Agnieszka Arthur

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.

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.

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

Implanted Adult Human Dental Pulp Stem Cells Induce Endogenous Axon Guidance

The human central nervous system has limited capacity for regeneration. Stem cell‐based therapies may overcome this through cellular mechanisms of neural replacement and/or through molecular mechanisms, whereby secreted factors induce change in the host tissue. To investigate these mechanisms, we used a readily accessible human cell population, dental pulp progenitor/stem cells (DPSCs) that can differentiate into functionally active neurons given the appropriate environmental cues. We hypothesized that implanted DPSCs secrete factors that coordinate axon guidance within a receptive host nervous system. An avian embryonic model system was adapted to investigate axon guidance in vivo after transplantation of adult human DPSCs. Chemoattraction of avian trigeminal ganglion axons toward implanted DPSCs was mediated via the chemokine, CXCL12, also known as stromal cell‐derived factor‐1, and its receptor, CXCR4. These findings provide the first direct evidence that DPSCs may induce neuroplasticity within a receptive host nervous system. STEM CELLS 2009;27:2229–2237

Enrichment for STRO-1 expression enhances the cardiovascular paracrine activity of human bone marrow-derived mesenchymal cell populations.

The cardiovascular therapeutic potential of bone marrow mesenchymal stromal/stem cells (MSC) is largely mediated by paracrine effects. Traditional preparation of MSC has involved plastic adherence‐isolation. In contrast, prospective immunoselection aims to improve cell isolation by enriching for mesenchymal precursor cells (MPC) at higher purity. This study compared the biological characteristics and cardiovascular trophic activity of plastic adherence‐isolated MSC (PA‐MSC) and MPC prepared from the same human donors by immunoselection for stromal precursor antigen‐1 (STRO‐1). Compared to PA‐MSC, STRO‐1‐MPC displayed greater (1) clonogenicity, (2) proliferative capacity, (3) multilineage differentiation potential, and (4) mRNA expression of mesenchymal stem cell‐related transcripts. In vitro assays demonstrated that conditioned medium from STRO‐1‐MPC had greater paracrine activity than PA‐MSC, with respect to cardiac cell proliferation and migration and endothelial cell migration and tube formation. In keeping with this, STRO‐1‐MPC exhibited higher gene and protein expression of CXCL12 and HGF. Inhibition of these cytokines attenuated endothelial tube formation and cardiac cell proliferation, respectively. Paracrine responses were enhanced by using supernatant from STRO‐1Bright MPC and diminished with STRO‐1Dim conditioned medium. Together, these findings indicate that prospective isolation gives rise to mesenchymal progeny that maintain a higher proportion of immature precursor cells compared to traditional plastic adherence‐isolation. Enrichment for STRO‐1 is also accompanied by increased expression of cardiovascular‐relevant cytokines and enhanced trophic activity. Immunoselection thus provides a strategy for improving the cardiovascular reparative potential of mesenchymal cells. J. Cell. Physiol. 223: 530–540, 2010.

A method to isolate and culture expand human dental pulp stem cells.

Dentinal repair in teeth occurs through the activity of specialized cells known as odontoblasts that are thought to be maintained by a precursor population associated with the perivascular cells within dental pulp tissue. We have previously isolated candidate dental pulp stem cells (DPSC) from adult human third molars, with the ability to generate clonogenic cell clusters (CFU-F: colony-forming units-fibroblastic), a high proliferation rate, and multi-potential differentiation in vitro. When cultured DPSC are transplanted into immunocompromised mice, they generated a dentin-like structure lined with human odontoblast-like cells that surrounded a pulp-like interstitial tissue, composed of collagen and a vascular network. The present protocol describes a methodology to generate highly purified preparations of human DPSC. This process involves the enzymatic digestion of fresh samples of human dental pulp tissue followed by the isolation of DPSC using magnetic bead cell separation, based on their expression of mesenchymal stem cell associated markers.

EphB/ephrin-B interactions mediate human MSC attachment, migration and osteochondral differentiation☆ , ☆☆

Bone marrow derived mesenchymal stem/stromal cells (MSC) contribute to skeletal tissue formation and the regulation of haematopoiesis. The Eph/ephrin family of receptor tyrosine kinases is potentially important in the maintenance of the stem cell niche within neural, intestinal and dental tissues and has recently been shown to play a role in regulating bone homeostasis. However, the contribution of EphB/ephrin-B molecules in human MSC function remains to be determined. In the present study, EphB and ephrin-B molecules were expressed by ex vivo expanded human MSC populations and within human bone marrow trephine samples. To elucidate the contribution of EphB/ephrin-B molecules in MSC recruitment, we performed functional spreading and migration assays and showed that reverse ephrin-B signalling inhibited MSC attachment and spreading by activating Src-, PI3Kinase- and JNK-dependent signalling pathways. In contrast, forward EphB2 signalling promoted MSC migration by activating the Src kinase- and Abl-dependent signalling pathways. Furthermore, activation of ephrin-B1 and/or ephrin-B2 molecules expressed by MSC was found to increase osteogenic differentiation, while ephrin-B1 activation promoted chondrogenic differentiation. These observations suggest that EphB/ephrin-B interactions may mediate the recruitment, migration and differentiation of MSC during bone repair.

EphB4 enhances the process of endochondral ossification and inhibits remodeling during bone fracture repair

Previous reports have identified a role for the tyrosine kinase receptor EphB4 and its ligand, ephrinB2, as potential mediators of both bone formation by osteoblasts and bone resorption by osteoclasts. In the present study, we examined the role of EphB4 during bone repair after traumatic injury. We performed femoral fractures with internal fixation in transgenic mice that overexpress EphB4 under the collagen type 1 promoter (Col1‐EphB4) and investigated the bone repair process up to 12 weeks postfracture. The data indicated that Col1‐EphB4 mice exhibited stiffer and stronger bones after fracture compared with wild‐type mice. The fractured bones of Col1‐EphB4 transgenic mice displayed significantly greater tissue and bone volume 2 weeks postfracture compared with that of wild‐type mice. These findings correlated with increased chondrogenesis and mineral formation within the callus site at 2 weeks postfracture, as demonstrated by increased safranin O and von Kossa staining, respectively. Interestingly, Col1‐EphB4 mice were found to possess significantly greater numbers of clonogenic mesenchymal stromal progenitor cells (CFU‐F), with an increased capacity to form mineralized nodules in vitro under osteogenic conditions, when compared with those of the wild‐type control mice. Furthermore, Col1‐EphB4 mice had significantly lower numbers of TRAP‐positive multinucleated osteoclasts within the callus site. Taken together, these observations suggest that EphB4 promotes endochondral ossification while inhibiting osteoclast development during callus formation and may represent a novel drug target for the repair of fractured bones.

Eph/ephrinB Mediate Dental Pulp Stem Cell Mobilization and Function

Damage to the dentin matrix instigates the proliferation and mobilization of dental progenitor cells to the injury site, the mechanisms of which are not defined. EphB receptors and ephrin-B ligands expressed within the perivascular niche of dental pulp have been implicated following tooth injury. We propose that elevated levels of ephrin-B1 following injury may prevent the proliferation and migration of dental pulp stem cell (DPSC), while EphB/ephrin-B interaction facilitates odontoblastic differentiation. The migration, proliferation, and differentiation of DPSC in response to Eph/ephrin-B molecules was assessed in an established ex vivo tooth injury model and by in vitro assays for the assessment of colony formation and differentiation. Analysis of our data demonstrated that EphB forward signaling promoted DPSC proliferation, while inhibiting migration. Conversely, reverse signaling enhanced DPSC mineral production. These observations suggest that EphB/ephrin-B molecules are important for perivascular DPSC migration toward the dentin surfaces and differentiation into functional odontoblasts, following damage to the dentin matrix.

EphB4 expressing stromal cells exhibit an enhanced capacity for hematopoietic stem cell maintenance

The tyrosine kinase receptor, EphB4, mediates cross‐talk between stromal and hematopoietic populations during bone remodeling, fracture repair and arthritis, through its interactions with the ligand, ephrin‐B2. This study demonstrated that transgenic EphB4 mice (EphB4 Tg), over‐expressing EphB4 under the control of collagen type‐1 promoter, exhibited higher frequencies of osteogenic cells and hematopoietic stem/progenitor cells (HSC), correlating with a higher frequency of long‐term culture‐initiating cells (LTC‐IC), compared with wild type (WT) mice. EphB4 Tg stromal feeder layers displayed a greater capacity to support LTC‐IC in vitro, where blocking EphB4/ephrin‐B2 interactions decreased LTC‐IC output. Similarly, short hairpin RNA‐mediated EphB4 knockdown in human bone marrow stromal cells reduced their ability to support high ephrin‐B2 expressing CD34+ HSC in LTC‐IC cultures. Notably, irradiated EphB4 Tg mouse recipients displayed enhanced bone marrow reconstitution capacity and enhanced homing efficiency of transplanted donor hematopoietic stem/progenitor cells relative to WT controls. Studies examining the expression of hematopoietic supportive factors produced by stromal cells indicated that CXCL12, Angiopoietin‐1, IL‐6, FLT‐3 ligand, and osteopontin expression were more highly expressed in EphB4 Tg stromal cells compared with WT controls. These findings indicate that EphB4 facilitates stromal‐mediated support of hematopoiesis, and constitute a novel component of the HSC niche. Stem Cells 2015;33:2838—2849