Development of pluripotent stem cells for vascular therapy

Abstract

Peripheral arterial disease (PAD) is characterized by reduced limb blood flow due to arterial obstruction. Current treatment includes surgical or endovascular procedures, the failure of which may result in amputation of the affected limb. An emerging therapeutic approach is cell therapy to enhance angiogenesis and tissue survival. Small clinical trials of adult progenitor cell therapies have generated promising results, although large randomized clinical trials using well-defined cells have not been performed. Intriguing pre-clinical studies have been performed using vascular cells derived from human embryonic stem cells (hESC) or human induced pluripotent stem cells (hiPSCs). In particular, hiPSC-derived vascular cells may be a superior approach for vascular regeneration. The regulatory roadmap to the clinic will be arduous, but achievable with further understanding of the reprogramming and differentiation processes; with meticulous attention to quality control; and perseverance.

Fig. 1

Generation of iPSCs and differentiation into ECs. IPSCs may be generated by overexpressing Oct4, Sox2, Klf 4 and cMyc (using retroviral, mmRNA, or cell-permeant peptide constructs). The iPSCs are characterized by staining for pluripotency markers and alkaline phosphatase activity. Other tests for quality of the reprogramming include spectral karyotyping, and bisulfite genomic sequencing to assess genomic and epigenomic quality. Endothelial differentiation is initiated by culturing hiPSCs for 14 days in differentiation media supplemented with 50 ng/ml bone morphogenetic protein 4 (BMP4) and 50 ng/ml vascular endothelial growth factor (VEGF). The heterogenous mixture of cells is purified by FACS using an antibody directed against CD31. The ECs can be characterized for functionality by looking at the ability of iPSC-ECs to proliferate (using BrdU), the ability to generate capillary-like structures (when grown in matrigel) and the ability to incorporate in a pre-existing EC network. Other functional tests include the ability of the IPSCs-ECs to incorporate acetylated-LDL cholesterol, to produce nitric oxide, and to express markers such as KDR, CD31, CD144, and eNOS.

Fig. 2

Development of therapeutic cells from human induced pluripotential cells. Readily accessible somatic cells (e.g. skin fibroblasts) are harvested from a patient, and expanded in culture. Cells are exposed to reprogramming transcriptional factors, in the form of cell permeant peptides or modified mRNA. The resulting iPSC colonies are differentiated into vascular progenitor cells, that are administered directly to patients with vascular disease, or which are incorporated into matrices as a biological conduit for surgical implantation.

Fig. 3

Roadmap to the clinic. Clinical derivation of the cell line of interest represents the initial stage of translating a cell therapy to the clinic. The ability to manufacture the stem cell product to current clinical expectations is addressed in process development before the clinical prototype cell is shown to be efficacious in animal models of disease. Note that significant late-stage changes to processes may result in rederivation and repeating key experiments. Based on advisory meetings with regulatory bodies (in this case the US FDA), release testing and safety/toxicology (tox) programs are initiated with the GMP manufactured cell product or equivalent. The regulatory dossier is compiled and further advice sought from the regulatory agency before submitting the Investigational New Drug (IND) application for a phase 1 clinical trial.