Andrew C.W. Zannettino

Molecular and cellular characterisation of highly purified stromal stem cells derived from human bone marrow

Previous studies have provided evidence for the existence of adult human bone marrow stromal stem cells (BMSSCs) or mesenchymal stem cells. Using a combination of cell separation techniques, we have isolated an almost homogeneous population of BMSSCs from adult human bone marrow. Lacking phenotypic characteristics of leukocytes and mature stromal elements, BMSSCs are non-cycling and constitutively express telomerase activity in vivo. This mesenchymal stem cell population demonstrates extensive proliferation and retains the capacity for differentiation into bone, cartilage and adipose tissue in vitro. In addition, clonal analysis demonstrated that individual BMSSC colonies exhibit a differential capacity to form new bone in vivo. These data are consistent with the existence of a second population of bone marrow stem cells in addition to those for the hematopoietic system. Our novel selection protocol provides a means to generate purified populations of BMSSCs for use in a range of different tissue engineering and gene therapy strategies.

Multipotential human adipose-derived stromal stem cells exhibit a perivascular phenotype in vitro and in vivo.

Mesenchymal stem‐like cells identified in different tissues reside in a perivascular niche. In the present study, we investigated the putative niche of adipose‐derived stromal/stem cells (ASCs) using markers, associated with mesenchymal and perivascular cells, including STRO‐1, CD146, and 3G5. Immunofluorescence staining of human adipose tissue sections, revealed that STRO‐1 and 3G5 co‐localized with CD146 to the perivascular regions of blood vessels. FACS was used to determine the capacity of the CD146, 3G5, and STRO‐1 specific monoclonal antibodies to isolate clonogenic ASCs from disassociated human adipose tissue. Clonogenic fibroblastic colonies (CFU‐F) were found to be enriched in those cell fractions selected with either STRO‐1, CD146, or 3G5. Flow cytometric analysis revealed that cultured ASCs exhibited similar phenotypic profiles in relation to their expression of cell surface markers associated with stromal cells (CD44, CD90, CD105, CD106, CD146, CD166, STRO‐1, alkaline phosphatase), endothelial cells (CD31, CD105, CD106, CD146, CD166), haematopoietic cells (CD14, CD31, CD45), and perivascular cells (3G5, STRO‐1, CD146). The immunoselected ASCs populations maintained their characteristic multipotential properties as shown by their capacity to form Alizarin Red positive mineralized deposits, Oil Red O positive lipid droplets, and Alcian Blue positive proteoglycan‐rich matrix in vitro. Furthermore, ASCs cultures established from either STRO‐1, 3G5, or CD146 selected cell populations, were all capable of forming ectopic bone when transplanted subcutaneously into NOD/SCID mice. The findings presented here, describe a multipotential stem cell population within adult human adipose tissue, which appear to be intimately associated with perivascular cells surrounding the blood vessels. J. Cell. Physiol. 214: 413–421, 2008.

Differential Cell Surface Expression of the STRO-1 and Alkaline Phosphatase Antigens on Discrete Developmental Stages in Primary Cultures of Human Bone Cells

Human osteoblast‐like cells can be readily cultured from explants of trabecular bone, reproducibly expressing the characteristics of cells belonging to the osteoblastic lineage. Dual‐color fluorescence‐activated cell sorting was employed to develop a model of bone cell development in primary cultures of normal human bone cells (NHBCs) based on the cell surface expression of the stromal precursor cell marker STRO‐1 and the osteoblastic marker alkaline phosphatase (ALP). Cells expressing the STRO‐1 antigen exclusively (STRO‐1+/ALP−), were found to exhibit qualities preosteoblastic in nature both functionally by their reduced ability to form a mineralized bone matrix over time, as measured by calcium release assay, and in the lack of their expression of various bone‐related markers including bone sialoprotein, osteopontin, and parathyroid hormone receptor based on reverse trancriptase polymerase chain reaction (PCR) analysis. The majority of the NHBCs which expressed the STRO‐1−/ALP+ and STRO‐1−/ALP− phenotypes appeared to represent fully differentiated osteoblasts, while the STRO‐1+/ALP+ subset represented an intermediate preosteoblastic stage of development. All STRO‐1/ALP NHBC subsets were also found to express the DNA‐binding transcription factor CBFA‐1, confirming that these cultures represent committed osteogenic cells. In addition, our primer sets yielded four distinct alternative splice variants of the expected PCR product for CBFA‐1 in each of the STRO‐1/ALP subsets, with the exception of the proposed preosteoblastic STRO‐1+/ALP− subpopulation. Furthermore, upon re‐culture of the four different STRO‐1/ALP subsets only the STRO‐1+/ALP− subpopulation was able to give rise to all of the four subsets yielding the same proportions of STRO‐1/ALP expression as in the original primary cultures. The data presented in this study demonstrate a hierarchy of bone cell development in vitro and facilitate the study of bone cell differentiation and function.

THE THERAPEUTIC APPLICATIONS OF MULTIPOTENTIAL MESENCHYMAL/STROMAL STEM CELLS IN SKELETAL TISSUE REPAIR

Four decades after the first isolation and characterization of clonogenic bone marrow stromal cells or mesenchymal stem cells (MSC) in the laboratory of Dr. Alexander Friedenstien, the therapeutic application of their progeny following ex vivo expansion are only now starting to be realized in the clinic. The multipotency, paracrine effects, and immune‐modulatory properties of MSC present them as an ideal stem cell candidate for tissue engineering and regenerative medicine. In recent years it has come to light that MSC encompass plasticity that extends beyond the conventional bone, adipose, cartilage, and other skeletal structures, and has expanded to the differentiation of liver, kidney, muscle, skin, neural, and cardiac cell lineages. This review will specifically focus on the skeletal regenerative capacity of bone marrow derived MSC alone or in combination with growth factors, biocompatible scaffolds, and following genetic modification. J. Cell. Physiol. 218: 237–245, 2009.

Concise Review: Mesenchymal Stromal Cells: Potential for Cardiovascular Repair

Cellular therapy for cardiovascular disease heralds an exciting frontier of research. Mesenchymal stromal cells (MSCs) are present in adult tissues, including bone marrow and adipose, from which they can be easily isolated and cultured ex vivo. Although traditional isolation of these cells by plastic adherence results in a heterogeneous composite of mature and immature cell types, MSCs do possess plasticity of differentiation and under appropriate in vitro culture conditions can be modified to adopt cardiomyocyte and vascular cell phenotypic characteristics. In vivo preclinical studies have demonstrated their capacity to facilitate both myocardial repair and neovascularization in models of cardiac injury. The mechanisms underlying these effects appear to be mediated predominantly through indirect paracrine actions, rather than direct regeneration of endogenous cells by transdifferentiation, especially because current transplantation strategies achieve only modest engraftment of cells in the host myocardium. Currently, published clinical trial experience of MSCs as cardiac therapy is limited, and the outcomes of ongoing studies are keenly anticipated. Of relevance to clinical application is the fact that MSCs are relatively immunoprivileged, potentially enabling their allogeneic therapeutic use, although this too requires further investigation. Overall, MSCs are an attractive adult‐derived cell population for cardiovascular repair; however, research is still required at both basic and clinical levels to resolve critical areas of uncertainty and to ensure continued development in cell culture engineering and cell transplantation technology.

RANKL expression is related to the differentiation state of human osteoblasts

Human osteoblast phenotypes that support osteoclast differentiation and bone formation are not well characterized. Osteoblast differentiation markers were examined in relation to RANKL expression. RANKL expression was induced preferentially in immature cells. These results support an important link between diverse osteoblast functions.

Positioning of bone marrow hematopoietic and stromal cells relative to blood flow in vivo: serially reconstituting hematopoietic stem cells reside in distinct nonperfused niches

Hematopoietic stem cell (HSC) niches have been reported at the endosteum or adjacent to bone marrow (BM) vasculature. To investigate functional attributes of these niches, mice were perfused with Hoechst 33342 (Ho) in vivo before BM cell collection in presence of pump inhibitors and antibody stained. We report that the position of phenotypic HSCs, multipotent and myeloid progenitors relative to blood flow, follows a hierarchy reflecting differentiation stage, whereas mesenchymal stromal cells are perivascular. Furthermore, during granulocyte colony-stimulating factor-induced mobilization, HSCs migrated closer to blood flow, whereas stromal cells did not. Interestingly, phenotypic Lin(-)Sca1(+)KIT(+)CD41(-)CD48(-)CD150(+) HSCs segregated into 2 groups (Ho(neg) or Ho(med)), based on degree of blood/Ho perfusion of their niche. HSCs capable of serial transplantation and long-term bromodeoxyuridine label retention were enriched in Ho(neg) HSCs, whereas Ho(med) HSCs cycled more frequently and only reconstituted a single host. This suggests that the most potent HSC niches are enriched in locally secreted factors and low oxygen tension due to negligible blood flow. Importantly, blood perfusion of niches correlates better with HSC function than absolute distance from vasculature. This technique enables prospective isolation of serially reconstituting HSCs distinct from other less potent HSCs of the same phenotype, based on the in vivo niche in which they reside.

Elevated Serum Levels of Stromal-Derived Factor-1α Are Associated with Increased Osteoclast Activity and Osteolytic Bone Disease in Multiple Myeloma Patients

Multiple myeloma (MM) is an incurable plasma cell (PC) malignancy able to mediate massive destruction of the axial and craniofacial skeleton. The aim of this study was to investigate the role of the potent chemokine, stromal-derived factor-1α (SDF-1α) in the recruitment of osteoclast precursors to the bone marrow. Our studies show that MM PC produce significant levels of SDF-1α protein and exhibit elevated plasma levels of SDF-1α when compared with normal, age-matched subjects. The level of SDF-1α positively correlated with the presence of multiple radiological bone lesions in individuals with MM, suggesting a potential role for SDF-1α in osteoclast precursor recruitment and activation. To examine this further, peripheral blood–derived CD14+ osteoclast precursors were cultured in an in vitro osteoclast-potentiating culture system in the presence of recombinant human SDF-1α. Although failing to stimulate an increase in TRAP+, multinucleated osteoclast formation, our studies show that SDF-1α mediated a dramatic increase in both the number and the size of the resorption lacunae formed. The increased osteoclast motility and activation in response to SDF-1α was associated with an increase in the expression of a number of osteoclast activation–related genes, including RANKL, RANK, TRAP, MMP-9, CA-II, and Cathepsin K. Importantly, the small-molecule CXCR4-specific inhibitor, 4F-Benzoyl-TE14011 (T140), effectively blocked osteoclast formation stimulated by the myeloma cell line, RPMI-8226. Based on these findings, we believe that the synthesis of high levels of SDF-1α by MM PC may serve to recruit osteoclast precursors to local sites within the bone marrow and enhance their motility and bone-resorbing activity. Therefore, we propose that inhibition of the CXCR4-SDF-1α axis may provide an effective means of treatment for MM-induced osteolysis.

TWIST Family of Basic Helix‐Loop‐Helix Transcription Factors Mediate Human Mesenchymal Stem Cell Growth and Commitment

The TWIST family of basic helix‐loop‐helix transcription factors, Twist‐1 and Dermo‐1 are known mediators of mesodermal tissue development and contribute to correct patterning of the skeleton. In this study, we demonstrate that freshly purified human bone marrow‐derived mesenchymal stromal/stem cells (MSC) express high levels of Twist‐1 and Dermo‐1 which are downregulated following ex vivo expansion. Enforced expression of Twist‐1 or Dermo‐1 in human MSC cultures increased expression of the MSC marker, STRO‐1, and the early osteogenic transcription factors, Runx2 and Msx2. Conversely, overexpression of Twist‐1 and Dermo‐1 was associated with a decrease in the gene expression of osteoblast‐associated markers, bone morphogenic protein‐2, bone sialoprotein, osteopontin, alkaline phosphatase and osteocalcin. High expressing Twist‐1 or Dermo‐1 MSC lines exhibited an enhanced proliferative potential of approximately 2.5‐fold compared with control MSC populations that were associated with elevated levels of Id‐1 and Id‐2 gene expression. Functional studies demonstrated that high expressing Twist‐1 and Dermo‐1 MSC displayed a decreased capacity for osteo/chondrogenic differentiation and an enhanced capacity to undergo adipogenesis. These findings implicate the TWIST gene family members as potential mediators of MSC self‐renewal and lineage commitment in postnatal skeletal tissues by exerting their effects on genes involved in the early stages of bone development. STEM CELLS 2009;27:2457–2468

A role for pericytes as microenvironmental regulators of human skin tissue regeneration

The cellular and molecular microenvironment of epithelial stem and progenitor cells is poorly characterized despite well-documented roles in homeostatic tissue renewal, wound healing, and cancer progression. Here, we demonstrate that, in organotypic cocultures, dermal pericytes substantially enhanced the intrinsically low tissue-regenerative capacity of human epidermal cells that have committed to differentiate and that this enhancement was independent of angiogenesis. We used microarray analysis to identify genes expressed by human dermal pericytes that could potentially promote epidermal regeneration. Using this approach, we identified as a candidate the gene LAMA5, which encodes laminin alpha5, a subunit of the ECM component laminin-511/521 (LM-511/521). LAMA5 was of particular interest as we had previously shown that it promotes skin regeneration both in vitro and in vivo. Analysis using immunogold localization revealed that pericytes synthesized and secreted LAMA5 in human skin. Consistent with this observation, coculture with pericytes enhanced LM-511/521 deposition in the dermal-epidermal junction of organotypic cultures. We further showed that skin pericytes could also act as mesenchymal stem cells, exhibiting the capacity to differentiate into bone, fat, and cartilage lineages in vitro. This study suggests that pericytes represent a potent stem cell population in the skin that is capable of modifying the ECM microenvironment and promoting epidermal tissue renewal from non-stem cells, a previously unsuspected role for pericytes.