Induced Pluripotent Stem Cells: Reprogramming Platforms and Applications in Cell Replacement Therapy

Abstract

The generation of induced pluripotent stem cells (iPSCs) from differentiated mature cells is one of the most promising technologies in the field of regenerative medicine. The ability to generate patient-specific iPSCs offers an invaluable reservoir of pluripotent cells, which could be genetically engineered and differentiated into target cells to treat various genetic and degenerative diseases once transplanted, hence counteracting the risk of graft versus host disease. In this context, we review the scientific research streams that lead to the emergence of iPSCs, the roles of reprogramming factors in reprogramming to pluripotency, and the reprogramming strategies. As iPSCs serve tremendous correction potentials for various diseases, we highlight the successes and challenges of iPSCs in cell replacement therapy and the synergy of iPSCs and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing tools in therapeutics research.

Keywords: OSKM; clinical applications; gene editing; iPSCs; reprogramming; viral and nonviral vectors.

FIG. 1.

Nuclear reprogramming strategy. The nucleus of a differentiated cell is transplanted into an enucleated egg in meiotic metaphase by nuclear transfer. The transplanted genome is reprogrammed into a pluripotent state, whereby the egg undergoes cell division and a cloned animal is produced.

FIG. 2.

Merger of three scientific research streams that facilitates the development of iPSCs. The first stream was initialized when Gurdon produced tadpoles from an unfertilized egg using a nucleus from frog intestinal cell in 1962. With more than three decades of research using the discovery of “master” transcription factors in the second stream and the research involving ESCs in the third stream, Wilmut's group demonstrated the first birth of live mammal created by nuclear transfer technology in 1997. Subsequently, Takahashi and Yamanaka reported the generation of iPSCs from somatic cells by transduction of four transcription factors in 2006. ESCs, embryonic stem cells; iPSCs, induced pluripotent stem cells.

FIG. 3.

Nuclear reprogramming strategy. Ectopic expression of the four defined transcription factors associated with pluripotency (Oct4, Sox2, Klf4, and c-Myc) reverses the unipotency state into a pluripotency state. c-Myc, cellular-Myelocytomatosis; Klf4, Krüppel-like factor 4; Oct4, octamer-binding transcription factor 4; Sox2, SRY (sex determining region Y)-box 2.

FIG. 4.

Various cell sources and transfer strategies for the generation of iPSCs. The iPSCs were initially derived from mouse embryonic and skin fibroblasts. Soon after, scientists have successfully used other somatic cells with improved reprogramming efficiency. Progress has been made in the choice of reprogramming factors, which include Yamanaka's factors, Nanog and Lin28. Integrating viral vectors like retrovirus and lentivirus were used to generate the first iPSC lines. Thereafter, nonintegrating viral vectors and plasmid systems were used. In recent times, successful reprogramming transfer strategies using recombinant and isolated proteins from ESCs have been demonstrated. Newer approaches, such as synthetic modified RNA or mRNA and miRNAs, have also been used to enhance reprogramming efficiency. miRNA, microRNA; mRNA, messenger RNA.

FIG. 5.

Coculture of ESCs or iPSCs on embryonic fibroblast feeder cell layers and feeder-free cell layers. Inactivated mouse and human-derived cells are traditionally utilized as feeder layers to retain the pluripotency of ESCs and iPSCs. Recently, a plethora of feeder-free layer systems was generated such as Matrigel^™, gelatin-coated substrates, and iMatrix-511 to maintain ESCs and iPSCs in undifferentiated state for long-term culture.

FIG. 6.

CRISPR/Cas9-mediated gene editing in the iPSCs derived from patient's somatic cells for cell replacement therapy to treat various genetic and degenerative diseases. Somatic cells isolated from a patient carrying mutation are reprogrammed into iPSCs by the introduction of Oct4, Sox2, Klf4, and c-Myc using either viral or nonviral gene transfer. The iPSCs are then genetically engineered to correct the mutation by the CRISPR/Cas9 technology. The corrected iPSCs are enriched and induced to differentiate into the target cells. Finally, the cells are reinfused into the patient to correct the disease condition. Cas9, CRISPR-associated 9; CRISPR, clustered regularly interspaced short palindromic repeats.