Mihaela Crisan

A Perivascular Origin for Mesenchymal Stem Cells in Multiple Human Organs

Mesenchymal stem cells (MSCs), the archetypal multipotent progenitor cells derived in cultures of developed organs, are of unknown identity and native distribution. We have prospectively identified perivascular cells, principally pericytes, in multiple human organs including skeletal muscle, pancreas, adipose tissue, and placenta, on CD146, NG2, and PDGF-Rbeta expression and absence of hematopoietic, endothelial, and myogenic cell markers. Perivascular cells purified from skeletal muscle or nonmuscle tissues were myogenic in culture and in vivo. Irrespective of their tissue origin, long-term cultured perivascular cells retained myogenicity; exhibited at the clonal level osteogenic, chondrogenic, and adipogenic potentials; expressed MSC markers; and migrated in a culture model of chemotaxis. Expression of MSC markers was also detected at the surface of native, noncultured perivascular cells. Thus, blood vessel walls harbor a reserve of progenitor cells that may be integral to the origin of the elusive MSCs and other related adult stem cells.

Prospective identification of myogenic endothelial cells in human skeletal muscle

We document anatomic, molecular and developmental relationships between endothelial and myogenic cells within human skeletal muscle. Cells coexpressing myogenic and endothelial cell markers (CD56, CD34, CD144) were identified by immunohistochemistry and flow cytometry. These myoendothelial cells regenerate myofibers in the injured skeletal muscle of severe combined immunodeficiency mice more effectively than CD56+ myogenic progenitors. They proliferate long term, retain a normal karyotype, are not tumorigenic and survive better under oxidative stress than CD56+ myogenic cells. Clonally derived myoendothelial cells differentiate into myogenic, osteogenic and chondrogenic cells in culture. Myoendothelial cells are amenable to biotechnological handling, including purification by flow cytometry and long-term expansion in vitro, and may have potential for the treatment of human muscle disease.

Perivascular Ancestors of Adult Multipotent Stem Cells

Independent studies by numerous investigators have shown that it is possible to harvest multipotent progenitor cells from diverse dissociated and cultured fetal, perinatal, and principally adult developed tissues. Despite the increasingly recognized medical value of these progenitor cells, the archetype of which remains the mesenchymal stem cell, this indirect extraction method has precluded the understanding of their native identity, tissue distribution, and frequency. Consistent with other researchers, we have hypothesized that blood vessels in virtually all organs harbor ubiquitous stem cells. We have identified, marked, and sorted to homogeneity by flow cytometry endothelial and perivascular cells in a large selection of human fetal, perinatal, and adult organs. Perivascular cells, including pericytes in the smallest blood vessels and adventitial cells around larger ones, natively express mesenchymal stem cell markers and produce in culture a long-lasting progeny of multilineage mesodermal progenitor cells. Herein, we review results from our and other laboratories that suggest a perivascular origin for mesenchymal stem cells and other adult progenitor cells. Recent experiments illustrate the therapeutic potential of human pericytes to regenerate skeletal muscle and promote functional recovery in the diseased heart and kidney.

Isolation of a slowly adhering cell fraction containing stem cells from murine skeletal muscle by the preplate technique

This protocol details a procedure, known as the modified preplate technique, which is currently used in our laboratory to isolate muscle cells on the basis of selective adhesion to collagen-coated tissue culture plates. By employing this technique to murine skeletal muscle, we have been able to isolate a rapidly adhering cell (RAC) fraction within the earlier stages of the process, whereas a slowly adhering cell (SAC) fraction containing muscle-derived stem cells is obtained from the later stages of the process. This protocol outlines the methods and materials needed to isolate RAC and SAC populations from murine skeletal muscle. The procedure involves mechanical and enzymatic digestion of skeletal muscle tissue with collagenase XI, dispase and trypsin followed by plating the resultant muscle slurry on collagen type I-coated flasks where the cells adhere at different rates. The entire preplate technique requires 5 d to obtain the final preplate SAC population. Two to three additional days are usually required before this population is properly established. We also detail additional methodologies designed to further enrich the resultant cell population by continuing the modified preplating process on the SAC population. This process is known as replating and requires further time.

Perivascular Multipotent Progenitor Cells in Human Organs

We have identified vascular pericytes in multiple human organs on expression of CD146, NG2, PDGF‐Rβ, and mesenchymal stem cell markers (CD44, CD73, CD90, CD105) and absence of blood, endothelial, and myogenic cell markers. Pericytes purified from all tissues were myogenic in culture and in vivo, sustained long‐term culture during which they expressed markers of mesenchymal stem cells, and exhibited, at the clonal level, osteogenic, chondrogenic, and adipogenic potentials. These results suggest that human capillary and microvessel walls all over the organism harbor a reserve of progenitor cells that are at the origin of the elusive mesenchymal stem cells, so far identified only retrospectively in primary tissue cultures.

A Reservoir of Brown Adipocyte Progenitors in Human Skeletal Muscle

Brown adipose tissue uncoupling protein‐1 (UCP1) plays a major role in the control of energy balance in rodents. It has long been thought, however, that there is no physiologically relevant UCP1 expression in adult humans. In this study we show, using an original approach consisting of sorting cells from various tissues and differentiating them in an adipogenic medium, that a stationary population of skeletal muscle cells expressing the CD34 surface protein can differentiate in vitro into genuine brown adipocytes with a high level of UCP1 expression and uncoupled respiration. These cells can be expanded in culture, and their UCP1 mRNA expression is strongly increased by cell‐permeating cAMP derivatives and a peroxisome‐proliferator‐activated receptor‐γ (PPARγ) agonist. Furthermore, UCP1 mRNA was detected in the skeletal muscle of adult humans, and its expression was increased in vivo by PPARγ agonist treatment. All the studies concerning UCP1 expression in adult humans have until now been focused on the white adipose tissue. Here we show for the first time the existence in human skeletal muscle and the prospective isolation of progenitor cells with a high potential for UCP1 expression. The discovery of this reservoir generates a new hope of treating obesity by acting on energy dissipation.

Human Placenta Is a Potent Hematopoietic Niche Containing Hematopoietic Stem and Progenitor Cells throughout Development

Hematopoietic stem cells (HSCs) are responsible for the life-long production of the blood system and are pivotal cells in hematologic transplantation therapies. During mouse and human development, the first HSCs are produced in the aorta-gonad-mesonephros region. Subsequent to this emergence, HSCs are found in other anatomical sites of the mouse conceptus. While the mouse placenta contains abundant HSCs at midgestation, little is known concerning whether HSCs or hematopoietic progenitors are present and supported in the human placenta during development. In this study we show, over a range of developmental times including term, that the human placenta contains hematopoietic progenitors and HSCs. Moreover, stromal cell lines generated from human placenta at several developmental time points are pericyte-like cells and support human hematopoiesis. Immunostaining of placenta sections during development localizes hematopoietic cells in close contact with pericytes/perivascular cells. Thus, the human placenta is a potent hematopoietic niche throughout development.

Perivascular cells for regenerative medicine.

Mesenchymal stem/stromal cells (MSC) are currently the best candidate therapeutic cells for regenerative medicine related to osteoarticular, muscular, vascular and inflammatory diseases, although these cells remain heterogeneous and necessitate a better biological characterization. We and others recently described that MSC originate from two types of perivascular cells, namely pericytes and adventitial cells and contain the in situ counterpart of MSC in developing and adult human organs, which can be prospectively purified using well defined cell surface markers. Pericytes encircle endothelial cells of capillaries and microvessels and express the adhesion molecule CD146 and the PDGFRβ, but lack endothelial and haematopoietic markers such as CD34, CD31, vWF (von Willebrand factor), the ligand for Ulex europaeus 1 (UEA1) and CD45 respectively. The proteoglycan NG2 is a pericyte marker exclusively associated with the arterial system. Besides its expression in smooth muscle cells, smooth muscle actin (αSMA) is also detected in subsets of pericytes. Adventitial cells surround the largest vessels and, opposite to pericytes, are not closely associated to endothelial cells. Adventitial cells express CD34 and lack αSMA and all endothelial and haematopoietic cell markers, as for pericytes. Altogether, pericytes and adventitial perivascular cells express in situ and in culture markers of MSC and display capacities to differentiate towards osteogenic, adipogenic and chondrogenic cell lineages. Importantly, adventitial cells can differentiate into pericyte‐like cells under inductive conditions in vitro. Altogether, using purified perivascular cells instead of MSC may bring higher benefits to regenerative medicine, including the possibility, for the first time, to use these cells uncultured.

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‐β, transforming growth factor‐β1 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. STEM CELLS2013;31:305–316

Perivascular multi-lineage progenitor cells in human organs: Regenerative units, cytokine sources or both?

Multi-lineage progenitors, e.g. mesenchymal stem cells, persist in adult developed organs, making a windfall for the cell therapist but an enigma for stem cell biologists. Recent results from our own and other laboratories show that the ancestor of these elusive adult stem cells is likely to be found in the perivascular area, explaining the ubiquitous distribution of these cells in the body. We have prospectively identified and purified vascular pericytes in multiple human organs and shown that these cells are potent mesodermal progenitors that give rise to genuine mesenchymal stem cells in culture. Pericytes can differentiate into diverse cell lineages, but also secrete multiple paracrine growth factors/cytokines, which likely explains in part their robust regenerative potential.