Abstract
Pluripotent stem cell-derived differentiated endothelial cells offer high potential in regenerative medicine in the cardiovascular system. With the aim of translating the use of a human stem cell-derived endothelial cell product (hESC-ECP) for treatment of critical limb ischemia (CLI) in man, we report a good manufacturing practice (GMP)-compatible protocol and detailed cell tracking and efficacy data in multiple preclinical models. The clinical-grade cell line RC11 was used to generate hESC-ECP, which was identified as mostly endothelial (60% CD31+/CD144+), with the remainder of the subset expressing various pericyte/mesenchymal stem cell markers. Cell tracking using MRI, PET, and qPCR in a murine model of limb ischemia demonstrated that hESC-ECP was detectable up to day 7 following injection. Efficacy in several murine models of limb ischemia (immunocompromised/immunocompetent mice and mice with either type I/II diabetes mellitus) demonstrated significantly increased blood perfusion and capillary density. Overall, we demonstrate a GMP-compatible hESC-ECP that improved ischemic limb perfusion and increased local angiogenesis without engraftment, paving the way for translation of this therapy.
Highlights
Peripheral arterial disease (PAD) is a common disorder and a major cause of morbidity and mortality, with 202 million people living with PAD globally in 2010.1 The most severely affected patients suffer from critical limb ischemia (CLI), characterized by rest pain, ulcerations, and/or gangrene, and have a very poor prognosis, with high rates of limb amputation and mortality.[2]
The pattern of staining fell into 3 groups (Figure 2B): markers typically observed on less mature endothelial cells and coexpressed on only CD144-positive cells (e.g., CD34, CD105, and CD309); mesenchymal stromal cells (MSCs) and pericyte markers on all cells (e.g., CD73, CD44, CD90, and CD146); and hematopoietic/earlier progenitors that were negative on all cells (e.g., CD14, CD45, CD56, and CD133)
Expression profiles of endothelial genes reflected the immature stage of the human embryonic stem cell (hESC)-EC product (ECP); in hESC-ECP, CD31 and CD144 increased over time (8 days) to levels similar to those in human umbilical vein endothelial cells (HUVECs), whereas expression levels of KDR and CD34 increased to levels that were significantly higher than in HUVECs (Figures 1C and S1A)
Summary
Peripheral arterial disease (PAD) is a common disorder and a major cause of morbidity and mortality, with 202 million people living with PAD globally in 2010.1 The most severely affected patients suffer from critical limb ischemia (CLI), characterized by rest pain, ulcerations, and/or gangrene, and have a very poor prognosis, with high rates of limb amputation and mortality.[2]. Pro-angiogenic, cell-based therapies have significant potential in the treatment of ischemic disease but have not yet showed a clear clinical success, with the majority of CLI clinical trials carried out to date utilizing autologous bone marrow- or peripheral blood-derived cells in small pilot trials.[5,6] A recent meta-analysis of randomized controlled trials (RCTs) (16 RCTs, involving 774 patients) demonstrated that cell therapy in CLI is associated with reduced risk of major amputation.[7] following reanalysis using placebocontrolled RCTs, these benefits were no longer significant This calls for the need to test for alternative sources of stem cells, expanding to allogenic approaches. A major focus of this field is to translate the use of pluripotent cell therapy into the clinic to assess its full potential
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