PDGFRA defines the mesenchymal stem cell Kaposi's sarcoma progenitors by enabling KSHV oncogenesis in an angiogenic environment

Kaposi’s sarcoma (KS) is an AIDS-defining cancer caused by the KS-associated herpesvirus (KSHV). Unanswered questions regarding KS are its cellular ontology and the conditions conducive to viral oncogenesis. We identify PDGFRA(+)/SCA-1(+) bone marrow-derived mesenchymal stem cells (Pα(+)S MSCs) as KS spindle-cell progenitors and found that pro-angiogenic environmental conditions typical of KS are critical for KSHV sarcomagenesis. This is because growth in KS-like conditions generates a de-repressed KSHV epigenome allowing oncogenic KSHV gene expression in infected Pα(+)S MSCs. Furthermore, these growth conditions allow KSHV-infected Pα(+)S MSCs to overcome KSHV-driven oncogene-induced senescence and cell cycle arrest via a PDGFRA-signaling mechanism; thus identifying PDGFRA not only as a phenotypic determinant for KS-progenitors but also as a critical enabler for viral oncogenesis.Fil: Naipauer, Julian. Miami University; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Sylvester Comprehensive Cancer Center and Miami Center for AIDS Research; Estados UnidosFil: Rosario, Santas. Miami University; Estados Unidos. Sylvester Comprehensive Cancer Center and Miami Center for AIDS Research; Estados UnidosFil: Gupta, Sachin. Miami University; Estados Unidos. Sylvester Comprehensive Cancer Center and Miami Center for AIDS Research; Estados UnidosFil: Premer, Courtney. Miami University; Estados UnidosFil: Méndez Solís, Omayra. Miami University; Estados Unidos. Sylvester Comprehensive Cancer Center and Miami Center for AIDS Research; Estados UnidosFil: Schlesinger, Mariana. Miami University; Estados Unidos. Sylvester Comprehensive Cancer Center and Miami Center for AIDS Research; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Ponzinibbio, Maria Virginia. Miami University; Estados Unidos. Sylvester Comprehensive Cancer Center and Miami Center for AIDS Research; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Jain, Vaibhav. University of Florida; Estados UnidosFil: Gay, Lauren. University of Florida; Estados UnidosFil: Renne, Rolf. University of Florida; Estados UnidosFil: Chan, Ho Lam. Miami University; Estados UnidosFil: Morey, Lluis. Miami University; Estados UnidosFil: Salyakina, Daria. Miami University; Estados Unidos. Sylvester Comprehensive Cancer Center and Miami Center for AIDS Research; Estados UnidosFil: Abba, Martín Carlos. Miami University; Estados Unidos. Universidad Nacional de La Plata. Facultad de Ciencias Médicas. Centro de Investigaciones Inmunológicas Básicas y Aplicadas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Williams, Sion. Miami University; Estados UnidosFil: Hare, Joshua M.. Miami University; Estados UnidosFil: Goldschmidt Clermont, Pascal. Miami University; Estados UnidosFil: Mesri, Enrique Alfredo. Sylvester Comprehensive Cancer Center and Miami Center for AIDS Research; Estados Unidos. Miami University; Estados Unido

PDGFRA defines the mesenchymal stem cell Kaposi's sarcoma progenitors by enabling KSHV oncogenesis in an angiogenic environment

Kaposi’s sarcoma (KS) is an AIDS-defining cancer caused by the KS-associated herpesvirus (KSHV). Unanswered questions regarding KS are its cellular ontology and the conditions conducive to viral oncogenesis. We identify PDGFRA(+)/SCA-1(+) bone marrow-derived mesenchymal stem cells (Pα(+)S MSCs) as KS spindle-cell progenitors and found that pro-angiogenic environmental conditions typical of KS are critical for KSHV sarcomagenesis. This is because growth in KS-like conditions generates a de-repressed KSHV epigenome allowing oncogenic KSHV gene expression in infected Pα(+)S MSCs. Furthermore, these growth conditions allow KSHV-infected Pα(+)S MSCs to overcome KSHV-driven oncogene-induced senescence and cell cycle arrest via a PDGFRA-signaling mechanism; thus identifying PDGFRA not only as a phenotypic determinant for KS-progenitors but also as a critical enabler for viral oncogenesis.Centro de Investigaciones Inmunológicas Básicas y Aplicada

MicroRNA-10A* and MicroRNA-21 Modulate Endothelial Progenitor Cell Senescence Via Suppressing High-Mobility Group A2

RATIONALE: Endothelial progenitor cells (EPCs) contribute to the regeneration of endothelium. Aging-associated senescence results in reduced number and function of EPCs, potentially contributing to increased cardiac risk, reduced angiogenic capacity, and impaired cardiac repair effectiveness. The mechanisms underlying EPC senescence are unknown. Increasing evidence supports the role of microRNAs in regulating cellular senescence. OBJECTIVE: We aimed to determine whether microRNAs regulated EPC senescence and, if so, what the underlying mechanisms are. METHODS AND RESULTS: To map the microRNA/gene expression signatures of EPC senescence, we performed microRNA profiling and microarray analysis in lineage-negative bone marrow cells from young and aged wild-type and apolipoprotein E–deficient mice. We identified 2 microRNAs, microRNA-10A* (miR-10A*), and miR-21, and their common target gene Hmga2 as critical regulators for EPC senescence. Overexpression of miR-10A* and miR-21 in young EPCs suppressed Hmga2 expression, caused EPC senescence, as evidenced by senescence-associated β–galactosidase upregulation, decreased self-renewal potential, increased p16(Ink4a)/p19(Arf) expression, and resulted in impaired EPC angiogenesis in vitro and in vivo, resembling EPCs derived from aged mice. In contrast, suppression of miR-10A* and miR-21 in aged EPCs increased Hmga2 expression, rejuvenated EPCs, resulting in decreased senescence-associated β–galactosidase expression, increased self-renewal potential, decreased p16(Ink4a)/p19(Arf) expression, and improved EPC angiogenesis in vitro and in vivo. Importantly, these phenotypic changes were rescued by miRNA-resistant Hmga2 cDNA overexpression. CONCLUSIONS: miR-10A* and miR-21 regulate EPC senescence via suppressing Hmga2 expression and modulation of microRNAs may represent a potential therapeutic intervention in improving EPC-mediated angiogenesis and vascular repair

VSX2 and ASCL1 Are Indicators of Neurogenic Competence in Human Retinal Progenitor Cultures

<div><p>Three dimensional (3D) culture techniques are frequently used for CNS tissue modeling and organoid production, including generation of retina-like tissues. A proposed advantage of these 3D systems is their potential to more closely approximate <i>in vivo</i> cellular microenvironments, which could translate into improved manufacture and/or maintenance of neuronal populations. Visual System Homeobox 2 (VSX2) labels all multipotent retinal progenitor cells (RPCs) and is known to play important roles in retinal development. In contrast, the proneural transcription factor Acheate scute-like 1 (ASCL1) is expressed transiently in a subset of RPCs, but is required for the production of most retinal neurons. Therefore, we asked whether the presence of VSX2 and ASCL1 could gauge neurogenic potential in 3D retinal cultures derived from human prenatal tissue or ES cells (hESCs). Short term prenatal 3D retinal cultures displayed multiple characteristics of human RPCs (hRPCs) found <i>in situ</i>, including robust expression of VSX2. Upon initiation of hRPC differentiation, there was a small increase in co-labeling of VSX2+ cells with ASCL1, along with a modest increase in the number of PKCα+ neurons. However, 3D prenatal retinal cultures lost expression of VSX2 and ASCL1 over time while concurrently becoming refractory to neuronal differentiation. Conversely, 3D optic vesicles derived from hESCs (hESC-OVs) maintained a robust VSX2+ hRPC population that could spontaneously co-express ASCL1 and generate photoreceptors and other retinal neurons for an extended period of time. These results show that VSX2 and ASCL1 can serve as markers for neurogenic potential in cultured hRPCs. Furthermore, unlike hESC-OVs, maintenance of 3D structure does not independently convey an advantage in the culture of prenatal hRPCs, further illustrating differences in the survival and differentiation requirements of hRPCs extracted from native tissue vs. those generated entirely <i>in vitro</i>.</p></div

VSX2+ hRPCs are abundant within short and long term cultures of 3D optic vesicles derived from hESCs.

<p>20 days after initiation of retinal differentiation [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135830#pone.0135830.ref020" target="_blank">20</a>], WA09 hESCs formed optic vesicle structures (OVs) comprised of VSX2+ hRPCs co-expressing (<b>A</b>) KI67, (<b>B</b>) NESTIN, and (<b>C</b>) SOX2. (<b>D-F</b>) At day 50, the majority of VSX2+ hRPCs remained KI67+. <b>(G)</b> VSX2+/KI67+ progenitors were also present at day 50 in hESC-OVs derived from the WA01 line. 50 day VSX2+ hRPCs continued to express (<b>H</b>) NESTIN and (<b>I</b>) SOX2. (<b>J</b>) At 20, 50, and 90 days of differentiation, the percentages of VSX2+ or KI67+ cells and (<b>K</b>) VSX2+ cells co-labeled with other progenitor markers were quantified. Nuclei were visualized with DAPI and cell count data is expressed as % immunopositive cells. Scale bar: 50 μm (panels A-D,H,I); 20 μm (panels E,F,G).</p

NOTCH inhibition augments production of PKCα+ neurons in short term prenatal human retinal neurospheres.

<p>The percentages of cells immunopositive for (<b>A</b>) ASCL1 and (<b>B</b>) other selected hRPC and neuronal markers following DAPT treatment was determined by immunocytochemistry. (<b>C</b>) RT-PCR analysis was used to evaluate expression of VSX2 and ASCL1 in prenatal human retinal tissue and short (1 week) and long (2 month) term prenatal retinal neurosphere cultures. (<b>D</b>) Phase photomicrographs of dissociated prenatal retinal neurospheres reveal profound cell morphology differences between short term and long term cultures. Cell counts are expressed as % immunopositive cells. *p<0.05; **p<0.01. Scale bars in panel E: 50 μm.</p

Inflammation, stem cells and atherosclerosis genetics

Atherosclerosis and its associated complications remain the primary cause of death in humans. Aging is the main contributor to atherosclerosis, compared with any other risk factor, yet the specific manner in which age increases risk (the 'aging-risk' mechanism) remains elusive. A novel concept for atherosclerosis risk implicates a lack of endothelial progenitor cell (EPC)-dependent arterial repair in the development of the disease that is secondary to exhaustion of repair-competent EPCs. Molecular evidence derived from genetic techniques indicates atherosclerotic lesions may begin to form as arterial repair fails, rather than merely following arterial injury. Thus, chronic arterial injury may overwhelm the ability of EPCs to maintain arterial homeostasis, particularly when EPCs capable of arterial repair become exhausted. Recent studies have reported genes identified using non-biased approaches (ie, genetic linkage studies and genome-wide association studies) that are associated with susceptibility for atherosclerosis and related thromboembolic disorders; these genes may be implicated in the control of arterial wall inflammation and EPC-mediated tissue repair. Most of the genes identified by using non-biased genomic techniques are associated with inflammation, immune response and stem cells. This review focuses on new genetic data in the field of atherosclerosis and arterial homeostasis

Short term cultures of human retinal neurospheres retain a robust population of VSX2+ proliferating progenitor cells from source prenatal retinal tissue.

<p>VSX2+/KI67+ proliferating hRPCs were observed in the outer neuroblastic layer of the developing retina at (<b>A</b>) 59 days, (<b>B</b>) 73 days, (<b>C</b>) 85 days, (<b>D</b>) 93 days, and (<b>E</b>) 108 days of gestation. (<b>F-I</b>) VSX2+/KI67+ co-labeled cells were also present in dissociated cells from short term prenatal retinal neurospheres established from retinal tissue of similar gestational ages. (<b>J</b>) Short term prenatal retinal neurospheres were dissociated and immunostained to determine the percentage of cells expressing VSX2 and/or KI67. Nuclei were visualized with DAPI. The insert is a 4X magnification of the indicated area in panel A. Scale bars: 100 μm (panel A); 50 μm (panels B-E); 20 μm (panels F-I).</p

Prenatal retinal neurospheres lose VSX2 expression over time in culture.

<p>KI67+ hRPCs in the outer neuroblastic layer of 96 day human prenatal retina co-express the neural stem cell markers (<b>A</b>) NESTIN and (<b>B</b>) SOX2. Short term prenatal retinal neurosphere cultures also contain abundant (<b>C</b>) KI67+/NESTIN+ and (<b>D</b>) KI67+/SOX2+ hRPCs. Nearly all VSX2+ hRPCs in short term prenatal retinal neurosphere cultures co-label with (<b>E</b>) NESTIN and (<b>F</b>) SOX2. Prenatal retinal neurosphere cultures (n = 5) from 79–108 day gestation tissue were sampled at 1 week, 1 month, and 2 months. After 2 months, very little VSX2 immunostaining is detected, although (<b>G</b>) NESTIN and (<b>H</b>) SOX2 remain highly expressed. The percentage of VSX2, KI67, NESTIN, and SOX2 immunopositive cells were quantified (<b>I</b>) in short term cultures and (<b>J</b>) over a 2 month period. Nuclei were visualized with DAPI and cell count data is expressed as % immunopositive cells. Scale bars: 50 μm (panels A,B); 20 μm (panels C-H).</p