Clonal and Scalable Endothelial Progenitor Cell Lines from Human Pluripotent Stem Cells

Abstract

Human pluripotent stem cells (hPSCs) can be used as a renewable source of endothelial cells for treating cardiovascular disease and other ischemic conditions. Here, we present the derivation and characterization of a panel of distinct clonal embryonic endothelial progenitor cells (eEPCs) lines that were differentiated from human embryonic stem cells (hESCs). The hESC line, ESI-017, was first partially differentiated to produce candidate cultures from which eEPCs were cloned. Endothelial cell identity was assessed by transcriptomic analysis, cell surface marker expression, immunocytochemical marker analysis, and functional analysis of cells and exosomes using vascular network forming assays. The transcriptome of the eEPC lines was compared to various adult endothelial lines as well as various non-endothelial cells including both adult and embryonic origins. This resulted in a variety of distinct cell lines with functional properties of endothelial cells and strong transcriptomic similarity to adult endothelial primary cell lines. The eEPC lines, however, were distinguished from adult endothelium by their novel pattern of embryonic gene expression. We demonstrated eEPC line scalability of up to 80 population doublings (pd) and stable long-term expansion of over 50 pd with stable angiogenic properties at late passage. Taken together, these data support the finding that hESC-derived clonal eEPC lines are a potential source of scalable therapeutic cells and cell products for treating cardiovascular disease. These eEPC lines offer a highly promising resource for the development of further preclinical studies aimed at therapeutic interventions.

Emerging Roles of Exosomes in Stroke Therapy

“…Recognizing that cellular source impacts the contents of exosomes raises the possibility that exosomes from other cell sources maybe more therapeutic for the treatment of stroke than those included in this review. For example, we have found that hESC derived endothelial progenitors produce more potent angiogenic exosomes than adult MSCs [90]. Additionally, while most studies included in this review used MSCs, there was variability in both the organism and the tissue from which MSCs were isolated, including rats [58,64], mini-pigs [66], and humans [21,66], and from either bone marrow [19,20,58] or adipose tissue [66].…”

“…Therefore, the source of exosomes with well-defined identity and homogeneity, stability, and scalability, all of which allow for optimal production and high potency, will be advantageous for usage of exosomes in future clinical applications. One potential solution to is the use of clonally pure hESC derived progenitor cells as a scalable source of exosomes [90,96]. It is critical to provide cell sources for large scale production and manufacturing process developed in Good Manufacturing Practices (cGMP) conditions, which includes in-process testing, quality control release procedures, the standard operating procedure (SOP) for production, and the development of product release criteria for the final exosome product.…”

Emerging Roles of Exosomes in Stroke Therapy

“…Recognizing that cellular source impacts the contents of exosomes raises the possibility that exosomes from other cell sources may be more therapeutic for the treatment of stroke than those included in this review. For example, we have found that hESC-derived endothelial progenitors produce more potent angiogenic exosomes than adult MSCs [ 94 ]. Additionally, while most studies included in this review used MSCs, there was variability in both the organism and the tissue from which MSCs were isolated, including rats [ 61 , 69 ], mini-pigs [ 62 ], and humans [ 21 , 62 ], and from either bone marrow [ 19 , 20 , 61 ] or adipose tissue [ 62 ].…”

“…Therefore, the source of exosomes with well-defined identity and homogeneity, stability, and scalability, all of which allow for optimal production and high potency, will be advantageous for usage of exosomes in future clinical applications. One potential solution is the use of clonally pure hESC-derived progenitor cells as a scalable source of exosomes [ 94 , 102 ]. It is critical to provide cell sources for large-scale production and a manufacturing process developed in Good Manufacturing Practices (cGMP) conditions, which includes in-process testing, quality control release procedures, the standard operating procedure (SOP) for production, and the development of product release criteria for the final exosome product.…”