Single-cell transcriptomic profiling and characterization of endothelial progenitor cells: new approach for finding novel markers

Background Endothelial progenitor cells (EPCs) are promising candidates for the cellular therapy of peripheral arterial and cardiovascular diseases. However, hitherto there is no specific marker(s) defining precisely EPCs. Herein, we are proposing a new in silico approach for finding novel EPC markers. Methods We assembled five groups of chosen EPC-related genes/factors using PubMed literature and Gene Ontology databases. This shortened database of EPC factors was fed into publically published transcriptome matrix to compare their expression between endothelial colony-forming cells (ECFCs), HUVECs, and two adult endothelial cell types (ECs) from the skin and adipose tissue. Further, the database was used for functional enrichment on Mouse Phenotype database and protein-protein interaction network analyses. Moreover, we built a digital matrix of healthy donors’ PBMCs (33 thousand single-cell transcriptomes) and analyzed the expression of these EPC factors. Results Transcriptome analyses showed that BMP2, 4, and ephrinB2 were exclusively highly expressed in EPCs; the expression of neuropilin-1 and VEGF-C were significantly higher in EPCs and HUVECs compared with other ECs; Notch 1 was highly expressed in EPCs and skin-ECs; MIR21 was highly expressed in skin-ECs; PECAM-1 was significantly higher in EPCs and adipose ECs. Moreover, functional enrichment of EPC-related genes on Mouse Phenotype and STRING protein database has revealed significant relations between chosen EPC factors and endothelial and vascular functions, development, and morphogenesis, where ephrinB2, BMP2, and BMP4 were highly expressed in EPCs and were connected to abnormal vascular functions. Single-cell RNA-sequencing analyses have revealed that among the EPC-regulated markers in transcriptome analyses, (i) ICAM1 and Endoglin were weekly expressed in the monocyte compartment of the peripheral blood; (ii) CD163 and CD36 were highly expressed in the CD14+ monocyte compartment whereas CSF1R was highly expressed in the CD16+ monocyte compartment, (iii) L-selectin and IL6R were globally expressed in the lymphoid/myeloid compartments, and (iv) interestingly, PLAUR/UPAR and NOTCH2 were highly expressed in both CD14+ and CD16+ monocytic compartments. Conclusions The current study has identified novel EPC markers that could be used for better characterization of EPC subpopulation in adult peripheral blood and subsequent usage of EPCs for various cell therapy and regenerative medicine applications.

Single-Cell Transcriptomic Profiling and Characterization of Endothelial Progenitor Cells: New Approach for Finding Novel Markers

Background: Endothelial progenitor cells (EPCs) are promising candidates for the cellular therapy of peripheral arterial & cardiovascular diseases. However, hitherto there is no specific marker(s) defining precisely EPCs. Herein, we are proposing a new in-silico approach for finding novel EPCs markers. Methods: we assembled five groups of chosen EPCs-related genes/factors using Pubmed literature & gene ontology databases. This shortened database of EPCs factors was fed into publically-published transcriptome matrix to compare their expression between endothelial colony-forming cells (ECFCs), HUVECs and two adult endothelial cell types (ECs) from skin and adipose tissue. Further, the database was used for functional enrichment on mouse phenotype database and protein-protein interaction network analyses. Moreover, we built a digital matrix of healthy donors' PBMCs (33 thousand single cell transcriptomes) and analyzed the expression of these EPCs factors Results: Transcriptome analyses showed that BMP2,4 & ephrinB2 were exclusively highly expressed in EPCs; the expression of neuropilin-1 & VEGF-C were significantly higher in EPCs & HUVECs compared with other ECs; Notch 1 was highly expressed in EPCs & skin-ECs; MIR21 was highly expressed in skin-ECs; PECAM-1 were significantly higher in EPCs & adipose ECs. Moreover, functional enrichment of EPCs-related genes on mouse phenotype and STRING protein database has revealed significant relations between chosen EPCs factors and endothelial & vascular functions, development and morphogenesis, where ephrinB2, BMP2 and BMP4 were highly expressed in EPCs and were connected to abnormal vascular functions. Single cell RNA-sequencing analyses has revealed that among the EPCs regulated markers in transcriptome analyses: i-ICAM1 & Endoglin were weekly expressed in the monocyte compartment of peripheral blood; ii-CD163 & CD36 were highly expressed in CD14+ monocyte compartment whereas CSF1R was highly expressed in CD16+ monocyte compartment; iii-L-selectin & IL6R were globally expressed in the lymphoid/myeloid compartments; iv-interestingly, PLAUR/UPAR & NOTCH2 were highly expressed in both CD14+ & CD16+ monocytic compartments. Conclusions: The current study has identified novel EPCs markers that could be used for better characterization of EPCs sub-population in adult peripheral blood and subsequent usage of EPCs for various cell therapy and regenerative medicine applications.

Single-Cell Transcriptomic Profiling and Characterization of Endothelial Progenitor Cells: New Approach for Finding Novel Markers

Background: Endothelial progenitor cells (EPCs) are promising candidates for the cellular therapy of peripheral arterial & cardiovascular diseases. However, hitherto there is no specific marker(s) defining precisely EPCs. Herein, we are proposing a new in-silico approach for finding novel EPCs markers. Methods: we assembled five groups of chosen EPCs-related genes/factors using Pubmed literature & gene ontology databases. This shortened database of EPCs factors was fed into publically-published transcriptome matrix to compare their expression between endothelial colony-forming cells (ECFCs), HUVECs and two adult endothelial cell types (ECs) from skin and adipose tissue. Further, the database was used for functional enrichment on mouse phenotype database and protein-protein interaction network analyses. Moreover, we built a digital matrix of healthy donors' PBMCs (33 thousand single cell transcriptomes) and analyzed the expression of these EPCs factorsResults: Transcriptome analyses showed that BMP2,4 & ephrinB2 were exclusively highly expressed in EPCs; the expression of neuropilin-1 & VEGF-C were significantly higher in EPCs & HUVECs compared with other ECs; Notch 1 was highly expressed in EPCs & skin-ECs; MIR21 was highly expressed in skin-ECs; PECAM-1 were significantly higher in EPCs & adipose ECs. Moreover, functional enrichment of EPCs-related genes on mouse phenotype and STRING protein database has revealed significant relations between chosen EPCs factors and endothelial & vascular functions, development and morphogenesis, where ephrinB2, BMP2 and BMP4 were highly expressed in EPCs and were connected to abnormal vascular functions. Single cell RNA-sequencing analyses has revealed that among the EPCs regulated markers in transcriptome analyses: i-ICAM1 & Endoglin were weekly expressed in the monocyte compartment of peripheral blood; ii-CD163 & CD36 were highly expressed in CD14+ monocyte compartment whereas CSF1R was highly expressed in CD16+ monocyte compartment; iii-L-selectin & IL6R were globally expressed in the lymphoid/myeloid compartments; iv-interestingly, PLAUR/UPAR & NOTCH2 were highly expressed in both CD14+ & CD16+ monocytic compartments. Conclusions: The current study has identified novel EPCs markers that could be used for better characterization of EPCs sub-population in adult peripheral blood and subsequent usage of EPCs for various cell therapy and regenerative medicine applications.

Minimal Consequences of CMAH and DBA/2J Background on a FKRP Deficient Model

Background: Muscular dystrophies (MD) are a large group of genetic diseases characterized by a progressive loss of muscle. The Latent TGFβ Binding Protein 4 (LTBP4) in the DBA/2 background and the Cytidine Monophosphate-sialic Acid Hydroxylase (CMAH) proteins were previously identified as genetic modifiers in severe MD. Objective: We investigated whether these modifiers could also influence a mild phenotype such as the one observed in a mouse model of Limb-Girdle MD2I (LGMD2I). Methods: The FKRPL276I mouse model was backcrossed onto the DBA/2 background, and in separate experiments the Cmah gene was inactivated in FKRPL276I mice by crossing with a Cmah-/- mouse and selecting the double-mutants. The mdx mouse was used as control for these two genome modifications. Consequences at the histological level as well as quantification of expression level by RT-qPCR of genes relevant for muscular dystrophy were then performed. Results: We observed minimal to no effect of the DBA/2 background on the mild FKRPL276I mouse phenotype, while this same background was previously shown to increase inflammation and fibrosis in the mdx mouse. Similarly, the Cmah-/- deletion had no observable effect on the FKRPL276I mouse phenotype whereas it was seen to increase features of regeneration in mdx mice. Conclusions: These modifiers were not observed to impact the severity of the presentation of the mild FKRPL276I model. An interesting association of the CMAH modifier with the regeneration process in the mdx model was seen and sheds new light on the influence of this protein on the dystrophic phenotype.