ZIC1 Dictates Osteogenesis Versus Adipogenesis in Human Mesenchymal Progenitor Cells Via a Hedgehog Dependent Mechanism.

Numerous intrinsic factors regulate mesenchymal progenitor commitment to a specific cell fate, such as osteogenic or adipogenic lineages. Identification and modulation of novel intrinsic regulatory factors represent an opportunity to harness the regenerative potential of mesenchymal progenitors. In the present study, the transcription factor (TF) ZIC1 was identified to be differentially expressed among adipose compared with skeletal-derived mesenchymal progenitor cells. We observed that ZIC1 overexpression in human mesenchymal progenitors promotes osteogenesis and prevents adipogenesis. ZIC1 knockdown demonstrated the converse effects on cell differentiation. ZIC1 misexpression was associated with altered Hedgehog signaling, and the Hedgehog antagonist cyclopamine reversed the osteo/adipogenic differentiation alterations associated with ZIC1 overexpression. Finally, human mesenchymal progenitor cells with or without ZIC1 overexpression were implanted in an ossicle assay in NOD-SCID gamma mice. ZIC1 overexpression led to significantly increased ossicle formation in comparison to the control, as assessed by radiographic and histologic measures. Together, these data suggest that ZIC1 represents a TF at the center of osteo/adipogenic cell fate determinations-findings that have relevance in the fields of stem cell biology and therapeutic regenerative medicine.

Bone-forming perivascular cells: Cellular heterogeneity and use for tissue repair.

Mesenchymal progenitor cells are broadly distributed across perivascular niches-an observation conserved between species. One common histologic zone with a high frequency of mesenchymal progenitor cells within mammalian tissues is the tunica adventitia, the outer layer of blood vessel walls populated by cells with a fibroblastic morphology. The diversity and functions of (re)generative cells present in this outermost perivascular niche are under intense investigation; we have reviewed herein our current knowledge of adventitial cell potential with a somewhat narrow focus on bone formation. Antigens of interest to functionally segregate adventicytes are discussed, including CD10, CD107a, aldehyde dehydrogenase isoforms, and CD140a, among others. Purified adventicytes (such as CD10+ , CD107alow , and CD140a+ cells) have stronger osteogenic potential and promote bone formation in vivo. Recent bone tissue engineering applications of adventitial cells are also presented. A better understanding of perivascular progenitor cell subsets may represent a beneficial advance for future efforts in tissue repair and bioengineering.

Perivascular Fibro-Adipogenic Progenitor Tracing during Post-Traumatic Osteoarthritis.

Perivascular mural cells surround capillaries and microvessels and have diverse regenerative or fibrotic functions after tissue injury. Subsynovial fibrosis is a well-known pathologic feature of osteoarthritis, yet transgenic animals for use in visualizing perivascular cell contribution to fibrosis during arthritic changes have not been developed. Here, inducible Pdgfra-CreERT2 reporter mice were subjected to joint-destabilization surgery to induce arthritic changes, and cell lineage was traced over an 8-week period with a focus on the joint-associated fat pad. Results showed that, at baseline, inducible Pdgfra reporter activity highlighted adventitial and, to a lesser extent, pericytic cells within the infrapatellar fat pad. Joint-destabilization surgery was associated with marked fibrosis of the infrapatellar fat pad, accompanied by an expansion of perivascular Pdgfra-expressing cellular descendants, many of which adopted α-smooth muscle actin expression. Gene expression analysis of microdissected infrapatellar fat pad confirmed enrichment in membrane-bound green fluorescent protein/Pdgfra-expressing cells, along with a gene signature that corresponded with injury-associated fibro-adipogenic progenitors. Our results highlight dynamic changes in joint-associated perivascular fibro-adipogenic progenitors during osteoarthritis.

PDGFRα marks distinct perivascular populations with different osteogenic potential within adipose tissue.

The perivascular niche within adipose tissue is known to house multipotent cells, including osteoblast precursors. However, the identity of perivascular subpopulations that may mineralize or ossify most readily is not known. Here, we utilize inducible PDGFRα (platelet-derived growth factor alpha) reporter animals to identify subpopulations of perivascular progenitor cells. Results showed that PDGFRα-expressing cells are present in four histologic niches within inguinal fat, including two perivascular locations. PDGFRα+ cells are most frequent within the tunica adventitia of arteries and veins, where PDGFRα+ cells populate the inner aspects of the adventitial layer. Although both PDGFRα+ and PDGFRα- fractions are multipotent progenitor cells, adipose tissue-derived PDGFRα+ stromal cells proliferate faster and mineralize to a greater degree than their PDGFRα- counterparts. Likewise, PDGFRα+ ectopic implants reconstitute the perivascular niche and ossify to a greater degree than PDGFRα- cell fractions. Adventicytes can be further grouped into three distinct groups based on expression of PDGFRα and/or CD34. When further partitioned, adventicytes co-expressing PDGFRα and CD34 represented a cell fraction with the highest mineralization potential. Long-term tracing studies showed that PDGFRα-expressing adventicytes give rise to adipocytes, but not to other cells within the vessel wall under homeostatic conditions. However, upon bone morphogenetic protein 2 (BMP2)-induced ossicle formation, descendants of PDGFRα+ cells gave rise to osteoblasts, adipocytes, and "pericyte-like" cells within the ossicle. In sum, PDGFRα marks distinct perivascular osteoprogenitor cell subpopulations within adipose tissue. The identification of perivascular osteoprogenitors may contribute to our improved understanding of pathologic mineralization/ossification.

CD10 expression identifies a subset of human perivascular progenitor cells with high proliferation and calcification potentials.

The tunica adventitia ensheathes arteries and veins and contains presumptive mesenchymal stem cells (MSCs) involved in vascular remodeling. We show here that a subset of human adventitial cells express the CD10/CALLA cell surface metalloprotease. Both CD10+ and CD10- adventitial cells displayed phenotypic features of MSCs when expanded in culture. However, CD10+ adventitial cells exhibited higher proliferation, clonogenic and osteogenic potentials in comparison to their CD10- counterparts. CD10+ adventitial cells increased expression of the cell cycle protein CCND2 via ERK1/2 signaling and osteoblastogenic gene expression via NF-κB signaling. CD10 expression was upregulated in adventitial cells through sonic hedgehog-mediated GLI1 signaling. These results suggest that CD10, which marks rapidly dividing cells in other normal and malignant cell lineages, plays a role in perivascular MSC function and cell fate specification. These findings also point to a role for CD10+ perivascular cells in vascular remodeling and calcification.

FOXP1 circular RNA sustains mesenchymal stem cell identity via microRNA inhibition.

Stem cell identity and plasticity are controlled by master regulatory genes and complex circuits also involving non-coding RNAs. Circular RNAs (circRNAs) are a class of RNAs generated from protein-coding genes by backsplicing, resulting in stable RNA structures devoid of free 5' and 3' ends. Little is known of the mechanisms of action of circRNAs, let alone in stem cell biology. In this study, for the first time, we determined that a circRNA controls mesenchymal stem cell (MSC) identity and differentiation. High-throughput MSC expression profiling from different tissues revealed a large number of expressed circRNAs. Among those, circFOXP1 was enriched in MSCs compared to differentiated mesodermal derivatives. Silencing of circFOXP1 dramatically impaired MSC differentiation in culture and in vivo. Furthermore, we demonstrated a direct interaction between circFOXP1 and miR-17-3p/miR-127-5p, which results in the modulation of non-canonical Wnt and EGFR pathways. Finally, we addressed the interplay between canonical and non-canonical Wnt pathways. Reprogramming to pluripotency of MSCs reduced circFOXP1 and non-canonical Wnt, whereas canonical Wnt was boosted. The opposing effect was observed during generation of MSCs from human pluripotent stem cells. Our results provide unprecedented evidence for a regulatory role for circFOXP1 as a gatekeeper of pivotal stem cell molecular networks.

Transcriptional Networks in Single Perivascular Cells Sorted from Human Adipose Tissue Reveal a Hierarchy of Mesenchymal Stem Cells.

Adipose tissue is a rich source of multipotent mesenchymal stem-like cells, located in the perivascular niche. Based on their surface markers, these have been assigned to two main categories: CD31- /CD45- /CD34+ /CD146- cells (adventitial stromal/stem cells [ASCs]) and CD31- /CD45- /CD34- /CD146+ cells (pericytes [PCs]). These populations display heterogeneity of unknown significance. We hypothesized that aldehyde dehydrogenase (ALDH) activity, a functional marker of primitivity, could help to better define ASC and PC subclasses. To this end, the stromal vascular fraction from a human lipoaspirate was simultaneously stained with fluorescent antibodies to CD31, CD45, CD34, and CD146 antigens and the ALDH substrate Aldefluor, then sorted by fluorescence-activated cell sorting. Individual ASCs (n = 67) and PCs (n = 73) selected from the extremities of the ALDH-staining spectrum were transcriptionally profiled by Fluidigm single-cell quantitative polymerase chain reaction for a predefined set (n = 429) of marker genes. To these single-cell data, we applied differential expression and principal component and clustering analysis, as well as an original gene coexpression network reconstruction algorithm. Despite the stochasticity at the single-cell level, covariation of gene expression analysis yielded multiple network connectivity parameters suggesting that these perivascular progenitor cell subclasses possess the following order of maturity: (a) ALDHbr ASC (most primitive); (b) ALDHdim ASC; (c) ALDHbr PC; (d) ALDHdim PC (least primitive). This order was independently supported by specific combinations of class-specific expressed genes and further confirmed by the analysis of associated signaling pathways. In conclusion, single-cell transcriptional analysis of four populations isolated from fat by surface markers and enzyme activity suggests a developmental hierarchy among perivascular mesenchymal stem cells supported by markers and coexpression networks. Stem Cells 2017;35:1273-1289.

The Wnt5a Receptor, Receptor Tyrosine Kinase-Like Orphan Receptor 2, Is a Predictive Cell Surface Marker of Human Mesenchymal Stem Cells with an Enhanced Capacity for Chondrogenic Differentiation.

Multipotent mesenchymal stem cells (MSCs) have enormous potential in tissue engineering and regenerative medicine. However, until now, their development for clinical use has been severely limited as they are a mixed population of cells with varying capacities for lineage differentiation and tissue formation. Here, we identify receptor tyrosine kinase-like orphan receptor 2 (ROR2) as a cell surface marker expressed by those MSCs with an enhanced capacity for cartilage formation. We generated clonal human MSC populations with varying capacities for chondrogenesis. ROR2 was identified through screening for upregulated genes in the most chondrogenic clones. When isolated from uncloned populations, ROR2+ve MSCs were significantly more chondrogenic than either ROR2-ve or unfractionated MSCs. In a sheep cartilage-repair model, they produced significantly more defect filling with no loss of cartilage quality compared with controls. ROR2+ve MSCs/perivascular cells were present in developing human cartilage, adult bone marrow, and adipose tissue. Their frequency in bone marrow was significantly lower in patients with osteoarthritis (OA) than in controls. However, after isolation of these cells and their initial expansion in vitro, there was greater ROR2 expression in the population derived from OA patients compared with controls. Furthermore, osteoarthritis-derived MSCs were better able to form cartilage than MSCs from control patients in a tissue engineering assay. We conclude that MSCs expressing high levels of ROR2 provide a defined population capable of predictably enhanced cartilage production. Stem Cells 2017;35:2280-2291.

Pericytes for Therapeutic Bone Repair.

Besides seminal functions in angiogenesis and blood pressure regulation, microvascular pericytes possess a latent tissue regenerative potential that can be revealed in culture following transition into mesenchymal stem cells. Endowed with robust osteogenic potential, pericytes and other related perivascular cells extracted from adipose tissue represent a potent and abundant cell source for refined bone tissue engineering and improved cell therapies of fractures and other bone defects. The use of diverse bone formation assays in vivo, which include mouse muscle pocket osteogenesis and calvaria replenishment, rat and dog spine fusion, and rat non-union fracture healing, has confirmed the superiority of purified perivascular cells for skeletal (re)generation. As a surprising observation though, despite strong endogenous bone-forming potential, perivascular cells drive bone regeneration essentially indirectly, via recruitment by secreted factors of local osteo-progenitors.

Adipose-derived Human Perivascular Stem Cells May Improve Achilles Tendon Healing in Rats.

BACKGROUND: Achilles tendon rupture is a common injury and the best treatment option remains uncertain between surgical and nonoperative methods. Biologic approaches using multipotent stem cells such as perivascular stem cells pose a possible treatment option, although there is currently a paucity of evidence regarding their clinical therapeutic use. QUESTIONS/PURPOSES: The purpose of this study was to determine whether injected perivascular stem cells (PSCs) would (1) improve histologic signs of tendon healing (such as percent area of collagen); and (2) improve biomechanical properties (peak load or stiffness) in a rat model of Achilles tendon transection. METHODS: Two subtypes of PSCs were derived from human adipose tissue: pericytes (CD146CD34CD45CD31) and adventitial cells (CD146CD34CD45CD31). Thirty-two athymic rats underwent right Achilles transection and were randomized to receive injection with saline (eight tendons), hydrogel (four tendons), pericytes in hydrogel (four tendons), or adventitial cells in hydrogel (eight tendons) 3 days postoperatively with the left serving as an uninjured control. Additionally, a subset of pericytes was labeled with CM-diI to track cell viability and localization. At 3 weeks, the rats were euthanized, and investigators blinded to treatment group allocation evaluated tendon healing by peak load and stiffness using biomechanical testing and percent area of collagen using histologic analysis with picrosirius red staining. RESULTS: Histologic analysis showed a higher mean percent area collagen for pericytes (30%) and adventitial cells (28%) than hydrogel (21%) or saline (26%). However, a nonparametric statistical analysis yielded no statistical difference. Mechanical testing demonstrated that the pericyte group had a higher peak load than the saline group (41 ± 7 N versus 26 ± 9 N; mean difference 15 N; 95% confidence interval [CI], 4-27 N; p = 0.003) and a higher peak load than the hydrogel group (41 ± 7 N versus 25 ± 3 N; mean difference 16; 95% CI, 8-24 N; p = 0.001). The pericyte group demonstrated higher stiffness than the hydrogel group (36 ± 12 N/mm versus 17 ± 6 N/mm; mean difference 19 N/mm; 95% CI, 5-34 N/mm; p = 0.005). CONCLUSIONS: Our results suggest that injection of PSCs improves mechanical but not the histologic properties of early Achilles tendon healing. CLINICAL RELEVANCE: This is a preliminary study that provides more insight into the use of adipose-derived PSCs as a percutaneous therapy in the setting of Achilles tendon rupture. Further experiments to characterize the function of these cells may serve as a pathway to development of minimally invasive intervention aimed at improving nonoperative management while avoiding the complications associated with surgical treatment down the line.

Neer Award 2018: Platelet-derived growth factor receptor α co-expression typifies a subset of platelet-derived growth factor receptor ß-positive progenitor cells that contribute to fatty degeneration and fibrosis of the murine rotator cuff.

BACKGROUND AND HYPOTHESIS: After massive tears, rotator cuff muscle often undergoes atrophy, fibrosis, and fatty degeneration. These changes can lead to high surgical failure rates and poor patient outcomes. The identity of the progenitor cells involved in these processes has not been fully elucidated. Platelet-derived growth factor receptor ß (PDGFRß) and platelet-derived growth factor receptor α (PDGFRα) have previously been recognized as markers of cells involved in muscle fibroadipogenesis. We hypothesized that PDGFRα expression identifies a fibroadipogenic subset of PDGFRß+ progenitor cells that contribute to fibroadipogenesis of the rotator cuff. METHODS: We created massive rotator cuff tears in a transgenic strain of mice that allows PDGFRß+ cells to be tracked via green fluorescent protein (GFP) fluorescence. We then harvested rotator cuff muscle tissues at multiple time points postoperatively and analyzed them for the presence and localization of GFP+ PDGFRß+ PDGFRα+ cells. We cultured, induced, and treated these cells with the molecular inhibitor CWHM-12 to assess fibrosis inhibition. RESULTS: GFP+ PDGFRß+ PDGFRα+ cells were present in rotator cuff muscle tissue and, after massive tears, localized to fibrotic and adipogenic tissues. The frequency of PDGFRß+ PDGFRα+ cells increased at 5 days after massive cuff tears and decreased to basal levels within 2 weeks. PDGFRß+ PDGFRα+ cells were highly adipogenic and significantly more fibrogenic than PDGFRß+ PDGFRα- cells in vitro and localized to adipogenic and fibrotic tissues in vivo. Treatment with CWHM-12 significantly decreased fibrogenesis from PDGFRß+ PDGFRα+ cells. CONCLUSION: PDGFRß+ PDGFRα+ cells directly contribute to fibrosis and fatty degeneration after massive rotator cuff tears in the mouse model. In addition, CWHM-12 treatment inhibits fibrogenesis from PDGFRß+ PDGFRα+ cells in vitro. Clinically, perioperative PDGFRß+ PDGFRα+ cell inhibition may limit rotator cuff tissue degeneration and, ultimately, improve surgical outcomes for massive rotator cuff tears.

Human kidney pericytes produce renin.

Pericytes, perivascular cells embedded in the microvascular wall, are crucial for vascular homeostasis. These cells also play diverse roles in tissue development and regeneration as multi-lineage progenitors, immunomodulatory cells and as sources of trophic factors. Here, we establish that pericytes are renin producing cells in the human kidney. Renin was localized by immunohistochemistry in CD146 and NG2 expressing pericytes, surrounding juxtaglomerular and afferent arterioles. Similar to pericytes from other organs, CD146+CD34-CD45-CD56- renal fetal pericytes, sorted by flow cytometry, exhibited tri-lineage mesodermal differentiation potential in vitro. Additionally, renin expression was triggered in cultured kidney pericytes by cyclic AMP as confirmed by immuno-electron microscopy, and secretion of enzymatically functional renin, capable of generating angiotensin I. Pericytes derived from second trimester human placenta also expressed renin in an inducible fashion although the renin activity was much lower than in renal pericytes. Thus, our results confirm and extend the recently discovered developmental plasticity of microvascular pericytes, and may open new perspectives to the therapeutic regulation of the renin-angiotensin system.

Human myocardial pericytes: multipotent mesodermal precursors exhibiting cardiac specificity.

Perivascular mesenchymal precursor cells (i.e., pericytes) reside in skeletal muscle where they contribute to myofiber regeneration; however, the existence of similar microvessel-associated regenerative precursor cells in cardiac muscle has not yet been documented. We tested whether microvascular pericytes within human myocardium exhibit phenotypes and multipotency similar to their anatomically and developmentally distinct counterparts. Fetal and adult human heart pericytes (hHPs) express canonical pericyte markers in situ, including CD146, NG2, platelet-derived growth factor receptor (PDGFR) ß, PDGFRα, alpha-smooth muscle actin, and smooth muscle myosin heavy chain, but not CD117, CD133, and desmin, nor endothelial cell (EC) markers. hHPs were prospectively purified to homogeneity from ventricular myocardium by flow cytometry, based on a combination of positive- (CD146) and negative-selection (CD34, CD45, CD56, and CD117) cell lineage markers. Purified hHPs expanded in vitro were phenotypically similar to human skeletal muscle-derived pericytes (hSkMPs). hHPs express mesenchymal stem/stromal cell markers in situ and exhibited osteo-, chondro-, and adipogenic potentials but, importantly, no ability for skeletal myogenesis, diverging from pericytes of all other origins. hHPs supported network formation with/without ECs in Matrigel cultures; hHPs further stimulated angiogenic responses under hypoxia, markedly different from hSkMPs. The cardiomyogenic potential of hHPs was examined following 5-azacytidine treatment and neonatal cardiomyocyte coculture in vitro, and intramyocardial transplantation in vivo. Results indicated cardiomyocytic differentiation in a small fraction of hHPs. In conclusion, human myocardial pericytes share certain phenotypic and developmental similarities with their skeletal muscle homologs, yet exhibit different antigenic, myogenic, and angiogenic properties. This is the first example of an anatomical restriction in the developmental potential of pericytes as native mesenchymal stem cells.

A Role for MOSPD1 in Mesenchymal Stem Cell Proliferation and Differentiation.

Mesenchymal stem cells (MSCs) isolated from many tissues including bone marrow and fat can be expanded in vitro and can differentiate into a range of different cell types such as bone, cartilage, and adipocytes. MSCs can also exhibit immunoregulatory properties when transplanted but, although a number of clinical trials using MSCs are in progress, the molecular mechanisms that control their production, proliferation, and differentiation are poorly understood. We identify MOSPD1 as a new player in this process. We generated MOSPD1-null embryonic stem cells (ESCs) and demonstrate that they are deficient in their ability to differentiate into a number of cell lineages including osteoblasts, adipocytes, and hematopoietic progenitors. The self-renewal capacity of MOSPD1-null ESCs was normal and they exhibited no obvious defects in early germ layer specification nor in epithelial to mesenchymal transition (EMT), indicating that MOSPD1 functions after these key steps in the differentiation process. Mesenchymal stem cell (MSC)-like cells expressing CD73, CD90, and CD105 were generated from MOSPD1-null ESCs but their growth rate was significantly impaired implying that MOSPD1 plays a role in MSC proliferation. Phenotypic deficiencies exhibited by MOSPD1-null ESCs were rescued by exogenous expression of MOSPD1, but not MOSPD3 indicating distinct functional properties of these closely related genes. Our in vitro studies were supported by RNA-sequencing data that confirmed expression of Mospd1 mRNA in cultured, proliferating perivascular pre-MSCs isolated from human tissue. This study adds to the growing body of knowledge about the function of this largely uncharacterized protein family and introduces a new player in the control of MSC proliferation and differentiation.

Brief Report: Human Perivascular Stem Cells and Nel-Like Protein-1 Synergistically Enhance Spinal Fusion in Osteoporotic Rats.

Autologous bone grafts (ABGs) are considered as the gold standard for spinal fusion. However, osteoporotic patients are poor candidates for ABGs due to limited osteogenic stem cell numbers and function of the bone microenvironment. There is a need for stem cell-based spinal fusion of proven efficacy under either osteoporotic or nonosteoporotic conditions. The purpose of this study is to determine the efficacy of human perivascular stem cells (hPSCs), a population of mesenchymal stem cells isolated from adipose tissue, in the presence and absence of NELL-1, an osteogenic protein, for spinal fusion in the osteoporosis. Osteogenic differentiation of hPSCs with and without NELL-1 was tested in vitro. The results indicated that NELL-1 significantly increased the osteogenic potential of hPSCs in both osteoporotic and nonosteoporotic donors. Next, spinal fusion was performed by implanting scaffolds with regular or high doses of hPSCs, with or without NELL-1 in ovariectomized rats (n = 41). Regular doses of hPSCs or NELL-1 achieved the fusion rates of only 20%-37.5% by manual palpation. These regular doses had previously been shown to be effective in nonosteoporotic rat spinal fusion. Remarkably, the high dose of hPSCs+NELL-1 significantly improved the fusion rates among osteoporotic rats up to approximately 83.3%. Microcomputed tomography imaging and quantification further confirmed solid bony fusion with high dose hPSCs+NELL-1. Finally, histologically, direct in situ involvement of hPSCs in ossification was shown using undecalcified samples. To conclude, hPSCs combined with NELL-1 synergistically enhances spinal fusion in osteoporotic rats and has great potential as a novel therapeutic strategy for osteoporotic patients.

Q&A: Mesenchymal stem cells - where do they come from and is it important?

Mesenchymal stem - or stromal - cells (MSCs) have been administered in hundreds of clinical trials for multiple indications, making them some of the most commonly used selected regenerative cells. Paradoxically, MSCs have also long remained the least characterized stem cells regarding native identity and natural function, being isolated retrospectively in long-term culture. Recent years have seen progress in our understanding of the natural history of these cells, and candidate native MSCs have been identified within fetal and adult organs. Beyond basic knowledge, deciphering the biology of innate MSCs may have important positive consequences for the therapeutic use of these cells.

Perivascular support of human hematopoietic stem/progenitor cells.

Hematopoietic stem and progenitor cells (HSPCs) emerge and develop adjacent to blood vessel walls in the yolk sac, aorta-gonad-mesonephros region, embryonic liver, and fetal bone marrow. In adult mouse bone marrow, perivascular cells shape a "niche" for HSPCs. Mesenchymal stem/stromal cells (MSCs), which support hematopoiesis in culture, are themselves derived in part from perivascular cells. In order to define their direct role in hematopoiesis, we tested the ability of purified human CD146(+) perivascular cells, as compared with unfractionated MSCs and CD146(-) cells, to sustain human HSPCs in coculture. CD146(+) perivascular cells support the long-term persistence, through cell-to-cell contact and at least partly via Notch activation, of human myelolymphoid HSPCs able to engraft primary and secondary immunodeficient mice. Conversely, unfractionated MSCs and CD146(-) cells induce differentiation and compromise ex vivo maintenance of HSPCs. Moreover, CD146(+) perivascular cells express, natively and in culture, molecular markers of the vascular hematopoietic niche. Unexpectedly, this dramatic, previously undocumented ability to support hematopoietic stem cells is present in CD146(+) perivascular cells extracted from the nonhematopoietic adipose tissue.

Natural history of mesenchymal stem cells, from vessel walls to culture vessels.

Mesenchymal stem/stromal cells (MSCs) can regenerate tissues by direct differentiation or indirectly by stimulating angiogenesis, limiting inflammation, and recruiting tissue-specific progenitor cells. MSCs emerge and multiply in long-term cultures of total cells from the bone marrow or multiple other organs. Such a derivation in vitro is simple and convenient, hence popular, but has long precluded understanding of the native identity, tissue distribution, frequency, and natural role of MSCs, which have been defined and validated exclusively in terms of surface marker expression and developmental potential in culture into bone, cartilage, and fat. Such simple, widely accepted criteria uniformly typify MSCs, even though some differences in potential exist, depending on tissue sources. Combined immunohistochemistry, flow cytometry, and cell culture have allowed tracking the artifactual cultured mesenchymal stem/stromal cells back to perivascular anatomical regions. Presently, both pericytes enveloping microvessels and adventitial cells surrounding larger arteries and veins have been described as possible MSC forerunners. While such a vascular association would explain why MSCs have been isolated from virtually all tissues tested, the origin of the MSCs grown from umbilical cord blood remains unknown. In fact, most aspects of the biology of perivascular MSCs are still obscure, from the emergence of these cells in the embryo to the molecular control of their activity in adult tissues. Such dark areas have not compromised intents to use these cells in clinical settings though, in which purified perivascular cells already exhibit decisive advantages over conventional MSCs, including purity, thorough characterization and, principally, total independence from in vitro culture. A growing body of experimental data is currently paving the way to the medical usage of autologous sorted perivascular cells for indications in which MSCs have been previously contemplated or actually used, such as bone regeneration and cardiovascular tissue repair.

Angiotensin-converting enzyme (CD143) specifies emerging lympho-hematopoietic progenitors in the human embryo.

Adult-type lympho-myeloid hematopoietic progenitors are first generated in the aorta-gonad-mesonephros region between days 27 and 40 of human embryonic development, but an elusive blood forming potential is present earlier in the underlying splanchnopleura. In the present study, we show that angiotensin-converting enzyme (ACE, also known as CD143), a recently identified cell-surface marker of adult human hematopoietic stem cells, is already expressed in all presumptive and developing blood-forming tissues of the human embryo and fetus: para-aortic splanchnopleura, yolk sac, aorta-gonad-mesonephros, liver, and bone marrow (BM). Fetal liver and BM-derived CD34(+)ACE(+) cells, but not CD34(+)ACE(-) cells, are endowed with long-term culture-initiating cell potential and sustain multilineage hematopoietic cell engraftment when transplanted into NOD/SCID mice. Furthermore, from 23-26 days of development, ACE expression characterizes rare CD34(-)CD45(-) cells concentrated in the hemogenic portion of the para-aortic splanchnopleura. ACE(+) cells sorted from the splanchnopleura generated colonies of hematopoietic cells more than 40 times more frequently than ACE(-) cells. These data suggest that, in addition to being a marker of adult human hematopoietic stem cells, ACE identifies embryonic mesodermal precursors responsible for definitive hematopoiesis, and we propose that this enzyme is involved in the regulation of human blood formation.

Human pericytes for ischemic heart repair.

Human microvascular pericytes (CD146(+)/34(-)/45(-)/56(-)) contain multipotent precursors and repair/regenerate defective tissues, notably skeletal muscle. However, their ability to repair the ischemic heart remains unknown. We investigated the therapeutic potential of human pericytes, purified from skeletal muscle, for treating ischemic heart disease and mediating associated repair mechanisms in mice. Echocardiography revealed that pericyte transplantation attenuated left ventricular dilatation and significantly improved cardiac contractility, superior to CD56+ myogenic progenitor transplantation, in acutely infarcted mouse hearts. Pericyte treatment substantially reduced myocardial fibrosis and significantly diminished infiltration of host inflammatory cells at the infarct site. Hypoxic pericyte-conditioned medium suppressed murine fibroblast proliferation and inhibited macrophage proliferation in vitro. High expression by pericytes of immunoregulatory molecules, including interleukin-6, leukemia inhibitory factor, cyclooxygenase-2, and heme oxygenase-1, was sustained under hypoxia, except for monocyte chemotactic protein-1. Host angiogenesis was significantly increased. Pericytes supported microvascular structures in vivo and formed capillary-like networks with/without endothelial cells in three-dimensional cocultures. Under hypoxia, pericytes dramatically increased expression of vascular endothelial growth factor-A, platelet-derived growth factor-ß, transforming growth factor-ß1 and corresponding receptors while expression of basic fibroblast growth factor, hepatocyte growth factor, epidermal growth factor, and angiopoietin-1 was repressed. The capacity of pericytes to differentiate into and/or fuse with cardiac cells was revealed by green fluorescence protein labeling, although to a minor extent. In conclusion, intramyocardial transplantation of purified human pericytes promotes functional and structural recovery, attributable to multiple mechanisms involving paracrine effects and cellular interactions.