Alessia Funari

No Identical "Mesenchymal Stem Cells" at Different Times and Sites: Human Committed Progenitors of Distinct Origin and Differentiation Potential Are Incorporated as Adventitial Cells in Microvessels

Summary A widely shared view reads that mesenchymal stem/stromal cells (“MSCs”) are ubiquitous in human connective tissues, can be defined by a common in vitro phenotype, share a skeletogenic potential as assessed by in vitro differentiation assays, and coincide with ubiquitous pericytes. Using stringent in vivo differentiation assays and transcriptome analysis, we show that human cell populations from different anatomical sources, regarded as “MSCs” based on these criteria and assumptions, actually differ widely in their transcriptomic signature and in vivo differentiation potential. In contrast, they share the capacity to guide the assembly of functional microvessels in vivo, regardless of their anatomical source, or in situ identity as perivascular or circulating cells. This analysis reveals that muscle pericytes, which are not spontaneously osteochondrogenic as previously claimed, may indeed coincide with an ectopic perivascular subset of committed myogenic cells similar to satellite cells. Cord blood-derived stromal cells, on the other hand, display the unique capacity to form cartilage in vivo spontaneously, in addition to an assayable osteogenic capacity. These data suggest the need to revise current misconceptions on the origin and function of so-called “MSCs,” with important applicative implications. The data also support the view that rather than a uniform class of “MSCs,” different mesoderm derivatives include distinct classes of tissue-specific committed progenitors, possibly of different developmental origin.

Transfer, analysis, and reversion of the fibrous dysplasia cellular phenotype in human skeletal progenitors

Human skeletal progenitors were engineered to stably express R201C mutated, constitutively active Gsα using lentiviral vectors. Long‐term transduced skeletal progenitors were characterized by an enhanced production of cAMP, indicating the transfer of the fundamental cellular phenotype caused by activating mutations of Gsα. Like skeletal progenitors isolated from natural fibrous dysplasia (FD) lesions, transduced cells could generate bone but not adipocytes or the hematopoietic microenvironment on in vivo transplantation. In vitro osteogenic differentiation was noted for the lack of mineral deposition, a blunted upregulation of osteocalcin, and enhanced upregulation of other osteogenic markers such as alkaline phosphatase (ALP) and bone sialoprotein (BSP) compared with controls. A very potent upregulation of RANKL expression was observed, which correlates with the pronounced osteoclastogenesis observed in FD lesions in vivo. Stable transduction resulted in a marked upregulation of selected phosphodiesterase (PDE) isoform mRNAs and a prominent increase in total PDE activity. This predicts an adaptive response in skeletal progenitors transduced with constitutively active, mutated Gsα. Indeed, like measurable cAMP levels, the differentiative responses of transduced skeletal progenitors were profoundly affected by inhibition of PDEs or lack thereof. Finally, using lentiviral vectors encoding short hairpin (sh) RNA interfering sequences, we demonstrated that selective silencing of the mutated allele is both feasible and effective in reverting the aberrant cAMP production brought about by the constitutively active Gsα and some of its effects on in vitro differentiation of skeletal progenitors.

In Vivo Osteoprogenitor Potency of Human Stromal Cells from Different Tissues Does Not Correlate with Expression of POU5F1 or Its Pseudogenes

Expression of “stemness” markers is widely used as a predictor of stem cell properties of mesenchymal stem cells (MSC). Here, we show that bone marrow‐derived (BM)‐MSC show stem cell‐like behavior in vivo; that is, they form ossicles with formation of bone, formation of adipocytes, and establishment of the murine hematopoietic microenvironment. Multipotent umbilical vein‐derived stromal cells (UVSC), on the other hand, do not form bone, nor do they give rise to adipocytes in vivo. Despite these differences in stem‐cell‐like behavior, BM‐MSC and UVSC express the two transcripts variants of POU5F1 at a similar level. Also, we found that in BM‐MSC and UVSC, POU5F1 is detectable. However, more than 89% of the POU5F1 transcripts correspond to the POU5F1P1, ‐P3, or ‐P4 pseudogene. Despite low‐level expression of POU5F1, we were unable to precipitate POU5F1 protein in either BM‐MSC or UVSC. These results demonstrate that MSC stemness does not correlate to expression of POU5F1 transcripts or its pseudogenes.

Lentiviral Transduction of Human Postnatal Skeletal (Stromal, Mesenchymal) Stem Cells: In Vivo Transplantation and Gene Silencing

Systems for gene transfer and silencing in human skeletal stem cells (hSSCs, also stromal or mesenchymal stem cells) are important for addressing critical issues in basic hSSC and skeletal biology and for developing gene therapy strategies for treatment of skeletal diseases. Whereas recent studies have shown the efficacy of lentiviral transduction for gene transfer in hSSCs in vitro, no study has yet proven that lentivector-transduced hSSCs retain their distinctive organogenic potential in vivo, as probed by in vivo transplantation assays. Therefore, in addition to analyzing the in vitro growth and differentiation properties of hSSCs transduced with advanced-generation lentivectors, we ectopically transplanted LV-eGFP-transduced hSSCs (along with an osteoconductive carrier) in the subcutaneous tissue of immunocompromised mice. eGFP-transduced cells formed heterotopic ossicles, generating osteoblasts, osteocytes, and stromal cells in vivo, which still expressed GFP at 2 months after transplantation. eGFP-expressing cells could be recovered from the ossicles 8 weeks posttransplantation and reestablished in culture as viable and proliferating cells. Further, we investigated the possibility of silencing individual genes in hSSCs using lentivectors encoding short hairpin precursors of RNA interfering sequences under the control of the Pol-III-dependent H1 promoter. Significant long-term silencing of both lamin A/C and GFP (an endogenous gene and a transgene, respectively) was obtained with lentivectors encoding shRNAs. These data provide the basis for analysis of the effect of gene knockdown during the organogenesis of bone in the in vivo transplantation system and for further studies on the silencing of alleles carrying dominant, disease-causing mutations.

Evaluation of the osteoconductive potential of bone substitutes embedded with schneiderian membrane‐ or maxillary bone marrow‐derived osteoprogenitor cells

AIM Sinus augmentation procedures commonly employ osteoconductive scaffolding materials to stimulate and support bone formation. The aim of this study was to develop a simple screening methodology for the evaluation of the osteoconductive potential of various bone graft materials prior to clinical use. MATERIALS AND METHODS Materials tested were Bio-Oss, Bi-Ostetic, OraGraft, and ProOsteon. These Simple and composite bone substitutes were embedded with osteoprogenitor cells derived from either the human maxillary sinus schneiderian membrane (hMSSM) or from maxillary tuberosity bone marrow and then monitored both in vitro and in vivo. RESULTS Cell adherence and proliferation was most pronounced in OraGraft, followed by ProOsteon. In vivo bone formation, within the bone graft, was also observed, with most marked results in OraGraft and ProOsteon grafts. CONCLUSIONS The proposed osteoconductivity testing method proved simple, informative, and reliable for the purpose of screening candidate biomaterials for sinus lifting or sinus augmentation.

Clonogenic, myogenic progenitors expressing MCAM/CD146 are incorporated as adventitial reticular cells in the microvascular compartment of human post-natal skeletal muscle

Recent observation identifies subendothelial (mural) cells expressing MCAM, a specific system of clonogenic, self-renewing, osteoprogenitors (a.k.a, “mesenchymal stem cells”) in the microvascular compartment of post-natal human bone marrow (BM). In this study, we used MCAM/CD146, as a marker to localize, isolate and assay subendothelial clonogenic cells from the microvasculature of postnatal human skeletal muscle. We show here that these cells share with their BM counterpart, anatomic position (subendothelial/adventitial) and ex vivo clonogenicity (CFU-Fs). When assayed under the stringent conditions, these cells display a high spontaneous myogenic potential (independent of co-culture with myoblasts or of in vivo fusion with local myoblasts), which is otherwise only attained in cultures of satellite cells. These muscle-derived mural cells activated a myogenic program in culture. Cultured CD146+ cells expressed the myogenic factors (Pax7, Pax3 and Myf5), NCAM/CD56, desmin as well as proteins characteristic of more advanced myogenic differentiation, such as myosin heavy chain. In vivo, these cells spontaneously generate myotubes and myofibrils. These data identify the anatomy and phenotype of a novel class of committed myogenic progenitor in human post-natal skeletal muscle of subendothelial cells associated with the abluminal surface of microvascular compartment distinct from satellite cells.