Tatjana Schilling

Plasticity in adipogenesis and osteogenesis of human mesenchymal stem cells.

We established a cell culture system of human mesenchymal stem cells that allows not only for osteogenic and adipogenic differentiation but also for transdifferentiation between both cell lineages. Committed osteoblasts were transdifferentiated into adipocytes with losing osteogenic but highly expressing adipogenic markers. Adipocytes were transdifferentiated into osteoblasts with most of the resulting cells showing osteogenic but some still displaying adipogenic markers apparently not responding to the reprogramming stimulus. Comparing transdifferentiated adipocytes with committed osteoblasts by microarray analysis revealed 258 regulated transcripts, many of them associated with signal transduction, metabolism, and transcription but mostly distinct from established inducing factors of normal adipogenic and osteogenic differentiation, respectively. The regulation pattern of 20 of 22 selected genes was confirmed by semiquantitative RT-PCR. Our results indicate that the plasticity between osteogenesis and adipogenesis extends into the differentiation pathways of both cell lineages and may contribute to the age-related expansion of adipose tissue in human bone marrow.

The transcriptional profile of mesenchymal stem cell populations in primary osteoporosis is distinct and shows overexpression of osteogenic inhibitors.

Primary osteoporosis is an age-related disease characterized by an imbalance in bone homeostasis. While the resorptive aspect of the disease has been studied intensely, less is known about the anabolic part of the syndrome or presumptive deficiencies in bone regeneration. Multipotent mesenchymal stem cells (MSC) are the primary source of osteogenic regeneration. In the present study we aimed to unravel whether MSC biology is directly involved in the pathophysiology of the disease and therefore performed microarray analyses of hMSC of elderly patients (79–94 years old) suffering from osteoporosis (hMSC-OP). In comparison to age-matched controls we detected profound changes in the transcriptome in hMSC-OP, e.g. enhanced mRNA expression of known osteoporosis-associated genes (LRP5, RUNX2, COL1A1) and of genes involved in osteoclastogenesis (CSF1, PTH1R), but most notably of genes coding for inhibitors of WNT and BMP signaling, such as Sclerostin and MAB21L2. These candidate genes indicate intrinsic deficiencies in self-renewal and differentiation potential in osteoporotic stem cells. We also compared both hMSC-OP and non-osteoporotic hMSC-old of elderly donors to hMSC of ∼30 years younger donors and found that the transcriptional changes acquired between the sixth and the ninth decade of life differed widely between osteoporotic and non-osteoporotic stem cells. In addition, we compared the osteoporotic transcriptome to long term-cultivated, senescent hMSC and detected some signs for pre-senescence in hMSC-OP. Our results suggest that in primary osteoporosis the transcriptomes of hMSC populations show distinct signatures and little overlap with non-osteoporotic aging, although we detected some hints for senescence-associated changes. While there are remarkable inter-individual variations as expected for polygenetic diseases, we could identify many susceptibility genes for osteoporosis known from genetic studies. We also found new candidates, e.g. MAB21L2, a novel repressor of BMP-induced transcription. Such transcriptional changes may reflect epigenetic changes, which are part of a specific osteoporosis-associated aging process.

Microarray analyses of transdifferentiated mesenchymal stem cells

The molecular events associated with the age‐related gain of fatty tissue in human bone marrow are still largely unknown. Besides enhanced adipogenic differentiation of mesenchymal stem cells (MSCs), transdifferentiation of osteoblast progenitors may contribute to bone‐related diseases like osteopenia. Transdifferentiation of MSC‐derived osteoblast progenitors into adipocytes and vice versa has previously been proven feasible in our cell culture system. Here, we focus on mRNA species that are regulated during transdifferentiation and represent possible control factors for the initiation of transdifferentiation. Microarray analyses comparing transdifferentiated cells with normally differentiated cells exhibited large numbers of reproducibly regulated genes for both, adipogenic and osteogenic transdifferentiation. To evaluate the relevance of individual genes, we designed a scoring scheme to rank genes according to reproducibility, regulation level, and reciprocity between the different transdifferentiation directions. Thereby, members of several signaling pathways like FGF, IGF, and Wnt signaling showed explicitly differential expression patterns. Additional bioinformatic analysis of microarray analyses allowed us to identify potential key factors associated with transdifferentiation of adipocytes and osteoblasts, respectively. Fibroblast growth factor 1 (FGF1) was scored as one of several lead candidate gene products to modulate the transdifferentiation process and is shown here to exert inhibitory effects on adipogenic commitment and differentiation. J. Cell. Biochem. 103: 413–433, 2008.

Functional Signature of Human Islet-Derived Precursor Cells Compared to Bone Marrow-Derived Mesenchymal Stem Cells

Pancreatic islet beta-cell replenishment can be driven by epithelial cells from exocrine pancreas via epithelial-mesenchymal transition (EMT) and the reverse process MET, while specified pancreatic mesenchymal cells control islet cell development and maintenance. The role of human islet-derived precursor cells (hIPCs) in regeneration and support of endocrine islets is under investigation. Here, we analyzed hIPCs as to their immunophenotype, multilineage differentiation capacity, and gene profiling, in comparison to human bone marrow-derived mesenchymal stem cells (hBM-MSCs). hIPCs and hBM-MSCs display a common mesenchymal character and express lineage-specific marker genes upon induction toward pancreatic endocrine and mesenchymal pathways of differentiation. hIPCs can go further along endocrine pathways while lacking some core mesenchymal differentiation attributes. Significance analysis of microarray (SAM) from 5 hBM-MSC and 3 hIPC donors mirrored such differences. Candidate gene cluster analysis disclosed differential expression of key lineage regulators, indicated a HoxA gene-associated positional memory in hIPCs and hBM-MSCs, and showed as well a clear transition state from mesenchyme to epithelium or vice versa in hIPCs. Our findings raise new research platforms to further clarify the potential of hIPCs to undergo complete MET thus contributing to islet cell replenishment, maintenance, and function.

Heparin affects human bone marrow stromal cell fate: Promoting osteogenic and reducing adipogenic differentiation and conversion

Heparins are broadly used for the prevention and treatment of thrombosis and embolism. Yet, osteoporosis is considered to be a severe side effect in up to one third of all patients on long-term treatment. However, the mechanisms underlying this clinical problem are only partially understood. To investigate if heparin affects differentiation of skeletal precursors, we examined the effects of heparin on the osteogenic and adipogenic lineage commitment and differentiation of primary human bone marrow stromal cells (hBMSCs). Due to the known inverse relationship between adipogenesis and osteogenesis and the capacity of pre-differentiated cells to convert into the respective other lineage, we also determined heparin effects on osteogenic conversion and adipogenic differentiation/conversion. Interestingly, heparin did not only significantly increase mRNA expression and enzyme activity of the osteogenic marker alkaline phosphatase (ALP), but it also promoted mineralization during osteogenic differentiation and conversion. Furthermore, the mRNA expression of the osteogenic marker bone morphogenic protein 4 (BMP4) was enhanced. In addition, heparin administration partly prevented adipogenic differentiation and conversion demonstrated by reduced lipid droplet formation along with a decreased expression of adipogenic markers. Moreover, luciferase reporter assays, inhibitor experiments and gene expression analyses revealed that heparin had putative permissive effects on osteogenic signaling via the BMP pathway and reduced the mRNA expression of the Wnt pathway inhibitors dickkopf 1 (DKK1) and sclerostin (SOST). Taken together, our data show a rather supportive than inhibitory effect of heparin on osteogenic hBMSC differentiation and conversion in vitro. Further studies will have to investigate the net effects of heparin administration on bone formation versus bone resorption in vivo to unravel the molecular mechanisms of heparin-associated osteoporosis and reconcile conflicting experimental data with clinical observations.

WISP 1 is an important survival factor in human mesenchymal stromal cells.

WNT-induced secreted protein 1 (WISP1/CCN4), a member of the CCN protein family, acts as a downstream factor of the canonical WNT signaling pathway. Its expression is known to affect proliferation and differentiation of human mesenchymal stromal cells (hMSCs), which are fundamental for the development and maintenance of the musculoskeletal system. Whereas a dysregulated, excessive expression of WISP1 often reflects its oncogenic potential via the inhibition of apoptosis, our study emphasizes the importance of WISP1 signaling for the survival of primary human cells. We have established the efficient and specific down-regulation of endogenous WISP1 transcripts by gene silencing in hMSCs and observed cell death as a consequence of WISP1 deficiency. This was confirmed by Annexin V staining for apoptotic cells. DNA microarray analyses of WISP1 down-regulated versus control samples revealed several clusters of differentially expressed genes important for apoptosis induction such as TNF-related apoptosis-inducing ligand 1 (TRAIL) and the corresponding apoptosis-inducing receptors TRAIL-R1 and -R2. An increased expression of TRAIL and its receptors TRAIL-R1 and -R2 in WISP1-deficient hMSCs was confirmed by immunocytofluorescence. Accordingly, WISP1 deficiency is likely to cause TRAIL-induced apoptosis. This is an important novel finding, which suggests that WISP1 is indispensable for the protection of healthy hMSCs against TRAIL-induced apoptosis.

Fibroblast growth factors 1 and 2 inhibit adipogenesis of human bone marrow stromal cells in 3D collagen gels

Multipotent human bone marrow stromal cells (hBMSCs) are the common progenitors of osteoblasts and adipocytes. A shift in hBMSC differentiation in favor of adipogenesis may contribute to the bone loss and marrow fat accumulation observed in aging and osteoporosis. Hence, the identification of factors modulating marrow adipogenesis is of great therapeutic interest. Fibroblast growth factors 1 (FGF1) and 2 (FGF2) play important roles in several cellular processes including differentiation. Their role in adipogenesis is, however, still unclear given the contradictory reports found in the literature. In this work, we investigated the effect of FGF signaling on hBMSC adipogenesis in a 3D collagen gel system to mimic the natural microenvironment. We successfully established adipogenic differentiation of hBMSC embedded in type I collagen gels. We found that exogenous FGF1 and FGF2 exerted an inhibitory effect on lipid droplet accumulation and gene expression of adipogenic markers, which was abolished by pharmacological blocking of FGF receptor (FGFR) signaling. FGF treatment also affected the expression of the matrix metalloproteinase 13 (MMP13) and the tissue inhibitor of metalloproteinases 1 (TIMP1), altering the MMP/TIMP balance, which modulates collagen processing and turnover. FGF1- and FGF2-mediated inhibition of differentiation was, however, not restricted to adipogenesis since FGF1 and FGF2 treatment also resulted in the inhibition of the osteogenic differentiation in collagen gels. We conclude that FGFR signaling inhibits the in vitro adipogenic commitment of hBMSCs, downregulating core differentiation markers and altering ECM composition.

Canonical FGFs Prevent Osteogenic Lineage Commitment and Differentiation of Human Bone Marrow Stromal Cells Via ERK1/2 Signaling.

Controlling the adipo‐osteogenic lineage decision of trabecular human bone marrow stromal cells (hBMSCs) in favor of osteogenesis represents a promising approach for osteoporosis therapy and prevention. Previously, Fibroblast Growth Factor 1 (FGF1) and its subfamily member FGF2 were scored as leading candidates to exercise control over skeletal precursor commitment and lineage decision albeit literature results are highly inconsistent. We show here that FGF1 and 2 strongly prevent the osteogenic commitment and differentiation of hBMSCs. Mineralization of extracellular matrix (ECM) and mRNA expression of osteogenic marker genes Alkaline Phosphatase (ALP), Collagen 1A1 (COL1A1), and Integrin‐Binding Sialoprotein (IBSP) were significantly reduced. Furthermore, master regulators of osteogenic commitment like Runt‐Related Transcription Factor 2 (RUNX2) and Bone Morphogenetic Protein 4 (BMP4) were downregulated. When administered under adipogenic culture conditions, canonical FGFs did not support osteogenic marker expression. Moreover despite the presence of osteogenic differentiation factors, FGFs even disabled the pro‐osteogenic lineage decision of pre‐differentiated adipocytic cells. In contrast to FGF Receptor 2 (FGFR2), FGFR1 was stably expressed throughout osteogenic and adipogenic differentiation and FGF addition. Moreover, FGFR1 and Extracellular Signal‐Regulated Kinases 1 and 2 (ERK1/2) were found to be responsible for underlying signal transduction using respective inhibitors. Taken together, we present new findings indicating that canonical FGFR‐ERK1/2 signaling entrapped hBMSCs in a pre‐committed state and arrested further maturation of committed precursors. Our results might aid in unraveling and controlling check points relevant for ageing‐associated aberrant adipogenesis with consequences for the treatment of degenerative diseases such as osteoporosis and for skeletal tissue engineering strategies. J. Cell. Biochem. 118: 263–275, 2017.

Influence of hormones on osteogenic differentiation processes of mesenchymal stem cells

Bone development, regeneration and maintenance are governed by osteogenic differentiation processes from mesenchymal stem cells through to mature bone cells, which are directed by local growth and differentiation factors and modulated strongly by hormones. Mesenchymal stem cells develop from both mesoderm and neural crest and can give rise to development, regeneration and maintenance of mesenchymal tissues, such as bone, cartilage, muscle, tendons and discs. There are only limited data regarding the effects of hormones on early events, such as regulation of stemness and maintenance of the mesenchymal stem cell pool. Hormones, such as estrogens, vitamin D-hormone and parathyroid hormone, besides others, are important modulators of osteogenic differentiation processes and bone formation, starting off with fate decision and the development of osteogenic offspring from mesenchymal stem cells, which end up in osteoblasts and osteocytes. Hormones are involved in fetal bone development and regeneration and, in childhood, adolescence and adulthood, they control adaptive needs for growth and reproduction, nutrition, physical power and crisis adaptation. As in other tissues, aging in mesenchymal stem cells and their osteogenic offspring is accompanied by the accumulation of genomic and proteomic damage caused by oxidative burden and insufficient repair. Failsafe programs, such as apoptosis and cellular senescence avoid tumorigenesis. Hormones can influence the pace of such events, thus supporting the quality of tissue regeneration in aging organisms in vivo; for example, by delaying osteoporosis development. The potential for hormones in systemic therapeutic strategies is well appreciated and some concepts are approved for clinical use already. Their potential for cell-based therapeutic strategies for tissue regeneration is probably underestimated and could enhance the quality of tissue-engineering constructs for transplantation and the concept of in situ-guided tissue regeneration.

Biology of Mesenchymal Stem Cells

Mesenchymal stem cells (MSC) are derived from mesodermal precursor and are committed towards mesenchymal differentiation. They are scattered all over the organism, situated in bone, cartilage, adipose tissue and accompany organs for tissue regeneration and structural and functional support. MSC populations are not homogenous, their signature is variable according to their localization. A process called “epithelial mesenchymal transition” is fundamental for the development of mesoderm. Epithelial-mesenchymal interactions specify MSC and this may influence their regeneration potential. Multipotent adult MSC are used for research in tissue regeneration and engineering. Crude mixtures of bone marrow- derived MSC are clinically applied for tissue healing, but complex transplantable tissue engineered constructs are still under development. The role and regeneration potential of MSC in inflammation and ageing organisms remains to be characterized. The establishment of reprogrammed homogenous MSC cultures of high plasticity might allow developing these cells towards multiple cell-based therapeutic strategies. Many applications can be envisioned, e.g. regeneration of bone, cartilage and tendon or engineering of beta cells and neurons. Since homogenous MSC with high plasticity represent a promising tool for the treatment of many diseases, research in this area of adult stem cells should be supported with high priority.