Enhancement of BMP4 inhibitor DMH1 on the efficiency of BiSF in human iPSC-induced differentiation into neurons

doi:10.3969/j.issn.1674-8115.2022.01.006

Abstract

Abstract: Objective

·To obtain an efficient way of promoting induced human pluripotent stem cells (hiPSCs) to differentiate into neurons by improving existing methods BiSF.

Methods

·Induction was initiated when hiPSCs reached 75% fusion, and BMP4 inhibitor (4-[6-[4-(1-methylethoxy)phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]quinoline,DMH1) was added based on this induction method. The growth state was observed under microscope, and the expression of neural stem cell (NSC)-specific genes was quantitatively detected by quantitative real-time PCR (qRT-PCR) and immunofluorescence analysis. The proliferation level of the induced cells was determined by cell counting kit-8 (CCK-8). Then the NSC-like cells were further induced into neurons, and the ability of differentiation was detected by qRT-PCR and immunofluorescence.

Results

·Microscopically, it was found that more spindle cells appeared around the cell mass of BiSF+DMH1 group on day 9, and a small amount of spindle cells appeared in the BiSF group with irregular gray cell clusters. CCK-8 growth curve showed that the cells derived from method BiSF+DMH1 were with a significantly higher proliferation on the next day (P=0.000). The cells derived from method BiSF+DMH1 achieved higher expression of nestin and PAX6 (P=0.019, P=0.011). The number of neurons with positive neuron-specific marker βⅢ-tubulin in the BiSF+ DMH1 group was significantly higher than that in BiSF group (P=0.003). The results of qRT-PCR showed that the relative expression of MAP2 in the BiSF+DMH1 group was significantly higher than that in the BiSF group (P=0.006).

Conclusion

·The synergistic effect of DMH1 can significantly improve the efficiency of hiPSCs to differentiate into neurons.

Key words: human induced pluripotent stem cell, neural stem cell, differentiation, neuron

CLC Number:

R331

Yanna LIU, Zhaorui REN, Jingbin YAN. Enhancement of BMP4 inhibitor DMH1 on the efficiency of BiSF in human iPSC-induced differentiation into neurons[J]. JOURNAL OF SHANGHAI JIAOTONG UNIVERSITY (MEDICAL SCIENCE), 2022, 42(1): 36-43.

Figures/Tables 6

Tab 1 Genes and primer sequences used for qRT-PCR analysis

Primer name	Forward （5'→3'）	Reverse （5'→3'）
GAPDH	TTCCAAGCATAAAAGAACGG	TACATCCTCATCACCACCCA
NANOG	TTTGTGGGGCTGAAGAAACT	AGGGCTGTCCTGAATAAGCAG
OCT4	GACGCCATCAACACCGAGTT	CTTTGTCGTTGGTTAGCTGGT
Nestin	CAGCGTTGGAACAGAGGTTGG	TGGCACAGGTGTCTCAAGGGTAG
PAX6	ATGTGTGAGTAAAATTCTGGGCA	GCTTACAACTTCTGGAGTCGCTA
MAP2	CCACCTGAGATTAAGGATCA	GGCTTACTTTGCTTCTC

Fig 1 hiPSC morphology and pluripotency identification

Fig 2 Cell morphologies were observed under microscope on day 9 of differentiation

Fig 3 Marker for NSC nestin and proliferation of NSCs

Fig 4 mRNA expression levels of pluripotency-related genes and NSC-related genes on day 13 of differentiation

Fig 5 Detection of neuron-specific markers

TrendMD

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