later life. Isoforms, whether molecular or cellular, have been identified by their structural differences, which allow separation and characterization of each variant. These isoforms play a central and controlling role in the continued and dynamic remodeling that takes place during development. Descriptions of the individual phases of the orderly replacement of one isoform for another provides an experimental context in which the process of development can be better understood.

Stem cells for tissue engineering of articular cartilage.

Articular cartilage injuries are one of the most common disorders in the musculo-skeletal system. Injured cartilage tissue cannot spontaneously heal and, if not treated, can lead to osteoarthritis of the affected joints. Although a variety of procedures are being employed to repair cartilage damage, methods that result in consistent durable repair tissue are not yet available. Tissue engineering is a recently developed science that merges the fields of cell biology, engineering, material science, and surgery to regenerate new functional tissue. Three critical components in tissue engineering of cartilage are as follows: first, sufficient cell numbers within the defect, such as chondrocytes or multipotent stem cells capable of differentiating into chondrocytes; second, access to growth and differentiation factors that modulate these cells to differentiate through the chondrogenic lineage; third, a cell carrier or matrix that fills the defect, delivers the appropriate cells, and supports cell proliferation and differentiation. Stem cells that exist in the embyro or in adult somatic tissues are able to renew themselves through cell division without changing their phenotype and are able to differentiate into multiple lineages including the chondrogenic lineage under certain physiological or experimental conditions. Here the application of stem cells as a cell source for cartilage tissue engineering is reviewed.

Separation of transcriptively active and inactive chromatin. Agarose gel chromatography.

Sheared chromatin fractionated by currently accepted methods of agarose gel exclusion chromatography, undergoes a limited and non-specific aggregation resulting from the high ionic strength and divalent cation concentration of the column elution buffer. Such aggregation causes the artifactual appearance of radioactively labeled, newly synthesized RNA within the column exclusion volume, erroneously suggesting an enrichment for actively transcribed chromatin. Claims for the efficacy of agarose gel exclusion as a method for separating template-active and -inactive chromatin are based largely on assays for active chromatin which rely on localization of specific molecular complexes of chromatin and nascent RNA. Under the conditions employed, the present studies invalidate this assay and thus cast considerable doubt on the agarose gel exclusion method itself.

Rapid hematopoietic recovery after coinfusion of autologous-blood stem cells and culture-expanded marrow mesenchymal stem cells in advanced breast cancer patients receiving high-dose chemotherapy.

PURPOSE: Multipotential mesenchymal stem cells (MSCs) are found in human bone marrow and are shown to secrete hematopoietic cytokines and support hematopoietic progenitors in vitro. We hypothesized that infusion of autologous MSCs after myeloablative therapy would facilitate engraftment by hematopoietic stem cells, and we investigated the feasibility, safety, and hematopoietic effects of culture-expanded MSCs in breast cancer patients receiving autologous peripheral-blood progenitor-cell (PBPC) infusion. PATIENTS AND METHODS: We developed an efficient method of isolating and culture-expanding a homogenous population of MSCs from a small marrow-aspirate sample obtained from 32 breast cancer patients. Twenty-eight patients were given high-dose chemotherapy and autologous PBPCs plus culture-expanded MSC infusion and daily granulocyte colony-stimulating factor. RESULTS: Human MSCs were successfully isolated from a mean +/- SD of 23.4 +/- 5.9 mL of bone marrow aspirate from all patients. Expansion cultures generated greater than 1 x 10(6) MSCs/kg for all patients over 20 to 50 days with a mean potential of 5.6 to 36.3 x 10(6) MSCs/kg after two to six passages, respectively. Twenty-eight patients were infused with 1 to 2.2 x 10(6) expanded autologous MSCs/kg intravenously over 15 minutes. There were no toxicities related to the infusion of MSCs. Clonogenic MSCs were detected in venous blood up to 1 hour after infusion in 13 of 21 patients (62%). Median time to achieve a neutrophil count greater than 500/microL and platelet count >/= 20,000/microL untransfused was 8 days (range, 6 to 11 days) and 8.5 days (range, 4 to 19 days), respectively. CONCLUSION: This report is the first describing infusion of autologous MSCs with therapeutic intent. We found that autologous MSC infusion at the time of PBPC transplantation is feasible and safe. The observed rapid hematopoietic recovery suggests that MSC infusion after myeloablative therapy may have a positive impact on hematopoiesis and should be tested in randomized trials.

Mesenchymal stem cells: building blocks for molecular medicine in the 21st century.

Mesenchymal stem sells (MSCs) are present in a variety of tissues during human development, and in adults they are prevalent in bone marrow. From that readily available source, MSCs can be isolated, expanded in culture, and stimulated to differentiate into bone, cartilage, muscle, marrow stroma, tendon, fat and a variety of other connective tissues. Because large numbers of MSCs can be generated in culture, tissue-engineered constructs principally composed of these cells could be re-introduced into the in vivo setting. This approach is now being explored to regenerate tissues that the body cannot naturally repair or regenerate when challenged. Moreover, MSCs can be transduced with retroviral and other vectors and are, thus, potential candidates to deliver somatic gene therapies for local or systemic pathologies. Untapped applications include both diagnostic and prognostic uses of MSCs and their descendents in healthcare management. Finally, by understanding the complex, multistep and multifactorial differentiation pathway from MSC to functional tissues, it might be possible to manipulate MSCs directly in vivo to cue the formation of elaborate, composite tissues in situ.

Bone marrow-derived mesenchymal stem cells remain host-derived despite successful hematopoietic engraftment after allogeneic transplantation in patients with lysosomal and peroxisomal storage diseases.

Human bone marrow contains mesenchymal stem cells (MSCs) that can differentiate into various cells of mesenchymal origin. We developed an efficient method of isolating and culture expanding a homogenous population of MSCs from bone marrow and determined that MSCs express alpha-L-iduronidase, arylsulfatase-A and B, glucocerebrosidase, and adrenoleukodystrophy protein. These findings raised the possibility that MSCs may be useful in the treatment of storage disorders. To determine if donor derived MSCs are transferred to the recipients with lysosomal or peroxisomal storage diseases by allogeneic hematopoietic stem cell (HSC) transplantation, we investigated bone marrow derived MSCs of 13 patients 1-14 years after allogeneic transplantation. Highly purified MSCs were genotyped either by fluorescence in situ hybridization using probes for X and Y-chromosomes in gender mis-matched recipients or by radiolabeled PCR amplification of polymorphic simple sequence repeats. Phenotype was determined by the measurement of disease specific protein/enzyme activity in purified MSCs. We found that MSCs isolated from recipients of allogeneic HSC transplantation are not of donor genotype and have persistent phenotypic defects despite successful donor type hematopoietic engraftment. Whether culture expanded normal MSCs can be successfully transplanted into patients with storage diseases and provide therapeutic benefit needs to be determined.

Stimulatory effects of basic fibroblast growth factor and bone morphogenetic protein-2 on osteogenic differentiation of rat bone marrow-derived mesenchymal stem cells.

Bone marrow stroma contains multipotential mesenchymal progenitor cells which can differentiate into osteoblastic cells; we refer to these cells as mesenchymal stem cells (MSCs). Basic fibroblast growth factor (bFGF) and bone morphogenetic protein-2 (BMP-2) have been implicated in the osteogenic regulatory process by virtue of their mitogenic and differentiation activities, respectively. This study examines and compares the effects of bFGF and BMP-2 on dexamethasone (Dex)-dependent in vitro osteogenic differentiation of rat marrow-derived MSCs. A 6-day exposure to bFGF markedly stimulated cell growth and induced osteoblastic differentiation as shown by osteocalcin mRNA expression (day 14), bone nodule formation (day 18), and calcium deposition (day 18). These results indicate that bFGF enhances both mitogenic activity and osteogenic development of Dex-treated marrow MSCs. In contrast, BMP-2 did not induce osteogenesis as strongly as bFGF. Thus, exposure to BMP-2 slightly increased bone nodule number and calcium content compared with the control. Exposure of MSCs to both BMP-2 and bFGF induced expression of osteocalcin mRNA and mineralizing bone-like nodules as early as day 11 and resulted in enhancement of bone formation more markedly than either factor alone. Consistent with these results, porous calcium phosphate ceramic cubes implanted in vivo, which were loaded with MSCs pre-exposed to both bFGF and BMP-2, showed higher histologic score for bone formation than those with MSCs pre-exposed to either bFGF or BMP-2 alone. These data indicate that combined treatment with bFGF and BMP-2 synergistically enhances the osteogenic potency of bFGF in rat marrow MSC culture.

Osteochondrogenic potential of marrow mesenchymal progenitor cells exposed to TGF-beta 1 or PDGF-BB as assayed in vivo and in vitro.

Mesenchymal progenitors cells can be isolated from rat bone marrow and mitotically expanded in vitro. When these cells, which we operationally call mesenchymal stem cells (MSCs), are placed in an appropriate environment, they have the capacity to differentiate into bone and/or cartilage. This capacity is called osteochondrogenic potential. In this study, preconfluent MSCs were exposed in vitro to 5 ng/ml transforming growth factor-beta 1 (TGF-beta 1) or platelet-derived growth factor, isoform BB (PDGF-BB) for a pulse of 48 h and assayed for cell proliferation, alkaline phosphatase activity, and osteochondrogenic potential; untreated MSC's served as controls. In these cell culture conditions, TGF-beta 1 or PDGF-BB had similar effects on proliferation and alkaline phosphatase activity. Both growth factors increased cell proliferation and decreased alkaline phosphatase activity of MSCs. Sister cultures of TGF-beta 1- or PDGF-BB-treated MSCs and untreated MSCs were trypsinized. For each type of culture, the trypsinised MSCs were split in two parts: one part was replated in an osteogenic medium to assess its in vitro osteogenic potential, whereas the other part was seeded into porous calcium phosphate ceramics and implanted subcutaneously in syngeneic rats to assess its in vivo osteochondrogenic potential. PDGF-pretreated MSCs showed no difference in in vivo and in vitro osteochondrogenesis from that of control MSCs, while TGF-beta 1 pretreatment blocked the osteochondrogenic potential of MSCs when assayed in vitro for bone nodule formation. However, when tested in vivo, TGF-beta 1-pretreated MSCs were able to form bone and cartilage. These data show that measurements of proliferation and alkaline phosphatase activity of preconfluent MSCs immediately after exposure to growth factor were not predictive of their subsequent osteochondrogenic potential. Moreover, the variation of the osteochondrogenic potential of MSCs after exposure to growth factor was further modulated by the environment in which the MSCs were assayed.

Bone morphogenetic protein 2 stimulates osteogenesis but does not affect chondrogenesis in osteochondrogenic differentiation of periosteum-derived cells.

The effects of recombinant human bone morphogenetic protein 2 (rhBMP-2) on osteochondrogenesis were examined in high-density cultures of periosteum-derived cells, which have the potential to differentiate into bone and hypertrophic cartilage in vitro. Proliferation of these cells was inhibited by treatment with rhBMP-2. The time course for alkaline phosphatase (ALP) expression was shortened and the mineralization of the culture was increased by supplementation with rhBMP-2. These stimulatory effects of rhBMP-2 were observed at doses of 10-100 ng/m. Bone Gla protein (BGP) was immunocytochemically detectable earlier in the culture treated with rhBMP-2, and the BGP-positive layer of the rhBMP-2-treated cultures was thicker than that of the control cultures. On the other hand, there was no difference in uronic acid content or the time course of alpha 1(II) collagen mRNA expression between the rhBMP-2-treated and the control cultures. These results indicate that rhBMP-2 shortens the time course of osteogenesis and increases the amount of bone formation, whereas chondrogenesis remains unaffected.