Lorenza Lazzari

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.

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.

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.

Molecular and phenotypic characterization of human amniotic fluid cells and their differentiation potential.

The main goal of the study was to identify a novel source of human multipotent cells, overcoming ethical issues involved in embryonic stem cell research and the limited availability of most adult stem cells. Amniotic fluid cells (AFCs) are routinely obtained for prenatal diagnosis and can be expanded in vitro; nevertheless current knowledge about their origin and properties is limited. Twenty samples of AFCs were exposed in culture to adipogenic, osteogenic, neurogenic and myogenic media. Differentiation was evaluated using immunocytochemistry, RT-PCR and Western blotting. Before treatments, AFCs showed heterogeneous morphologies. They were negative for MyoD, Myf-5, MRF4, Myogenin and Desmin but positive for osteocalcin, PPARgamma2, GAP43, NSE, Nestin, MAP2, GFAP and beta tubulin III by RT-PCR. The cells expressed Oct-4, Rex-1 and Runx-1, which characterize the undifferentiated stem cell state. By immunocytochemistry they expressed neural-glial proteins, mesenchymal and epithelial markers. After culture, AFCs differentiated into adipocytes and osteoblasts when the predominant cellular component was fibroblastic. Early and late neuronal antigens were still present after 2 week culture in neural specific media even if no neuronal morphologies were detectable. Our results provide evidence that human amniotic fluid contains progenitor cells with multi-lineage potential showing stem and tissue-specific gene/protein presence for several lineages.

Oct-4 Expression in Adult Human Differentiated Cells Challenges Its Role as a Pure Stem Cell Marker

The Oct‐4 transcription factor, a member of the POU family that is also known as Oct‐3 and Oct3/4, is expressed in totipotent embryonic stem cells (ES) and germ cells, and it has a unique role in development and in the determination of pluripotency. ES may have their postnatal counterpart in the adult stem cells, recently described in various mammalian tissues, and Oct‐4 expression in putative stem cells purified from adult tissues has been considered a real marker of stemness. In this context, normal mature adult cells would not be expected to show Oct‐4 expression. On the contrary, we demonstrated, using reverse transcription‐polymerase chain reaction (PCR) (total RNA, Poly A+), real‐time PCR, immunoprecipitation, Western blotting, band shift, and immunofluorescence, that human peripheral blood mononuclear cells, genetically stable and mainly terminally differentiated cells with well defined functions and a limited lifespan, express Oct‐4. These observations raise the question as to whether the role of Oct‐4 as a marker of pluripotency should be challenged. Our findings suggest that the presence of Oct‐4 is not sufficient to define a cell as pluripotent, and that additional measures should be used to avoid misleading results in the case of an embryonic‐specific gene with a large number of pseudogenes that may contribute to false identification of Oct‐4 in adult stem cells. These unexpected findings may provide new insights into the role of Oct‐4 in fully differentiated cells.

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

Abstract 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.

Life-Sparing Effect of Human Cord Blood-Mesenchymal Stem Cells in Experimental Acute Kidney Injury

In search for new sources of mesenchymal stem cells (MSCs) for renal repair in acute kidney injury (AKI), we investigated the potential of human cord blood (CB)‐MSCs to cure mice with AKI. Infusion of CB‐MSCs in immunodeficient mice with cisplatin‐induced AKI ameliorated both renal function and tubular cell injury, and prolonged survival. Transplanted CB‐MSCs localized in peritubular areas, limited capillary alterations and neutrophil infiltration. Apoptosis reduced and tubular cell proliferation increased by virtue of stem cell capacity to produce growth factors. The reno‐protective effect of CB‐MSCs was further confirmed by their ability to inhibit oxidative damage and to induce the prosurvival factor Akt in tubular cells. The evidence that CB‐MSCs in vitro increased the production of growth factors and inhibited IL‐1β and TNFα synthesis when cocultured with damaged proximal tubular cells indicates a regenerative and anti‐inflammatory action of stem cell treatment. Altogether these results highlight the potential of human CB‐MSCs as future cell therapy for testing in human AKI. STEM CELLS 2010;28:513–522

Short-term, long-term and paracrine effect of human umbilical cord-derived stem cells in lung injury prevention and repair in experimental bronchopulmonary dysplasia

Background Bronchopulmonary dysplasia (BPD) remains a main complication of extreme prematurity and currently lacks efficient treatment. Rat bone marrow-derived mesenchymal stem cells (MSC) prevent lung injury in an oxygen-induced model of BPD. Human cord is an advantageous source of stem cells that is especially appealing for the treatment of neonatal diseases. The therapeutic benefit after established lung injury and long-term safety of cord-derived stem cells is unknown. Methods Human cord-derived perivascular cells (PCs) or cord blood-derived MSCs were delivered prophylactically or after established alveolar injury into the airways of newborn rats exposed to hyperoxia, a well-established BPD model. Results Rat pups exposed to hyperoxia showed the characteristic arrest in alveolar growth with air space enlargement and loss of lung capillaries. PCs and MSCs partially prevented and rescued lung function and structure. Despite therapeutic benefit, cell engraftment was low, suggesting that PCs and MSCs act via a paracrine effect. Accordingly, cell free-derived conditioned media from PCs and MSCs also exerted therapeutic benefit when used either prophylactically or therapeutically. Finally, long-term (6 months) assessment of stem cell or conditioned media therapy showed no adverse lung effects of either strategy, with persistent improvement in exercise capacity and lung structure. Conclusions Human umbilical cord-derived PCs and MSCs exert short- and long-term therapeutic benefit without adverse lung effects in this experimental model and offer new therapeutic options for lung diseases characterised by alveolar damage.

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.

Purification and Long-Term Culture of Multipotent Progenitor Cells Affiliated with the Walls of Human Blood Vessels : Myoendothelial Cells and Pericytes

We have identified with molecular markers and purified by flow cytometry two populations of cells that are developmentally and anatomically related to blood vessel walls in human tissues: myoendothelial cells, found in skeletal muscle and coexpressing markers of endothelial and myogenic cells, and pericytes--aka mural cells--which surround endothelial cells in capillaries and microvessels. Purified myoendothelial cells and pericytes exhibit multilineage developmental potential and differentiate, in culture and in vivo, into skeletal myofibers, bone, cartilage, and adipocytes. Myoendothelial cells and pericytes can be cultured on the long term with sustained marker expression and differentiation potential and clonal populations thereof have been derived. Yet, these blood vessel wall-derived progenitors exhibit no tendency to malignant transformation upon extended culture. Our results suggest that multipotent progenitor cells, such as mesenchymal stem cells, previously isolated retrospectively from diverse cultured adult tissues are derived from a subset of perivascular cells. We present in this chapter the main strategies and tactics used to purify, culture on the long term, and phenotypically characterize these novel multipotent cells.