The role of post-natal skeletal stem cells in health and disease

Dental & Craniofacial Research

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Abstract

G-protein coupled receptors on human BMSCs[unreadable] [unreadable] Cell signaling is mediated by a large number of extracellular agents and receptors that respond to them. Among these receptors are those that interact with intracellular signal cascades via trimeric G-proteins. These G-protein coupled receptors (GPCRs) are involved in numerous physiological and pathological processes. Approximately 30% of all drugs in use today modulate GPCR activity. Since these drugs target only about 10% of all GPCRs; it seems likely that agonists or antagonists of additional receptors will prove beneficial in the future. However, these receptors are encoded by scarce mRNAs. Therefore, detecting their transcripts with conventional microarrays has been difficult, if not impossible. We designed a method based on multiplex PCR and array detection of amplicons to assay GPCR gene expression using as little as 1mg of total RNA. As an example of the power of the method, we profiled human bone marrow stromal cells (BMSCs). Among the 162 receptor mRNAs detected in these cells, some were reported previously; but most had not been seen (or sought) before. The parathyroid hormone 1 (PTHR1) and prostaglandin E2 (PTGER4) receptors, both of which were expressed at low levels, stimulate osteoblastic commitment. The opposite is true of FZD1, the message for which was abundant. Stimulation of this receptor with Wnt3a increases cell proliferation and suppresses osteogenesis. Other GPCR mRNAs detected in BMSCs have been described in their progeny: osteoblasts, adipocytes or chondrocytes. The calcitonin (CALCRL) and oxytocin (OXTR) receptors, relatively abundant transcripts, stimulate osteogenic proliferation; and the proton sensing receptors (GPR68, GPR132), which have recently been discovered on osteoblasts, may play a role in pH homeostasis. GPR41 and GPR109a mRNAs, both moderately abundant, are expressed in adipose tissue. The former stimulates adipogenesis and leptin production; the latter, is lipolytic. FZD7, a relatively abundant transcript, is activated by Wnt5a and regulates chondrocyte proliferation and differentiation. We also detected a large number of receptor mRNAs, several of them still orphans, which have not been reported previously. Studies of these receptors should reveal more about the signals to which BMSCs respond and the nature of their responses. [unreadable] [unreadable] Circulating connective tissue stem cells[unreadable] [unreadable] Using a variety of cell separation techniques and cultivation conditions, we sought to extend our previous studies to further characterize adherent, clonogenic cells connective tissue cells in peripheral blood from humans and guinea pigs. Circulating connective tissue stem cells capable of forming bone upon in vivo transplantation were found in only 3 donors out of 66, including patients with multiple fractures, demonstrating that these precursors are extremely rare in postnatal human blood. Unlike in humans, guinea pig blood shows much more reproducible connective tissue colony formation. Consequently, we studied the differentiation potential of guinea pig adherent blood-derived clonogenic cells. Out of 22 single colony-derived strains of various morphologies, only 5 spindle-shaped strains showed extensive proliferative capacity in vitro. None of these strains formed bone upon in vivo transplantation, whereas two strains formed cartilage in high-density pellet cultures in vitro. Both chondrogenic strains included cells expressing the cartilage specific protein, aggrecan, while non-chondrogenic strains did not. Out of 4 polyclonal strains studied, one formed both cartilage and abundant bone accompanied by hematopoiesis-supporting stroma. Evidently, there are cells in adult guinea pig blood capable of both extensive proliferation and differentiation towards cartilage: circulating chondrogenic precursors. While some of these cells lack osteogenic potential and therefore represent committed chondrogenic precursors, others may be multipotential and consequently can be classified as skeletal stem cells. This is the first demonstration of postnatal circulating chondrogenic precursors, as well as of precursor cells with chondrogenic but not osteogenic potential. [unreadable] [unreadable] Imprinting of GNAS transcripts in normal and fibrous dysplastic skeletal progenitors [unreadable] [unreadable] At a time when significant attention is devoted worldwide to stem cells as a potential tool for curing incurable diseases, Fibrous Dysplasia of bone (FD)/McCune-Albright Syndrome (MAS) provides a paradigm of stem cell disease. Caused by activating missense mutations in the GNAS gene coding for Gs alpha that are not transmitted via the germline, the disease reflects, in its most common multi-organ expressions, the dysfunction of tissues emanating from the three different germ layers: ectoderm (skin hyperpigmentation, craniofacial FD), mesoderm (axial and appendicular FD) and endoderm (hyperfunctioning endocrine systems). In the vast majority of clinical cases of FD, and in all cases MAS, a pluripotent, pre-gastrulation cell (that is, a cell within the inner cell mass) generates the mutated clone that distributes to different organs and tissues. In this cell, the mutation arises in conjunction with specific, early embryonic, epigenetic events. Bone disease in the post-natal life, in turn, emanates from the dysfunction of post-natal skeletal stem cells, which generate dysfunctional osteoblasts. [unreadable] [unreadable] Epigenetic changes such as imprinting (transcription from either the paternal or maternal allele of a gene), involve chromatin and DNA modifications (methylation and demethylation) that do not involve changes in the underlying DNA sequence. Of note, GNAS is a complex, imprinted gene, with at least four different transcripts, two of which are expressed exclusively from the paternal allele (XLas, exon 1A), one of which is expressed from the maternal allele (NESP55), and one that can be monoallelic or biallelic (Gs alpha). [unreadable] [unreadable] Patients with FD/MAS are extremely heterogeneous in their pattern of pathological phenotypes. This clinical heterogeneity is assumed to reflect the post-zygotic origin of the mutation. However, the pattern of imprinting of Gs alpha in some human post-natal tissues suggests that parental-dependent epigenetic mechanisms may also play a role in the phenotypic effect of the mutated GNAS genotype. FD lesions are generated by mutated clonogenic osteoprogenitors that reside, along with their normal counterparts, in FD bone marrow stroma. We analyzed the allelic expression pattern of Gs alpha and the other GNAS alternative transcripts in the progeny of normal and mutated clonogenic stromal cells isolated in vitro from a series of informative FD/MAS patients. We found, for the first time, that the two Gsa alleles are unequally expressed in both normal and FD-mutated stromal clones. However, in contrast to imprinting, the ratio of Gs alpha allelic expression is randomly established in different clones from the same patient. This result suggests that a parental-independent modulation of Gs alpha expression occurs in clonogenic osteoprogenitor cells, and at the single cell level, may impact on the severity of an FD lesion. Furthermore, we showed that normal and mutated clonogenic stromal cells express the other GNAS alternative transcripts in addition to the common Gs alpha, some of which may be relevant to the development of FD. [unreadable] [unreadable] Human embryonic stem cells[unreadable] [unreadable] We continue to gain expertise in routinely growing HSF6 cells. These cells have been cultured in a number of different ways to induce their differentiation into mesoderm and into osteogenic tissues. Such cells have been transplanted in vivo using a number of different scaffolds to determine the efficacy of the differentiation conditions that have been utilized.