Mirko Corselli

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.

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

Summary 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 PDGFRβ), 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.

Abstract 2015: Multiparameter flow cytometry analysis of breast cancer cell lines MDA-MB-231 and MDA-MB-468 to simultaneously assess cell proliferation, apoptosis, and DNA damage in response to treatment with camptothecin

In cell analysis, separate functional assays are often utilized to monitor multiple functional states. To simultaneously interrogate multiple functional states, we have optimized simple flow cytometric panels that use up to four parameters. As a result, more detailed biological correlations between distinct cellular functions can be obtained from a single sample. In this study, multiparameter panels were used to analyze breast cancer cell lines MDA-MB-231 and MDA-MB-468 for differences in proliferation, apoptosis, and DNA damage in response to drug treatment. Cells were treated in culture with 20 μM camptothecin, and then analyzed on a two-laser BD Accuri™ C6 flow cytometer. In the first panel, we analyzed mitochondrial membrane potential (TMRE), phosphatidylserine exposure (Annexin V), and viability (7-AAD) to quantify early or late apoptotic and necrotic cells. In the second panel, we assessed double-stranded DNA breaks (phosphorylated H2AX), caspase activity (cleaved PARP), DNA synthesis (BrdU incorporation), and DNA content (7-AAD) to quantify DNA damage, apoptosis, and cell cycle status. In the third panel, we performed a more detailed cell cycle analysis by detection of DNA synthesis (BrdU incorporation), DNA content (7-AAD), M-phase associated histone expression (phosphorylated histone H3), and cyclin content (cyclin A or B). In response to camptothecin treatment, both cell lines showed decreased proliferation, increased DNA damage responses, increased apoptosis, and decreased viability. The presence of mitochondrial depolarization, caspase activity, and phosphatidylserine exposure suggests an apoptotic death mechanism for both cell types. Compared to MDA-MB-231 cells, MDA-MB-468 cells showed higher levels of mitochondrial depolarization, phosphatidylserine exposure, caspase activity, and loss of viability indicating increased drug susceptibility. Additionally, while both MDA-MB-231 and MDA-MB-468 cells showed decreased proliferation and an increased DNA damage response, the cell lines showed different cyclin expression across cell cycle compartments, potentially suggesting different cellular responses to drug treatment. Multiparameter flow cytometry enables the simultaneous analysis of cell proliferation, viability status, and other cellular states at the single cell level and serves as an invaluable and tool to understand the response of heterogeneous cancer cells to drug treatment. When combined with an automated platform for sample acquisition, this method can provide an effective tool for high throughput drug screening applications. Citation Format: Lissette Wilensky, Stacey Roys, Mirko Corselli, Alice Wang, Nil Emre. Multiparameter flow cytometry analysis of breast cancer cell lines MDA-MB-231 and MDA-MB-468 to simultaneously assess cell proliferation, apoptosis, and DNA damage in response to treatment with camptothecin. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2015. doi:10.1158/1538-7445.AM2015-2015

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

Summary 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.

Abstract 5082: A quantitative and multi-parameter flow cytometry assay to simultaneously assess cell migration and immunophenotype

Cell migration occurs during physiological and pathological processes, which include wound healing and tumor metastasis. In vitro migration assays are necessary to understand the mechanism underlying cell migration and also to identify inhibitory or stimulatory molecules. The Boyden chamber assay is commonly used to measure cell motility in vitro where the number of cells that migrate through the transwell membrane is quantitated manually using an inverted microscope. Alternatively, cells can be detached from the membrane and stained with a fluorescent probe, prior to counting cells using a plate reader. These conventional assays are limited by an inability to simultaneously characterize individual cells while monitoring migration. To address this, we have developed a multi-parameter flow cytometry-based migration assay for the quantitative measurement of cell migration and simultaneous immunophenotypic analysis of the migrated cells. Invasive HT-1080 and non-invasive MCF-7 cancer cell lines were plated in the upper layer of a cell permeable membrane, as per the standard Boyden chamber assay. BD Horizon™ Fixable Viability Stain (FVS) was used to determine the optimal detachment conditions providing maximum yield with minimal impact on cell viability. Migrated cells were also co-stained with multiple conjugated antibodies to assess cell surface marker expression. Cells were analyzed on BD Accuri™ flow cytometer for rapid quantitation of migrated cells and analysis of up to 4 parameters. Alternatively, BD FACSCelesta™ flow cytometer was used for higher parameter analysis. Migrated cells were identified based on light scatter properties and viability staining with FVS, while immunophenotype was assessed in live cells. Our results demonstrate that this flow cytometric assay can be used to rapidly quantify changes in the migratory ability of different cell lines in the presence of inhibitors or stimulators. Additionally, we were able to assess the expression of hallmark cancer cell markers (CD44, CD24, CD326 (EpCAM)) before and after migration. The ability to quantify the number of migrating cells in response to specific stimuli and to simultaneously define cell immunophenotype represents a significant advancement, as compared to conventional cell motility assays. Our results demonstrate that this novel approach allows for a deeper analysis of cell migration that could enable complex drug discovery studies and the discovery of new cell signatures associated with metastatic progression. Citation Format: Mirko Corselli, Xiao Wang, Lissette Wilensky, Aaron Middlebrook, Smita Ghanekar, Jacob Rabenstein, Nil Emre. A quantitative and multi-parameter flow cytometry assay to simultaneously assess cell migration and immunophenotype. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5082.

Multiparameter Flow Cytometry Applications for Analyzing and Isolating Neural Cell Populations Derived from Human Pluripotent Stem Cells

Pluripotent stem cells (PSCs) have been successfully differentiated to many cell types relevant to human neurodegenerative diseases. Moreover, induced pluripotent stem cell technology offers an unprecedented opportunity to study these diseases in a patient-specific manner. One impediment to this research is that neural differentiations from PSCs often yield heterogeneous cell cultures, which can hamper or preclude studies requiring pure cell populations. Researchers have partially overcome this hurdle by identifying cell surface signatures of neural stem cells, neurons, glia, and neural crest cells and utilized flow cytometry to isolate near-pure populations of these cells. These advancements have enabled groundbreaking research models of neurodegenerative diseases and have been instrumental in animal models of spinal cord injury. In addition, flow cytometry has been employed to facilitate quantification of neural differentiation cultures. The identification of novel cell surface signatures has also been instrumental in a more comprehensive characterization of pluripotent stem cell derivatives, as well as adult and cancer stem cells. All together, these studies have demonstrated that multiparameter flow cytometry is an indispensable tool for studying neurogenesis and other cell lineages utilizing human PSCs.