Int J Stem Cells.  2019 Nov;12(3):474-483. 10.15283/ijsc19075.

Direct Reprogramming to Human Induced Neuronal Progenitors from Fibroblasts of Familial and Sporadic Parkinson’s Disease Patients

• Lee M
• Sim H
• Ahn H
• Ha J
• Baek A
• Jeon YJ
• Son MY
• Kim J

• KMID: 2465888
• DOI: http://doi.org/10.15283/ijsc19075

Abstract

In Parkinson's disease (PD) research, human neuroblastoma and immortalized neural cell lines have been widely used as in vitro models. The advancement in the field of reprogramming technology has provided tools for generating patient-specific induced pluripotent stem cells (hiPSCs) as well as human induced neuronal progenitor cells (hiNPCs). These cells have revolutionized the field of disease modeling, especially in neural diseases. Although the direct reprogramming to hiNPCs has several advantages over differentiation after hiPSC reprogramming, such as the time required and the simple procedure, relatively few studies have utilized hiNPCs. Here, we optimized the protocol for hiNPC reprogramming using pluripotency factors and Sendai virus. In addition, we generated hiNPCs of two healthy donors, a sporadic PD patient, and a familial patient with the LRRK2 G2019S mutation (L2GS). The four hiNPC cell lines are highly proliferative, expressed NPC markers, maintained the normal karyotype, and have the differentiation potential of dopaminergic neurons. Importantly, the patient hiNPCs show different apoptotic marker expression. Thus, these hiNPCs, in addition to hiPSCs, are a favorable option to study PD pathology.

Keyword

Reprogramming; Direct reprogramming; Induced neuronal progenitor cells; Pluripotency factors; Parkinson’s disease

MeSH Terms

Cell Line
Dopaminergic Neurons
Fibroblasts*
Humans*
In Vitro Techniques
Induced Pluripotent Stem Cells
Karyotype
Neuroblastoma
Neurons*
Pathology
Sendai virus
Stem Cells
Tissue Donors

Figure

• 

  Fig. 1 Direct reprogramming to generate hiNPCs. (a) Schematic diagram to show direct reprogramming of fibroblasts to hiNPCs. (b) Representative bright field images of fibroblasts, a reprogrammed hiNPC colony, clonally expanded hiNPCs, and spontaneously differentiated cells from hiNPCs. Scale bars represent 100 μm.

• 

  Fig. 2 Characterization of hiNPC lines. (a) Flow cytometry to detect ploidy of PI stained hiNPCs. Human fibroblasts from healthy donors were used as a 2n control. WT1, WT2, FPD, and SPD represent AG02261-hiNPC, GM01680-hiNPC, ND38262-hiNPC, and AG20446-hiNPC, respectively. (b) Immunocytochemistry for key NPC markers in hiNPCs. Ho. represents Hoechst33342 for staining nuclei. Scale bars represent 50 μm. (c) Mutation analysis of generated hiNPCs and the parental fibroblasts. The arrow indicates the G2019S mutation site in LRRK2. The red arrow indicates heterozygosity of G and A. (d) Immunocytochemistry of differentiated cells from hiNPCs with representative markers for pan-neurons, dopaminergic neurons, mature neurons, and glia. All hiNPCs were differentiated for 21 days. Scale bars represent 50 μm. (e) mRNA expression of MAP2, NEUN, SYNAPSIN1, GRIN1, GRIA2, and S100B in undifferentiated and differentiated hiNPCs.

• 

  Fig. 3 Quality check of hiNPC lines before cryopreservation. (a) Karyotypes of established hiNPC lines at passage 9, 13, 8, and 13 of WT1-, WT2-, FPD-, and SPD-hiNPC, respectively. (b) STR analysis comparing starting fibroblasts and their corresponding hiNPCs. (c) Mycoplasma test by PCR. A 100 bp ladder was used.

• 

  Fig. 4 hiNPCs as a PD model. (a) Schematic diagram for PD modeling. (b) Representative bright field images of hiNPCs after treatment with MG132. Scale bars represent 200 μm. (c) WST based cell viability assay with DMSO or MG132 treatment. All values indicate relative level of its corresponding DMSO control groups. (d) Immunoblot of cCASP3 in hiNPCs with or without MG132 treatment. GAPDH was used as an internal control. (e) Quantification of the band intensities. All values indicate relative level of cCASP3 to GAPDH. ** represents p<0.01; *** represents p<0.001 using Student’s t-test.

Reference

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