Single cell multiomic analysis of T cell exhaustion in vitro.

T-cell activation is a key step in the amplification of an immune response. Over the course of an immune response, cells may be chronically stimulated, with some proportion becoming exhausted; an enormous number of molecules are involved in this process. There remain a number of questions about the process, namely: (1) what degree of heterogeneity and plasticity do T-cells exhibit during stimulation? (2) how many unique cell states define chronic stimulation? and (3) what markers discriminate activated from exhausted cells? We addressed these questions by performing single-cell multiomic analysis to simultaneously measure expression of 38 proteins and 399 genes in human T cells expanded in vitro. This approach allowed us to study -with unprecedented depth-how T cells change over the course of chronic stimulation. Comprehensive immunophenotypic and transcriptomic analysis at day 0 enabled a refined characterization of T-cell maturational states and the identification of a donor-specific subset of terminally differentiated T-cells that would have been otherwise overlooked using canonical cell classification schema. As expected, activation downregulated naïve-cell markers and upregulated effector molecules, proliferation regulators, co-inhibitory and co-stimulatory receptors. Our deep kinetic analysis further revealed clusters of proteins and genes identifying unique states of activation, defined by markers temporarily expressed upon 3 days of stimulation (PD-1, CD69, LTA), markers constitutively expressed throughout chronic activation (CD25, GITR, LGALS1), and markers uniquely up-regulated upon 14 days of stimulation (CD39, ENTPD1, TNFDF10); expression of these markers could be associated with the emergence of short-lived cell types. Notably, different ratios of cells expressing activation or exhaustion markers were measured at each time point. These data reveal the high heterogeneity and plasticity of chronically stimulated T cells. Our study demonstrates the power of a single-cell multiomic approach to comprehensively characterize T-cells and to precisely monitor changes in differentiation, activation, and exhaustion signatures during cell stimulation.

Flow cytometric detection of most proteins in the cell surface proteome is unaffected by trypsin treatment.

Flow cytometry is often performed on adherent cells or solid tissues that have been released from their growth substrate or disaggregated by enzymatic digestion. Although detection of strongly expressed cell surface proteins following such procedures indicates that many survive treatment with proteolytic enzymes, applications such as cell surface proteomics involve assessment of the expression of more than 200 proteins and it is important to know how to interpret negative results. To address this problem, we performed flow cytometry-based cell surface proteomic analysis on two non-adherent cell lines, THP1 and K562, after mock and authentic trypsin treatment, according to a widely used protocol to remove adherent cells (0.25% trypsin, 2.21 mM EDTA, 37°C, 5 min). In a single screening experiment, we examined the effect of treatment on mean fluorescence intensity and on the percent of positive cells and determined the false negative rate. Of 164 determinations that were ≥20% positive after mock treatment, 13 (7.9%) were <20% positive after trypsin treatment. Four proteins were chosen for time-course studies (performed in triplicate), confirming initial sensitivity results but revealing significant variability in the magnitude of the trypsin effect. When trypsin sensitivity of individual proteins was examined as a function of the number of predicted high probability extracellular trypsin cleavage sites, we found that the markers that yielded false negatives all had high numbers of sites (>30), but even so, the majority of proteins with high numbers of trypsin sites could still be detected after mild trypsin treatment. We conclude that the great majority of cell surface proteins can be detected after mild trypsin treatment, but that negative results should not be over-interpreted, due to the possibility of false negatives.

Proteomic Profiling of Native Unpassaged and Culture-Expanded Mesenchymal Stromal Cells (MSC).

Human culture-expanded mesenchymal stromal cells (MSC) are being considered for multiple therapeutic applications because of their regenerative and anti-inflammatory properties. Although a large number of MSC can be propagated from a small initial sample, several lines of evidence indicate that MSC lose their immunosuppressive and regenerative potency aftaer multiple passages. In this report, we use the FACSCAP Lyoplate proteomic analysis system to detect changes in cell surface protein expression of CD45- /CD31- /CD34- /CD73+ /CD105+ stromal cells in unpassaged bone marrow (BM) and through 10 serial culture passages. We provide for the first time a detailed characterization of native unpassaged BM MSC (0.08% of BM mononuclear cells) as well as the changes that occur during the initial expansion. Adipogenic and osteogenic differentiative potential was determined though the serial passages and correlated with immunophenotypic changes and senescence. Among the most prominent were striking decreases in Fas ligand, CD98, CD205, and CD106, accompanied by a gain in the expression of CD49c, CD63, CD98, and class 1 and class 2 major histocompatibility complex (MHC) molecules. Other molecules that are down-modulated with later passage include CD24, CD54, CD59, CD243/P-glycoprotein, and CD273/PD-L2. Early senescence, as defined by the loss of replicative capacity occurring with the loss of differentiative capacity, increase in CDKN2A p16, and increased time to confluence, was accompanied by loss of the motility-associated metalloproteinase CD10 and the proliferation-associated transferrin receptor CD71. Among the strongest statistical associations were loss of MAC-inhibitory protein/CD59, loss of ICAM-1/CD54, and increase in CDKN2A as a function of increasing passage, as well as increased CD10 expression with adipogenic and osteogenic capacities. The data provide a clear set of markers that can be used to assess MSC quality. We suggest that clinically relevant numbers of highly functional low passage MSC can be manufactured starting with large quantities of BM, which are readily available from cadaveric organ donors.

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.

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.

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.

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.

Identification of perivascular mesenchymal stromal/stem cells by flow cytometry.

Mesenchymal stem/stromal cells (MSCs) are adult multipotent progenitors of great promise for cell therapy. MSCs can mediate tissue regeneration, immunomodulation, and hematopoiesis support. Despite the unique properties of MSCs and their broad range of potential clinical applications, the very nature of these cells has been uncertain. Furthermore, MSCs are heterogeneous and only defined subpopulations of these are endowed with the particular abilities to sustain hematopoietic stem cells, regulate immune responses, or differentiate into mesodermal cell lineages. It is becoming evident that current criteria used to define cultured polyclonal MSCs (expression of nonspecific markers and in vitro mesodermal differentiation) are not sufficient to fully understand and exploit the potential of these cells. Here, we describe how flow cytometry has been used to reveal a perivascular origin of MSCs. As a result, the prospective purification of MSCs and specialized subsets thereof is now possible, and the clinical use of purified autologous MSCs is now within reach.

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 PDGFRß, 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 blood-vessel-derived stem cells for tissue repair and regeneration.

Multipotent stem/progenitor cells with similar developmental potentials have been independently identified from diverse human tissue/organ cultures. The increasing recognition of the vascular/perivascular origin of mesenchymal precursors suggested blood vessels being a systemic source of adult stem/progenitor cells. Our group and other laboratories recently isolated multiple stem/progenitor cell subsets from blood vessels of adult human tissues. Each of the three structural layers of blood vessels: intima, media, and adventitia has been found to include at least one precursor population, that is, myogenic endothelial cells (MECs), pericytes, and adventitial cells (ACs), respectively. MECs and pericytes efficiently regenerate myofibers in injured and dystrophic skeletal muscles as well as improve cardiac function after myocardial infarction. The applications of ACs in vascular remodeling and angiogenesis/vasculogenesis have been examined. Our recent finding that MECs and pericytes can be purified from cryogenically banked human primary muscle cell culture further indicates their potential applications in personalized regenerative medicine.

Endothelial colony-forming cells from patients with chronic myeloproliferative disorders lack the disease-specific molecular clonality marker.

Two putative types of circulating endothelial progenitor cells have been recently identified in vitro: (1) endothelial colony-forming cell (ECFC) and (2) colony-forming unit-endothelial cell (CFU-EC). Only the former is now recognized to belong to endothelial lineage. We have used the ECFC and CFU-EC assays to readdress the issue of the clonal relation between endothelial progenitor cells and hematopoietic stem cells in patients with Philadelphia-positive and Philadelphia-negative chronic myeloproliferative disorders. Both ECFCs and CFU-ECs were cultured from peripheral blood mononuclear cells, and either BCR-ABL rearrangement or JAK2-V617F mutation were assessed in both types of endothelial colonies. We found that ECFCs lack the disease-specific markers, which are otherwise present in CFU-ECs, thus reinforcing the concept that the latter belongs to the hematopoietic lineage, and showing that in chronic myeloproliferative disorders the cell that gives rise to circulating ECFC has a distinct origin from the cell of the hematopoietic malignant clone.

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.

Diabetes impairs the vascular recruitment of normal stem cells by oxidant damage, reversed by increases in pAMPK, heme oxygenase-1, and adiponectin.

BACKGROUND: Atherosclerosis progression is accelerated in diabetes mellitus (DM) by either direct endothelial damage or reduced availability and function of endothelial progenitor cells (EPCs). Both alterations are related to increased oxidant damage. AIM: We examined if DM specifically impairs vascular signaling, thereby reducing the recruitment of normal EPCs, and if increases in antioxidant levels by induction of heme oxygenase-1 (HO-1) can reverse this condition. METHODS: Control and diabetic rats were treated with the HO-1 inducer cobalt protoporphyrin (CoPP) once a week for 3 weeks. Eight weeks after the development of diabetes, EPCs harvested from the aorta of syngenic inbred normal rats and labeled with technetium-99m-exametazime were infused via the femoral vein to estimate their blood clearance and aortic recruitment. Circulating endothelial cells (CECs) and the aortic expression of thrombomodulin (TM), CD31, and endothelial nitric oxide synthase (eNOS) were used to measure endothelial damage. RESULTS: DM reduced blood clearance and aortic recruitment of EPCs. Both parameters were returned to control levels by CoPP treatment without affecting EPC kinetics in normal animals. These abnormalities of EPCs in DM were paralleled by reduced serum adiponectin levels, increased numbers of CECs, reduced endothelial expression of phosphorylated eNOS, and reduced levels of TM, CD31, and phosphorylated AMP-activated protein kinase (pAMPK). CoPP treatment restored all of these parameters to normal levels. CONCLUSION: Type II DM and its related oxidant damage hamper the interaction between the vascular wall and normal EPCs by mechanisms that are, at least partially, reversed by the induction of HO-1 gene expression, adiponectin, and pAMPK levels.

Adoptive immunotherapy mediated by ex vivo expanded natural killer T cells against CD1d-expressing lymphoid neoplasms.

BACKGROUND: CD1d is a monomorphic antigen presentation molecule expressed in several hematologic malignancies. Alpha-galactosylceramide (alpha-GalCer) is a glycolipid that can be presented to cytotoxic CD1d-restricted T cells. These reagents represent a potentially powerful tool for cell mediated immunotherapy. DESIGN AND METHODS: We set up an experimental model to evaluate the use of adoptively transferred cytotoxic CD1d-restricted T cells and alpha-GalCer in the treatment of mice engrafted with CD1d(+) lymphoid neoplastic cells. To this end the C1R cell line was transfected with CD1c or CD1d molecules. In addition, upon retroviral infection firefly luciferase was expressed on C1R transfected cell lines allowing the evaluation of tumor growth in xenografted immunodeficient NOD/SCID mice. RESULTS: The C1R-CD1d cell line was highly susceptible to specific CD1d-restricted T cell cytotoxicity in the presence alpha-GalCer in vitro. After adoptive transfer of CD1d-restricted T cells and alpha-GalCer to mice engrafted with both C1R-CD1c and C1R-CD1d, a reduction in tumor growth was observed only in CD1d(+) masses. In addition, CD1d-restricted T-cell treatment plus alpha-GalCer eradicated small C1R-CD1d(+) nodules. Immunohistochemical analysis revealed that infiltrating NKT cells were mainly observed in CD1d nodules. CONCLUSIONS: Our results indicate that ex vivo expanded cytotoxic CD1d-restricted T cells and alpha-GalCer may represent a new immunotherapeutic tool for treatment of CD1d(+) hematologic malignancies.

Clinical scale ex vivo expansion of cord blood-derived outgrowth endothelial progenitor cells is associated with high incidence of karyotype aberrations.

OBJECTIVE: Endothelial progenitor cells (EPCs) are involved in neovessel formation. So far, therapeutic angiogenesis is hampered by the low frequency and limited proliferative potential of these cells isolated from peripheral blood. Recently, it has been shown that cord blood-derived EPCs (CB EPCs) can be ex vivo expanded on a clinical scale. In this study, we evaluated the expansion potential of CB EPCs together with their phenotypic, functional, and chromosomal stability over time. MATERIALS AND METHODS: Flow cytometry, in vitro tube formation, and proliferation assays were performed to characterize CB EPC-derived cells. Chromosomal stability was evaluated by karyotype analysis. In vitro and in vivo tumorigenicity was evaluated by soft agar assay and injection into nonobese diabetic/severe combined immunodeficient mice, respectively. RESULTS: We showed that CB EPC-derived cells displayed phenotypic and functional features of EPCs, although a process of maturation was observed over time. Although we confirmed that CB EPCs have a greater expansion potential compared to peripheral blood EPCS, we observed a high incidence of cytogenetic alterations (71%) in the expanded endothelial cell population, even at early times of culture. In two cases, spontaneous transformation in vitro was documented, but none of the samples tested showed tumorigenic potential in vivo. Conversely, no karyotype alterations have been observed on peripheral blood EPCs-derived cells. CONCLUSIONS: We confirm that CB represents a good source for clinical ex vivo expansion of EPCs. However, because of high frequency of karyotype alterations, these cells cannot be considered free of risk in clinical application.

Induction and survival of binucleated Purkinje neurons by selective damage and aging.

Fusion of bone marrow-derived cells with adult Purkinje cells in the cerebellum gives rise to binucleated Purkinje cells. Whether fusion can be modulated by epigenetic factors and whether fused neurons are stable has remained unclear. Here, we show that in mice and rats, partial ablation of Purkinje cells and local microglial activation in the absence of structural damage to the cerebellum increase the rate of fusion. Moreover, mouse Purkinje cells once fused with bone marrow-derived cells are viable for at least 7 months. We also show that cerebellar irradiation is unnecessary for the generation of binucleated Purkinje cells after bone marrow grafting. Moreover, binucleated Purkinje cells can be found in aged mice that did not receive any treatment, suggesting that fusion events occasionally occur throughout the whole lifespan of healthy, unmanipulated individuals. However, in aged chimeric mice that, after bone marrow transplant, have the majority of their nucleated blood cells fluorescent, the number of binucleated fluorescent Purkinje cells is two orders of magnitude less than the total number of binucleated Purkinje cells. This suggests that, in the majority of heterokaryons, either the incoming nucleus is quickly inactivated or fusion is not the only way to generate a binucleated Purkinje cell.

Mesenchymal stem cells protective effect in adriamycin model of nephropathy.

Mesenchymal stem cells (MSCs) may be of value in regeneration of renal tissue after damage; however, lack of biological knowledge and variability of results in animal models limit their utilization. We studied the effects of MSCs on podocytes in vitro and in vivo utilizing adriamycin (ADR) as a model of renal toxicity. The in vivo experimental approach was carried out in male Sprague-Dawley rats (overall 60 animals) treated with different ADR schemes to induce acute and chronic nephrosis. MSCs were given a) concomitantly to ADR in tail vein or b) in aorta and c) in tail vein 60 days after ADR. Homing was assessed with PKH26-MSCs. MSCs rescued podocytes from apoptosis induced by ADR in vitro. The maximal effect (80% rescue) was obtained with MSCs/podocytes coculture ratio of 1:1 for 72 h. All rats treated with ADR developed nephrosis. MSCs did not modify the clinical parameters (i.e., proteinuria, serum creatinine, lipids) but protected the kidney from severe glomerulosclerosis when given concomitantly to ADR. Rats given MSCs 60 days after ADR developed the same severe renal damage. Only a few MSCs were found in renal tubule-interstitial areas 1-24 h after injection and no MSCs were detected in glomeruli. MSCs reduced apoptosis of podocytes treated with ADR in vitro. Early and repeated MSCs infusion blunted glomerular damage in chronic ADR-induced nephropathy. MSCs did not modify proteinuria and progression to renal failure, which implies lack of regenerative potential in this model.

Transcriptionally and Functionally Distinct Mesenchymal Subpopulations Are Generated from Human Pluripotent Stem Cells.

Various mesenchymal cell types have been identified as critical components of the hematopoietic stem/progenitor cell (HSPC) niche. Although several groups have described the generation of mesenchyme from human pluripotent stem cells (hPSCs), the capacity of such cells to support hematopoiesis has not been reported. Here, we demonstrate that distinct mesenchymal subpopulations co-emerge from mesoderm during hPSC differentiation. Despite co-expression of common mesenchymal markers (CD73, CD105, CD90, and PDGFRß), a subset of cells defined as CD146hiCD73hi expressed genes associated with the HSPC niche and supported the maintenance of functional HSPCs ex vivo, while CD146loCD73lo cells supported differentiation. Stromal support of HSPCs was contact dependent and mediated in part through high JAG1 expression and low WNT signaling. Molecular profiling revealed significant transcriptional similarity between hPSC-derived CD146++ and primary human CD146++ perivascular cells. The derivation of functionally diverse types of mesenchyme from hPSCs opens potential avenues to model the HSPC niche and develop PSC-based therapies.

Cell-Surface Marker Signature for Enrichment of Ventricular Cardiomyocytes Derived from Human Embryonic Stem Cells.

To facilitate understanding of human cardiomyocyte (CM) subtype specification, and the study of ventricular CM biology in particular, we developed a broadly applicable strategy for enrichment of ventricular cardiomyocytes (VCMs) derived from human embryonic stem cells (hESCs). A bacterial artificial chromosome transgenic H9 hESC line in which GFP expression was driven by the human ventricular-specific myosin light chain 2 (MYL2) promoter was generated, and screened to identify cell-surface markers specific for MYL2-GFP-expressing VCMs. A CD77+/CD200- cell-surface signature facilitated isolation of >97% cardiac troponin I-positive cells from H9 hESC differentiation cultures, with 65% expressing MYL2-GFP. This study provides a tool for VCM enrichment when using some, but not all, human pluripotent stem cell lines. Tools generated in this study can be utilized toward understanding CM subtype specification, and enriching for VCMs for therapeutic applications.

Isolation of blood-vessel-derived multipotent precursors from human skeletal muscle.

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization of MSCs have been obscured by their retrospective isolation in culture. Recently, using fluorescence-activated cell sorting (FACS), we and other researchers prospectively identified and purified three subpopulations of multipotent precursor cells associated with the vasculature of human skeletal muscle. These three cell populations: myogenic endothelial cells (MECs), pericytes (PCs), and adventitial cells (ACs), are localized respectively to the three structural layers of blood vessels: intima, media, and adventitia. All of these human blood-vessel-derived stem cell (hBVSC) populations not only express classic MSC markers but also possess mesodermal developmental potentials similar to typical MSCs. Previously, MECs, PCs, and ACs have been isolated through distinct protocols and subsequently characterized in separate studies. The current isolation protocol, through modifications to the isolation process and adjustments in the selective cell surface markers, allows us to simultaneously purify all three hBVSC subpopulations by FACS from a single human muscle biopsy. This new method will not only streamline the isolation of multiple BVSC subpopulations but also facilitate future clinical applications of hBVSCs for distinct therapeutic purposes.