Treatment of murine partial thickness scald injuries with multipotent adult progenitor cells decreases inflammation and promotes angiogenesis leading to improved burn injury repair.

Stem cells have been shown to have potential as a new therapy for burns and promote wound healing through decreasing inflammation and increasing angiogenesis. Multipotent adult progenitor cells (MAPC® cells) are a subpopulation of bone marrow-derived stem cells with outstanding self-renewal and differentiation capacity. MAPC cells also secrete a wide range of cytokines which can affect cellular activities. This article aimed to examine the effects of MAPC cells treatment on burn injury repair using a mouse model of partial thickness burn injury. The immunomodulatory effect of MAPC cells was investigated in vitro using a simultaneous T-cell proliferation assay. Partial thickness burns were created on the dorsal surface of mice and MAPC cells were administered via intradermal injection to the wound margins 24 h post-burn injury. The burn tissues were analysed macroscopically to determine wound area and histologically assessed to determine wound width and rate of re-epithelialisation. Immunohistochemistry and ELISA were employed to assess cell proliferation, inflammation and angiogenesis and collagen deposition in the burn area. MAPC cells inhibit the proliferation of stimulated T cells in culture. Burns intradermally injected with MAPC cells showed a significant reduction in the macroscopic wound area, histologic wound width and had an increased rate of re-epithelialisation. Immunohistochemistry and ELISA analysis of burn tissues showed dampened inflammation evidenced by a reduction in neutrophilic infiltration and modulation of inflammatory cytokines. Angiogenesis within the burn area was also improved in MAPC cell treated mice. However, no significant effect of MAPC cell treatment was observed on extracellular matrix production. Treatment of burns with MAPC cells improved burn injury repair with reduced time to healing, decreased inflammation and increased angiogenesis. These findings demonstrate the promising effects of MAPC cells on burn injury repair and suggest MAPC cells as a candidate source for clinical cell therapies.

Multipotent adult progenitor cells grown under xenobiotic-free conditions support vascularization during wound healing.

BACKGROUND: Cell therapy has been evaluated pre-clinically and clinically as a means to improve wound vascularization and healing. While translation of this approach to clinical practice ideally requires the availability of clinical grade xenobiotic-free cell preparations, studies proving the pre-clinical efficacy of the latter are mostly lacking. Here, the potential of xenobiotic-free human multipotent adult progenitor cell (XF-hMAPC®) preparations to promote vascularization was evaluated. METHODS: The potential of XF-hMAPC cells to support blood vessel formation was first scored in an in vivo Matrigel assay in mice. Next, a dose-response study was performed with XF-hMAPC cells in which they were tested for their ability to support vascularization and (epi) dermal healing in a physiologically relevant splinted wound mouse model. RESULTS: XF-hMAPC cells supported blood vessel formation in Matrigel by promoting the formation of mature (smooth muscle cell-coated) vessels. Furthermore, XF-hMAPC cells dose-dependently improved wound vascularization associated with increasing wound closure and re-epithelialization, granulation tissue formation, and dermal collagen organization. CONCLUSIONS: Here, we demonstrated that the administration of clinical-grade XF-hMAPC cells in mice represents an effective approach for improving wound vascularization and healing that is readily applicable for translation in humans.

Human multipotent adult progenitor cell-conditioned medium improves wound healing through modulating inflammation and angiogenesis in mice.

BACKGROUND: Stem cell therapies have been widely investigated for their healing effects. However, the translation of these therapies has been hampered by the requirement to deliver live allogeneic or autologous cells directly to the wound in a clinical setting. Multipotent adult progenitor cells (MAPC® cells) are a subpopulation of bone marrow-derived adherent stem cells that secrete a wide range of factors known to accelerate the wound healing process. The aim of this study was to determine the impact of MAPC cells secretome on healing outcomes without the presence of MAPC cells. METHODS: The effect of MAPC-conditioned medium (MAPC-CM) on the capacity of keratinocytes, fibroblasts and endothelial cells to migrate and proliferate was determined in vitro using scratch wound closure and WST1 assay, respectively. The effect of MAPC-CM on collagen deposition and angiogenesis was also assessed using in vitro methods. Additionally, two excisional wounds were created on the dorsal surface of mice (n = 8/group) and 100 µL of 20× MAPC-CM were intradermally injected to the wound margins. Wound tissues were collected at 3, 7 and 14 days post-wounding and stained with H&E for microscopic analysis. Immunohistochemistry was performed to investigate inflammation, angiogenesis and collagen deposition in the wounds. RESULTS: Skin fibroblasts, keratinocytes and endothelial cells treated with MAPC-CM all showed improved rates of scratch closure and increased cellular proliferation. Moreover, fibroblasts treated with MAPC-CM deposited more collagens I and III and endothelial cells treated with MAPC-CM showed increased capillary tube formation. Murine excisional wounds intradermally injected with MAPC-CM showed a significant reduction in the wound area and an increase in the rate of reepithelialisation. The results also showed that inflammatory cell infiltration was decreased while an increase in angiogenesis, as well as collagens I and III expressions, was observed. CONCLUSION: These findings suggest that factors produced by MAPC cells can have an important effect on cutaneous wound healing by affecting skin cell proliferation and migration, balancing inflammation and improving the formation of extracellular matrix and angiogenesis. Development of stem cell-free therapy for the treatment of wounds may be a more clinically translatable approach for improving healing outcomes.

Systemic multipotent adult progenitor cells improve long-term neurodevelopmental outcomes after preterm hypoxic-ischemic encephalopathy.

There is an urgent need for therapies that could reduce the disease burden of preterm hypoxic-ischemic encephalopathy. Here, we evaluate the long-term effects of multipotent adult progenitor cells (MAPC) on long-term behavioral outcomes in a preterm rat model of perinatal asphyxia. Rats of both sexes were treated with two doses of MAPCs within 24 h after the insult. Locomotor, cognitive and psychiatric impairments were evaluated starting at 1.5 (juvenile) and 6 months (adult). Hypoxia-ischemia affected locomotion, cognition, and anxiety in a sex-dependent manner, with higher vulnerability observed in males. The MAPC therapy partially attenuated deficits in object recognition memory in females of all tested ages, and in the adult males. The hypoxic insult caused delayed hyperactivity in adult males, which was corrected by MAPC therapy. These results suggest that MAPCs may have long-term benefits for neurodevelopmental outcome after preterm birth and global hypoxia-ischemia, which warrants further preclinical exploration.

Alginate encapsulated multipotent adult progenitor cells promote corneal stromal cell activation via release of soluble factors.

To reduce the increasing need for corneal transplantation, attempts are currently aiming to restore corneal clarity, one potent source of cells are multipotent adult progenitor cells (MAPC®). These cells release a powerful cocktail of paracrine factors that can guide wound healing and tissue regeneration. However, their role in corneal regeneration has been overlooked. Thus, we sought to explore the potential of combining the cytoprotective storage feature of alginate, with MAPC to generate a storable cell-laden gel for corneal wound healing. 72 hours following hypothermic storage, alginate encapsulation was shown to maintain MAPC viability at either 4 or 15°C. Encapsulated MAPC (2 x106 cells/mL) stored at 15°C presented the optimum temperature that allowed for cell recovery. These cells had the ability to reattach to tissue culture plastic whilst exhibiting normal phenotype and this was maintained in serum-free and xenobiotic-free medium. Furthermore, corneal stromal cells presented a significant decrease in scratch-wounds in the presence of alginate encapsulated MAPC compared to a no-cell control (p = 0.018). This study shows that immobilization of MAPC within an alginate hydrogel does not hinder their ability to affect a secondary cell population via soluble factors and that these effects are successfully retained following hypothermic storage.

Eye lens dosimetry in anesthesiology: a prospective study.

The eye lens is one of the most sensitive organs for radiation injury and exposure might lead to radiation induced cataract. Eye lens dosimetry in anesthesiology has been published in few clinical trials and an active debate about the causality of radiation induced cataract is still ongoing. Recently, the International Commission on Radiological Protection (ICRP) recommended a reduction in the annual dose limit for occupational exposure for the lens of the eye from 150 to 20 mSv, averaged over a period of 5 years, with the dose in a single year not exceeding 50 mSv. This prospective study investigated eye lens dosimetry in anesthesiology practice during a routine year of professional activity. The radiation exposure measured represented the exposure in a normal working schedule of a random anesthesiologist during 1 month and this cumulative eye lens dose was extrapolated to 1 year. Next, eye lens doses were measured in anesthesiology during neuro-embolisation procedures, radiofrequency ablations or vertebroplasty/kyphoplasty procedures. The eye lens doses are measured in terms of the dose equivalent H p(3) with the Eye-D dosimeter (Radcard, Poland) close to the right eye (on the temple). In 16 anesthesiologists, the estimated annual eye lens doses range from a minimum of 0.4 mSv to a maximum of 3.5 mSv with an average dose of 1.33 mSv. Next, eye lens doses were measured for nine neuro-embolisation procedures, ten radiofrequency ablations and six vertebroplasty/kyphoplasty procedures. Average eye lens doses of 77 ± 76 µSv for neuro-embolisations, 38 ± 34 µSv for cardiac ablations and 40 ± 44 µSv for vertebro-/kyphoplasty procedures were recorded. The maximum doses were respectively 264, 97 and 122 µSv. This study demonstrated that the estimated annual eye lens dose is well below the revised ICRP's limit of 20 mSv/year. However, we demonstrated high maximum and average doses during neuro-embolisation, cardiac ablation and vertebro-/kyphoplasty procedures. With radiation induced cataract being explained as a possible stochastic effect, without a threshold dose, anesthesiologists who regularly work in a radiological environment should remain vigilant and maintain radiation safety standards at all times. This includes adequately protective equipment (protection shields, apron, thyroid shield and leaded eye wear), keeping distance, routine monitoring and appropriate education.

Human multipotent adult progenitor cells enhance islet function and revascularisation when co-transplanted as a composite pellet in a mouse model of diabetes.

AIMS/HYPOTHESIS: Hypoxia in the initial days after islet transplantation leads to considerable loss of islet mass and contributes to disappointing outcomes in the clinical setting. The aim of the present study was to investigate whether co-transplantation of human non-endothelial bone marrow-derived multipotent adult progenitor cells (MAPCs), which are non-immunogenic and can secrete angiogenic growth factors during the initial days after implantation, could improve islet engraftment and survival. METHODS: Islets (150) were co-transplanted, with or without human MAPCs (2.5 × 105) as separate or composite pellets, under the kidney capsule of syngeneic alloxan-induced diabetic C57BL/6 mice. Blood glucose levels were frequently monitored and IPGTTs were carried out. Grafts and serum were harvested at 2 and 5 weeks after transplantation to assess outcome. RESULTS: Human MAPCs produced high amounts of angiogenic growth factors, including vascular endothelial growth factor, in vitro and in vivo, as demonstrated by the induction of neo-angiogenesis in the chorioallantoic membrane assay. Islet-human MAPC co-transplantation as a composite pellet significantly improved the outcome of islet transplantation as measured by the initial glycaemic control, diabetes reversal rate, glucose tolerance and serum C-peptide concentration compared with the outcome following transplantation of islets alone. Histologically, a higher blood vessel area and density in addition to a higher vessel/islet ratio were detected in recipients of islet-human MAPC composites. CONCLUSIONS/INTERPRETATION: The present data suggest that co-transplantation of mouse pancreatic islets with human MAPCs, which secrete high amounts of angiogenic growth factors, enhance islet graft revascularisation and subsequently improve islet graft function.

Using miRNA-mRNA Interaction Analysis to Link Biologically Relevant miRNAs to Stem Cell Identity Testing for Next-Generation Culturing Development.

UNLABELLED: Therapeutic benefit of stem cells has been demonstrated in multiple disease models and clinical trials. Robust quality assurance is imperative to make advancements in culturing procedures to enable large-scale cell manufacturing without hampering therapeutic potency. MicroRNAs (miRNAs or miRs) are shown to be master regulators of biological processes and are potentially ideal quality markers. We determined miRNA markers differentially expressed under nonclinical multipotent adult progenitor cell (MAPC) and mesenchymal stem cell (MSC) culturing conditions that regulate important stem cell features, such as proliferation and differentiation. These bone marrow-derived stem cell types were selected because they both exert therapeutic functions, but have different proliferative and regenerative capacities. To determine cell-specific marker miRNAs and assess their effects on stem cell qualities, a miRNA and mRNA profiling was performed on MAPCs and MSCs isolated from three shared donors. We applied an Ingenuity Pathway Analysis-based strategy that combined an integrated RNA profile analysis and a biological function analysis to determine the effects of miRNA-mRNA interactions on phenotype. This resulted in the identification of important miRNA markers linked to cell-cycle regulation and development, the most distinctive being MAPC marker miR-204-5p and MSC marker miR-335-5p, for which we provide in vitro validation of its function in differentiation and cell cycle regulation, respectively. Importantly, marker expression is maintained under xeno-free conditions and during bioreactor isolation and expansion of MAPC cultures. In conclusion, the identified biologically relevant miRNA markers can be used to monitor stem cell stability when implementing variations in culturing procedures. SIGNIFICANCE: Human adult marrow stromal stem cells have shown great potential in addressing unmet health care needs. Quality assurance is imperative to make advancements in large-scale manufacturing procedures. MicroRNAs are master regulators of biological processes and potentially ideal quality markers. MicroRNA and mRNA profiling data of two human adult stem cell types were correlated to biological functions in silico. Doing this provided evidence that differentially expressed microRNAs are involved in regulating specific stem cell features. Furthermore, expression of a selected microRNA panel was maintained in next-generation culturing platforms, demonstrating the robustness of microRNA profiling in stem cell comparability testing.

Suppression of IL-7-dependent Effector T-cell Expansion by Multipotent Adult Progenitor Cells and PGE2.

T-cell depletion therapy is used to prevent acute allograft rejection, treat autoimmunity and create space for bone marrow or hematopoietic cell transplantation. The evolved response to T-cell loss is a transient increase in IL-7 that drives compensatory homeostatic proliferation (HP) of mature T cells. Paradoxically, the exaggerated form of this process that occurs following lymphodepletion expands effector T-cells, often causing loss of immunological tolerance that results in rapid graft rejection, autoimmunity, and exacerbated graft-versus-host disease (GVHD). While standard immune suppression is unable to treat these pathologies, growing evidence suggests that manipulating the incipient process of HP increases allograft survival, prevents autoimmunity, and markedly reduces GVHD. Multipotent adult progenitor cells (MAPC) are a clinical grade immunomodulatory cell therapy known to alter γ-chain cytokine responses in T-cells. Herein, we demonstrate that MAPC regulate HP of human T-cells, prevent the expansion of Th1, Th17, and Th22 effectors, and block the development of pathogenic allograft responses. This occurs via IL-1ß-primed secretion of PGE2 and activates T-cell intrinsic regulatory mechanisms (SOCS2, GADD45A). These data provide proof-of-principle that HP of human T-cells can be targeted by cellular and molecular therapies and lays a basis for the development of novel strategies to prevent immunopathology in lymphodepleted patients.

Culturing protocols for human multipotent adult stem cells.

Culture procedures are presented that support the initiation and controlled expansion of the multipotent adult progenitor cell (MAPC) population within the human bone marrow derived multipotent mesenchymal stromal cell compartment. Culture procedures or conditions and characterization assays that maintain and survey the distinctive primitive MAPC properties are discussed in the context of cell culturing platforms that facilitate controlled expansion of clinical grade human MAPC product to levels required for mid to late stage clinical testing.

Impaired of a non-DNA dependent methylation status decides the fat decision of bone marrow-derived C3H10T1/2 stem cell.

A decrease in the lineage commitment of multipotent Mesenchymal stem cells (MSC) to the bone forming osteoblast lineage and an increase in the commitment to the fat forming adipocyte lineage is more common in bone marrow of elderly persons. A link between methylation status and MSC differentiation remains unclear. Therefore, we hypothesize that hypomethylation may decide the fate decisions of MSC. In the current study, murine bone marrow derived-C3H10T1/2 stem cell was used to examine the role of methylation mechanism on the differentiation potential of stem cells into osteoblasts or adipocytes. C3H10T1/2 cells were treated with Periodate oxidized adenosine (Adox), an inhibitor of S-adenosylhomocysteine-dependent hydrolase (SAHH), which in turn block the non-DNA methylation pathway. The effect of hypomethylation on C3H10T1/2 stem cell differentiation was determined by measuring the alkaline phosphates activity and the degree of mineralization as well as Oil-red O staining and lipid content. The ratio of S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) was determined as a metabolic indicator of cellular methylation potential. It was clearly observed that hypomethylation significantly (P < 0.05) reduces SAM: SAH ratio, alkaline phosphates activity, calcification and thereby, osteoblast differentiation. Conversely, adipocyte differentiation was stimulated by hypomethylation. Altogether, our data suggest that non-DNA hypomethylation changes the differentiation potential of C3H10T1/2 stem cells for less osteogenic and more adipogenic.

Clinical-grade multipotent adult progenitor cells durably control pathogenic T cell responses in human models of transplantation and autoimmunity.

A major goal of immunotherapy remains the control of pathogenic T cell responses that drive autoimmunity and allograft rejection. Adherent progenitor cells, including mesenchymal stromal cells (MSCs) and multipotent adult progenitor cells (MAPCs), represent attractive immunomodulatory cell therapy candidates currently active in clinical trials. MAPCs can be distinguished from MSCs on the basis of cellular phenotype, size, transcriptional profile, and expansion capacity. However, despite their ongoing evaluation in autoimmune and allogeneic solid organ transplantation settings, data supporting the immune regulatory potential of clinical-grade MAPCs are limited. In this study, we used allogeneic islet transplantation as a model indication to assess the ability of clinical-grade MAPCs to control T cell responses that drive immunopathology in human autoimmune disease and allograft rejection. MAPCs suppressed T cell proliferation and Th1 and Th17 cytokine production while increasing secretion of IL-10 and were able to suppress effector functions of bona fide autoreactive T cells from individuals with type 1 diabetes mellitus, including killing of human islets. Furthermore, MAPCs favored the proliferation of regulatory T cells during homeostatic expansion driven by γ-chain cytokines and exerted a durable, yet reversible, control of T cell function. MAPC suppression required licensing and proceeded via IDO-mediated tryptophan catabolism. Therefore, the common immune modulatory characteristics of clinical-grade MAPCs shown in this study suggest that they can be regarded as an alternative source of adult progenitor cells with similar clinical usefulness to MSCs. Taken collectively, these findings may guide the successful deployment of both MSCs and MAPCs for the amelioration of human autoimmunity and allograft rejection.

Application of MultiStem(®) Allogeneic Cells for Immunomodulatory Therapy: Clinical Progress and Pre-Clinical Challenges in Prophylaxis for Graft Versus Host Disease.

The last decade has seen much progress in adjunctive cell therapy for immune disorders. Both corporate and institutional Phase III studies have been run using mesenchymal stromal cells (MSC) for treatment of Graft versus Host Disease (GvHD), and product approval has been achieved for treatment of pediatric GvHD in Canada and New Zealand (Prochymal(®); Osiris Therapeutics). This effectiveness has prompted the prophylactic use of adherent stem cells at the time of allogeneic hematopoietic stem cell transplantation (HSCT) to prevent occurrence of GvHD and possibly provide stromal support for hematopoietic recovery. The MultiStem(®) product is an adult adherent stem cell product derived from bone marrow which has significant clinical exposure. MultiStem cells are currently in phase II clinical studies for treatment of ischemic stroke and ulcerative colitis, with Phase I studies completed in acute myocardial infarction and for GvHD prophylaxis in allogeneic HSCT, demonstrating that MultiStem administration was well tolerated while the incidence and severity of GvHD was reduced. In advancing this clinical approach, it is important to recognize that alternate models exist based on clinical manufacturing strategies. Corporate sponsors exploit the universal donor properties of adherent stem cells and manufacture at large scale, with many products obtained from one or limited donors and used across many patients. In Europe, institutional sponsors often produce allogeneic product in a patient designated context. For this approach, disposable bioreactors producing <10 products/donor in a closed system manner are very well suited. In this review, the use of adherent stem cells for GvHD prophylaxis is summarized and the suitability of disposable bioreactors for MultiStem production is presented, with an emphasis on quality control parameters, which are critical with a multiple donor approach for manufacturing.

Inhibition of methylation decreases osteoblast differentiation via a non-DNA-dependent methylation mechanism.

S-adenosylmethionine (SAM)-dependent methylation of biological molecules including DNA and proteins is rapidly being uncovered as a critical mechanism for regulation of cellular processes. We investigated the effects of reduced SAM-dependent methylation on osteoblast differentiation by using periodate oxidized adenosine (ADOX), an inhibitor of SAM-dependent methyltransferases. The capacity of this agent to modulate osteoblast differentiation was analyzed under non-osteogenic control conditions and during growth factor-induced differentiation and compared with the effect of inhibition of DNA methylation by 5-Aza-2'-deoxycytidine (5-Aza-CdR). Without applying specific osteogenic triggers, both ADOX and 5-Aza-CdR induced mRNA expression of the osteoblast markers Alp, Osx, and Ocn in murine C2C12 cells. Under osteogenic conditions, ADOX inhibited differentiation of both human mesenchymal stem cells and C2C12 cells. Gene expression analysis of early (Msx2, Dlx5, Runx2) and late (Alp, Osx, Ocn) osteoblast markers during bone morphogenetic protein 2-induced C2C12 osteoblast differentiation revealed that ADOX only reduced expression of the late phase Runx2 target genes. By using a Runx2-responsive luciferase reporter (6xOSE), we showed that ADOX reduced the activity of Runx2, while 5-Aza-CdR had no effect. Taken together, our data suggest that decreased SAM-dependent methyltransferase activity leads to impaired osteoblast differentiation via non-DNA-dependent methylation mechanisms and that methylation is a regulator of Runx2-controlled gene expression.

Vitamin B(12) deficiency stimulates osteoclastogenesis via increased homocysteine and methylmalonic acid.

The risk of nutrient deficiencies increases with age in our modern Western society, and vitamin B(12) deficiency is especially prevalent in the elderly and causes increased homocysteine (Hcy) and methylmalonic acid (MMA) levels. These three factors have been recognized as risk factors for reduced bone mineral density and increased fracture risk, though mechanistic evidence is still lacking. In the present study, we investigated the influence of B(12), Hcy, and MMA on differentiation and activity of bone cells. B(12) deficiency did not affect the onset of osteoblast differentiation, maturation, matrix mineralization, or adipocyte differentiation from human mesenchymal stem cells (hMSCs). B(12) deficiency caused an increase in the secretion of Hcy and MMA into the culture medium by osteoblasts, but Hcy and MMA appeared to have no effect on hMSC osteoblast differentiation. We further studied the effect of B(12), Hcy, and MMA on the formation of multinucleated tartrate-resistant acid phosphatase-positive osteoclasts from mouse bone marrow. We observed that B(12) did not show an effect on osteoclastogenesis. However, Hcy as well as MMA were found to induce osteoclastogenesis in a dose-dependent manner. On the basis of these results, we conclude that B(12) deficiency may lead to decreased bone mass by increased osteoclast formation due to increased MMA and Hcy levels.

Microarray analysis on Runx2-deficient mouse embryos reveals novel Runx2 functions and target genes during intramembranous and endochondral bone formation.

A major challenge in developmental biology is to correlate genome-wide gene expression modulations with developmental processes in vivo. In this study, we analyzed the role of Runx2 during intramembranous and endochondral bone development, by comparing gene expression profiles in 14.5 dpc wild-type and Runx2 (-/-) mice. A total of 1277, 606 and 492 transcripts were found to be significantly modulated by Runx2 in calvaria, forelimbs and hindlimbs, respectively. Bioinformatics analysis indicated that Runx2 not only controls the processes of osteoblast differentiation and chondrocyte maturation, but may also play a role in axon formation and hematopoietic cell commitment during bone development. A total of 41 genes are affected by the Runx2 deletion in both intramembranous and endochondral bone, indicating common pathways between these two developmental modes of bone formation. In addition, we identified genes that are specifically involved in endochondral ossification. In conclusion, our data show that a comparative genome-wide expression analysis of wild-type and mutant mouse models allows the examination of mutant phenotypes in complex tissues.

Microarray analysis reveals expression regulation of Wnt antagonists in differentiating osteoblasts.

Wnt signaling has been implicated in regulating bone formation by controlling osteoblast proliferation and function. Although stabilization of beta-catenin by Wnt has been shown to increase alkaline phosphatase expression and osteoblast differentiation, the precise role of Wnt signaling during the process of osteoblast differentiation is largely unknown. In this study, we used microarray technology to investigate expression regulation of Wnt signaling components during in vitro osteoblast differentiation. Expression was analyzed during bone morphogenetic protein 2 (BMP2)-induced osteoblast differentiation of murine C2C12 and MC3T3 cells and data were compared with expression in BMP2-treated NIH3T3 fibroblasts. During osteoblast differentiation, particularly strong expression regulation of the Wnt antagonists Sfrp2 (secreted frizzled related protein 2) and Wif1 (Wnt inhibitory factor 1) was observed in the late phase of differentiation. In situ expression analysis in murine tail vertebrae supported Wif1 expression during late phase bone cell differentiation, since Wif1 was found to be expressed in vivo in trabecular, but not in cortical bone. We further analyzed the effects of continuous activation of Wnt signaling by lithium chloride and observed that osteoblast differentiation was reduced, as measured by expression of osteoblast marker genes encoding alkaline phosphatase, osteocalcin, and osterix, as well as by the amount of calcium release. Taken together, our data indicate that endogenous expression of Wnt antagonists by osteoblasts provides a negative Wnt feedback loop which is essential in controlling osteoblast maturation.

Identification of novel regulators associated with early-phase osteoblast differentiation.

UNLABELLED: Key regulatory components of the BMP-induced osteoblast differentiation cascade remain to be established. Microarray and subsequent expression analyses in mice identified two transcription factors, Hey1 and Tcf7, with in vitro and in vivo expression characteristics very similar to Cbfa1. Transfection studies suggest that Tcf7 modulates BMP2-induced osteoblast differentiation. This study contributes to a better definition of the onset of BMP-induced osteoblast differentiation. INTRODUCTION: Elucidation of the genetic cascade guiding mesenchymal stem cells to become osteoblasts is of extreme importance for improving the treatment of bone-related diseases such as osteoporosis. The aim of this study was to identify regulators of the early phases of bone morphogenetic protein (BMP)2-induced osteoblast differentiation. MATERIALS AND METHODS: Osteoblast differentiation of mouse C2C12 cells was induced by treatment with BMP2, and regulation of gene expression was studied during the subsequent 24 h using high-density microarrays. The regulated genes were grouped by means of model-based clustering, and protein functions were assigned. Real-time quantitative RT-PCR analysis was used to validate BMP2-induced gene expression patterns in C2C12 cells. Osteoblast specificity was studied by comparing these expression patterns with those in C3H10T1/2 and NIH3T3 cells under similar conditions. In situ hybridization of mRNA in embryos at embryonic day (E)14.5 and E16.5 of gestation and on newborn mouse tails were used to study in vivo expression patterns. Cells constitutively expressing the regulated gene Tcf7 were used to investigate its influence on BMP-induced osteoblast differentiation. RESULTS AND CONCLUSIONS: A total of 184 genes and expressed sequence tags (ESTs) were differentially expressed in the first 24 h after BMP2 treatment and grouped in subsets of immediate early, intermediate early, and late early response genes. Signal transduction regulatory factors mainly represented the subset of immediate early genes. Regulation of expression of these genes was direct, independent of de novo protein synthesis and independent of the cell type studied. The intermediate early and late early genes consisted primarily of genes related to processes that modulate morphology, basement membrane formation, and synthesis of extracellular calcified matrix. The late early genes require de novo protein synthesis and show osteoblast specificity. In vivo and in vitro experiments showed that the transcription factors Hey1 and Tcf7 exhibited expression characteristics and cell type specificity very similar to those of the osteoblast specific transcription factor Cbfa1, and constitutive expression of Tcf7 in C2C12 cells differentially regulated osteoblast differentiation marker genes.

Comprehensive microarray analysis of bone morphogenetic protein 2-induced osteoblast differentiation resulting in the identification of novel markers for bone development.

Osteoblasts are cells responsible for matrix deposition during bone development and although temporal expression of many genes has been related to osteoblast differentiation, a complete description of osteoblast-specific gene regulation will lead to a better understanding of osteoblast function. In this study, microarray technology was used to analyze gene expression on a broad scale during osteoblast differentiation. Expression analysis of 9596 sequences revealed 342 genes and expressed sequence tags (ESTs) to be modulated differentially during a time course experiment in which murine C2C12 mesenchymal progenitor cells were induced to differentiate into mature osteoblasts by treatment with bone morphogenetic protein 2 (BMP-2). By means of hierarchical clustering, these genes were grouped by similarities in their expression profiles, resulting in subsets of early, intermediate, and late response genes, which are representative of the distinct stages of osteoblast differentiation. To identify new bone markers, the bone specificity of the late response genes was determined by comparing BMP-induced expression in C2C12 and MC3T3 osteoblasts with that in NIH3T3 fibroblasts. This resulted in the identification of nine novel genes and ESTs that were induced specifically in osteoblasts, in addition to the well-known markers ALP and osteocalcin. For at least one of these novel genes, Wnt inhibitory factor 1, and two of the ESTs, expression in developing bone was verified in vivo by in situ hybridization of E16.5 mouse embryos. In conclusion, by a combination of in vitro and in vivo screening approaches, a set of new genes related to osteoblast differentiation and skeletal development has been identified.