Exploring analytical proteomics platforms toward the definition of human cardiac stem cells receptome

Human cardiac stem cells (hCSC) express a portfolio of plasma membrane receptors that are involved in the regulatory auto/paracrine feedback loop mechanism of activation of these cells, and consequently contribute to myocardial regeneration. In order to attain a comprehensive description of hCSC receptome and overcoming the inability demonstrated by other technologies applied in receptor identification, mainly due to the transmembrane nature, high hydrophobic character and relative low concentration of these proteins, we have exploited and improved a proteomics workflow. This approach was based on the enrichment of hCSC plasma membrane fraction and addition of prefractionation steps prior to MS analysis. More than 100 plasma membrane receptors were identified. The data reported herein constitute a valuable source of information to further understand cardiac stem cells activation mechanisms and the subsequent cardiac repair process. All MS data have been deposited in the ProteomeXchange with identifier PXD001117 (http://proteomecentral.proteomexchange.org/dataset/PXD001117).Authors acknowledge FP7 EU project CARE-MI (HEALTH-2009_242038) and the Portuguese Foundation for Science and Technology (PTDC/BBBBIO/1414) for financial support. PGA is a recipient of the FCT fellowship SFRH/BPD/86513/2012. MALDI-TOF/TOF analyses were performed at the Mass Spectrometry Unit (UniMS), ITQB/iBET, Oeiras, Portugal. The data deposition to the ProteomeXchange Consortium was supported by PRIDETeam, EBI.S

CXCL6 is an important paracrine factor in the pro-angiogenic human cardiac progenitor-like cell secretome

Studies in recent years have established that the principal effects in cardiac cell therapy are associated with paracrine/autocrine factors. We combined several complementary techniques to define human cardiac progenitor cell (CPC) secretome constituted by 914 proteins/genes; 51% of these are associated with the exosomal compartment. To define the set of proteins specifically or highly differentially secreted by CPC, we compared human mesenchymal stem cells and dermal fibroblasts; the study defined a group of growth factors, cytokines and chemokines expressed at high to medium levels by CPC. Among them, IL-1, GROa (CXCL1), CXCL6 (GCP2) and IL-8 are examples whose expression was confirmed by most techniques used. ELISA showed that CXCL6 is significantly overexpressed in CPC conditioned medium (CM) (18- to 26-fold) and western blot confirmed expression of its receptors CXCR1 and CXCR2. Addition of anti-CXCL6 completely abolished migration in CPC-CM compared with anti-CXCR2, which promoted partial inhibition, and anti-CXCR1, which was inefficient. Anti-CXCL6 also significantly inhibited CPC CM angiogenic activity. In vivo evaluation also supported a relevant role for angiogenesis. Altogether, these results suggest a notable angiogenic potential in CPC-CM and identify CXCL6 as an important paracrine factor for CPC that signals mainly through CXCR2.This study was supported by funding from the European Commission (HEALTH-2009_242038) and by grants from the Spanish Ministry of Science and Innovation (SAF2012-34327 and SAF2015-70882-R to AB and BIO2012-37926 and BIO2015-67580-P to JV), the Research Program of the Comunidad Autónoma de Madrid (S2010/BMD-2420) and the Instituto de Salud Carlos III (RETICS-RD12/0019/0018 to AB and RETICS-RD12/0042/0056 to JV).S

The catalytic domain of pp56(lck), but not its regulatory domain, is sufficient for inducing IL-2 production

The lymphoid src kinase pp56lck has been shown to be essential for the induction of different T lymphocyte responses, including CD4-mediated enhancement of Ag-induced T cell activation, early T cell differentiation, induction of IL-2 production, and cytotoxicity. It is assumed that pp56lck acts on these processes by phosphorylating substrates. However, it has been recently reported that the NH2 regulatory domain is sufficient to mediate CD4 accessory function. In this report we address the contribution of the regulatory and catalytic domains of pp56lck to another function of this enzyme independent of CD4: TCR-induced IL-2 production. Two pp56lck mutants lacking either the entire catalytic domain or the entire NH2 regulatory domain were generated, and their abilities to trigger transactivation of the TCR-regulated nuclear factor of activated T cells (NF-AT) region of the IL-2 promoter were compared. Only the catalytic, but not the NH2 regulatory, domain of pp56lck was able to induce NF-AT region transactivation on its own and to cooperate with other intracellular signals to trigger this response. Moreover, the catalytic domain of pp56lck was able to induce IL-2 cytokine production to an extent similar to that of wild-type pp56lck. We conclude that different domains of the pp56lck molecule contribute to regulate distinct biologic functions. In fact, while the NH2 regulatory domain is sufficient to mediate CD4 accessory function, we show here that the catalytic domain of pp56lck is sufficient for induction of IL-2 production, mimicking TCR ligation.This work was supported by the CICyT, Pharmacia, Consejo Superiro de Investigaciones Científicas, and the José Carreras International Foundation.Peer Reviewe

Definition of a cell surface signature for human cardiac progenitor cells after comprehensive comparative transcriptomic and proteomic characterization

Adult cardiac progenitor/stem cells (CPC/CSC) are multipotent resident populations involved in cardiac homeostasis and heart repair. Assisted by complementary RNAseq analysis, we defined the fraction of the CPC proteome associable with specific functions by comparison with human bone marrow mesenchymal stem cells (MSC), the reference population for cell therapy, and human dermal fibroblasts (HDF), as a distant reference. Label-free proteomic analysis identified 526 proteins expressed differentially in CPC. iTRAQ analysis confirmed differential expression of a substantial proportion of those proteins in CPC relative to MSC, and systems biology analysis defined a clear overrepresentation of several categories related to enhanced angiogenic potential. The CPC plasma membrane compartment comprised 1,595 proteins, including a minimal signature of 167 proteins preferentially or exclusively expressed by CPC. CDH5 (VE-cadherin),  OX2G (OX-2 membrane glycoprotein; CD200), GPR4 (G protein-coupled receptor 4), CACNG7 (calcium voltage-gated channel auxiliary subunit gamma 7) and F11R (F11 receptor; junctional adhesion molecule A; JAM-A; CD321) were selected for validation. Their differential expression was confirmed both in expanded CPC batches and in early stages of isolation, particularly when compared against cardiac fibroblasts. Among them, GPR4 demonstrated the highest discrimination capacity between all cell lineages analyzed.This study was supported by funding from the European Commission (HEALTH-2009_242038), and by grants to AB from the Spanish Ministry of Science and Innovation (SAF2012-34327; SAF2015-70882-R), the Research Program of the Comunidad Autónoma de Madrid (S2011/BMD-2420), the Instituto de Salud Carlos III (RETICS-RD12/0019/0018), and grants from the Portuguese Foundation for Science and Technology (PTDC/ BBB-BIO/1414) to PMA. iNOVA4Health - UID/Multi/04462/2013, financially supported by FCT/Ministério da Educação e Ciência, through national funds and co-funded by FEDER under the PT2020 Partnership Agreement is also acknowledged. JL Abad, I Palacios and LR Borlado were employees of Coretherapix; Coretherapix is part of Tigenix Group since July 2015. The other authors declare no conflict of interest.S

Deregulated expression of Cdc6 in the skin facilitates papilloma formation and affects the hair growth cycle.

Cdc6 encodes a key protein for DNA replication, responsible for the recruitment of the MCM helicase to replication origins during the G1 phase of the cell division cycle. The oncogenic potential of deregulated Cdc6 expression has been inferred from cellular studies, but no mouse models have been described to study its effects in mammalian tissues. Here we report the generation of K5-Cdc6, a transgenic mouse strain in which Cdc6 expression is deregulated in tissues with stratified epithelia. Higher levels of CDC6 protein enhanced the loading of MCM complexes to DNA in epidermal keratinocytes, without affecting their proliferation rate or inducing DNA damage. While Cdc6 overexpression did not promote skin tumors, it facilitated the formation of papillomas in cooperation with mutagenic agents such as DMBA. In addition, the elevated levels of CDC6 protein in the skin extended the resting stage of the hair growth cycle, leading to better fur preservation in older mice.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Definition of a cell surface signature for human cardiac progenitor cells after comprehensive comparative transcriptomic and proteomic characterization

© The Author(s) 2019.Adult cardiac progenitor/stem cells (CPC/CSC) are multipotent resident populations involved in cardiac homeostasis and heart repair. Assisted by complementary RNAseq analysis, we defined the fraction of the CPC proteome associable with specific functions by comparison with human bone marrow mesenchymal stem cells (MSC), the reference population for cell therapy, and human dermal fibroblasts (HDF), as a distant reference. Label-free proteomic analysis identified 526 proteins expressed differentially in CPC. iTRAQ analysis confirmed differential expression of a substantial proportion of those proteins in CPC relative to MSC, and systems biology analysis defined a clear overrepresentation of several categories related to enhanced angiogenic potential. The CPC plasma membrane compartment comprised 1,595 proteins, including a minimal signature of 167 proteins preferentially or exclusively expressed by CPC. CDH5 (VE-cadherin), OX2G (OX-2 membrane glycoprotein; CD200), GPR4 (G protein-coupled receptor 4), CACNG7 (calcium voltage-gated channel auxiliary subunit gamma 7) and F11R (F11 receptor; junctional adhesion molecule A; JAM-A; CD321) were selected for validation. Their differential expression was confirmed both in expanded CPC batches and in early stages of isolation, particularly when compared against cardiac fibroblasts. Among them, GPR4 demonstrated the highest discrimination capacity between all cell lineages analyzed.This study was supported by funding from the European Commission (HEALTH-2009_242038), and by grants to AB from the Spanish Ministry of Science and Innovation (SAF2012-34327; SAF2015-70882-R), the Research Program of the Comunidad Autónoma de Madrid (S2011/BMD-2420), the Instituto de Salud Carlos III (RETICS-RD12/0019/0018), and grants from the Portuguese Foundation for Science and Technology (PTDC/ BBB-BIO/1414) to PMA. iNOVA4Health - UID/Multi/04462/2013, fnancially supported by FCT/Ministério da Educação e Ciência, through national funds and co-funded by FEDER under the PT2020 Partnership Agreement is also acknowledged. JL Abad, I Palacios and LR Borlado were employees of Coretherapix; Coretherapix is part of Tigenix Group since July 2015