From Bench to Bedside: iPSCs in Modern Biomedicine

1. Research on the Mechanism of Parkinson's Disease

Construction of PARK2-KO-iPSC Model for the Study of Parkinson's Disease

Parkinson's disease (PD) is an incurable neurodegenerative disease characterized by progressive loss of midbrain dopaminergetic neurons in the substantia nigra pars compacta (SNc), which leads to the subsequent depletion of the striatum dopamine. The mechanisms of sporadic and familial PD are largely due to mitochondrial dysfunction and oxidative stress. Mutations in the PARK2 gene (which has important impacts on mitochondrial function) can cause autosomal dominant familial Parkinson's disease.

Research into the mechanisms of PD has used Parkinson's gene knockout animal models, which only show mild disease phenotype. Induced pluripotent stem cells (iPSCs) from familial PD patients or cells with PARK2 mutations introduced by genome editing can be used to study Parkinson's functional impairments in human dopamine neurons in vitro. PD patient iPSC-derived neurons with PARK2 mutations show increased oxidative stress, accumulation of α-synuclein, and irregular mitochondrial morphology and dysfunction.

The authors generated PARK2-KO-iPSCs by knocking out PARK2 on the basis of WT-iPSCs, and then differentiated both WT-iPSCs and PARK2-KO-iPSCs into dopamine neurons. The pathway analysis of protein changes in both iPSCs showed that PARK2 KO neurons had disrupted regulation of the cell cycle, oxidative stress, and energy metabolism. Additionally, various experiments showed that PARK2-KO neurons had abnormal mitochondria and morphology, insufficient glycolysis and lactic acid metabolism, and reduced cell proliferation and survival rate.^[1]

2. Stem Cell Therapy for Myocardial Infarction

Stem cell therapy for myocardial infarction: Over-expression of CCND2 can enhance the proliferation ability of heart muscle cells derived from hiPSCs.

In recent decades, there has been a significant improvement in the treatment of end-stage congestive heart failure (CHF), but success is still limited by the ability of heart muscle cells to regenerate. The molecular and cellular basis of progressive heart failure is the inability of damaged and dying heart muscle cells to be replaced. Strategies to promote heart muscle regeneration include reprogramming resident heart fibroblasts into heart muscle cell-like cells, transplanting stem/progenitor cells derived from body stem cells, and treatments aimed at recruiting endogenous stem cells or promoting cell cycle activity and proliferation in endogenous heart muscle cells.

The authors previously demonstrated that overexpression of CCND2 can enhance the proliferation of heart muscle cells derived from hiPSCs. The authors then differentiated human induced pluripotent stem cells (hiPSCs) into heart muscle cells (CCND2WT-CMs or CCND2OE-CMs) with WT-CCND2 or OE-CCND2 and transplanted them into infarcted pig hearts. By evaluating heart muscle function, heart muscle cell proliferation, angiogenesis in the lesion area, and heart muscle cell oxygen tolerance, the authors found that compared to wild-type (WT) CCND2 human heart muscle cells, overexpression (OE) of CCND2 human heart muscle cells had stronger proliferation and heart repair abilities, a reduction in the size of the infarcted area, and improvement in heart function, demonstrating good therapeutic effects.^[2]

3. Allogeneic stem cell immunotherapy

Allogeneic stem cell immunotherapy: iPSCs can be indefinitely cultured in vitro and successfully differentiate into lymphoid lineages with high induction efficiency.

Adoptive cellular therapy refers to the practice of isolating immune cells, followed by in vitro (ex vivo) manipulation, and subsequent delivery into patients as a therapeutic intervention. An area of interest is the exploration of cellular or immunotherapeutic approaches for the treatment of hematologic and oncologic diseases, including using chimeric antigen receptors (CARs). The universal (allogeneic) cell immunotherapy approach includes CAR-T, TCR-T, and NK cells that can be used to treat entire patient populations. These therapies have broad application prospects and therapeutic effects, but also face some challenges. However, the combination of iPSCs and gene editing can solve some of these difficulties.

The treatment principle of CAR-T therapy is to express CAR molecules on the surface of T cells that match the surface antigens of tumor cells, thus specifically killing tumor cells. CAR-T therapy is generally edited and transformed using the patient's T cells, but there are some problems such as limited expansion and function of the patient's T cells, difficulty in editing T cells, and time-consuming preparation of CAR-T cells, which delay the patient's treatment. The principle of NK therapy is similar to that of CAR-T therapy, which separates autologous or allogeneic immune effector cells, activates them in vitro, and infuses them into patients to directly kill tumors or stimulate the host's anti-tumor immune response. However, NK cells offer limited proliferative capacity and are difficult to genetically modify.

The combination of iPSCs and our gene editing technology can solve these problems so iPSCs can be easily genetically transformed in vitro. By editing human leukocyte antigen (HLA) and T cell receptor (TCR) genes related to immunity, the development of a universal CAR-T cell can effectively reduce immunogenicity, increase its applicability, efficiency, and durability. iPSCs can be cultured indefinitely in vitro and successfully differentiated into lymphoid-like cells, solving the problem of limited number of primary cells and difficulty in expansion. The success rate of inducing iPSCs into NK cells is high and their functional phenotype is mature.^[3]

4. Alzheimer's Disease Drug Screening

Alzheimer's disease drug screening: establishment of Aβ42-OE-iPSCs can be used to screen inhibitors of Aβ42.

Alzheimer's disease (AD) is a degenerative disorder of the central nervous system characterized by progressive cognitive and behavioral impairment, typically in late life. Currently, it is believed that the excessive accumulation of beta-amyloid protein (Aβ42) in the brain is the cause of the disease. To screen for drugs that inhibit Aβ42, one can establish Aβ42-OE-iPSCs (induced pluripotent stem cells overexpressing Aβ42) and differentiate them into neuronal cells. High-throughput screening can then be performed to identify effective inhibitors of Aβ42.

Reference

[1] Bogetofte H, Jensen P, Ryding M, et al. PARK2 Mutation Causes Metabolic Disturbances and Impaired Survival of Human iPSC-Derived Neurons. Front Cell Neurosci. 2019;13:297. Published 2019 Jul 5. doi:10.3389/fncel.2019.00297

[2] Zhao M, Nakada Y, Wei Y, et al. Cyclin D2 Overexpression Enhances the Efficacy of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes for Myocardial Repair in a Swine Model of Myocardial Infarction. Circulation. 2021;144(3):210-228. doi:10.1161/CIRCULATIONAHA.120.049497

[3] Nianias A, Themeli M. Induced Pluripotent Stem Cell (iPSC)-Derived Lymphocytes for Adoptive Cell Immunotherapy: Recent Advances and Challenges. Curr Hematol Malig Rep. 2019;14(4):261-268. doi:10.1007/s11899-019-00528-6