Amplification of chromosome 1q impairs neural differentiation ability of human embryonic stem cells

doi:10.3877/cma.j.issn.2095-1221.2022.03.001

Abstract

Abstract:

Objective

To explore the effects of long arm of chromosome 1 (1q) amplification on neural differentiation and to establish a cellular model for studying neural diseases.

Methods

The cell line harboring chromosome 1q amplification was isolated from H9 human embryonic stem cells (hESCs) by subcloning. The number of chromosomes was determined by chromosome counting.Chromosome aberration was analyzed by karyotyping. Whole genome sequencing (WGS) was used to analyze genome copy number variations. Alkaline phosphatase (AP) staining was used to evaluate the state of stem cells. RT-qPCR and immunofluorescence staining were used to detect the expression of ESCs markers OCT4, SOX2, NANOG, REX1 and SSEA4. Embryoid body (EB) formation was used to detect the undirected differentiation abilities of hESCs. The ectoderm differentiation was induced by all-trans retinoic acid (RA) . Neural progenitor cell (NPC) differentiation was induced by using STEMdiff? SMADi Neural Induction Kit. RT-qPCR, AP staining and immunofluorescence were used to determine the differentiation abilities of hESCs. Student's t test was used for comparison between two groups.

Results

One aneuploid cell line harboring isochromosome 1q gain was isolated and named as Amp (1q) hESCs. The karyotype of Amp (1q) hESCs is 47, XX, +i (1q) . Amp (1q) cells showed positive AP staining and expressed hESCs markers OCT4, SOX2, NANOG, REX1 and SSEA4, and had the ability of self-renewal. During EB differentiation, Amp (1q) hESCs showed impaired ectoderm differentiation, and the expression of MAP2 (29.67±1.53 vs 66.67±1.15) and PAX6 (8001.00±567.09 vs 28308.00±1692.50) decreased compared with H9 cells (all P < 0.05) . The defective ectoderm differentiation of Amp (1q) hESCs were confirmed by RA-induced differentiation, and the expression of MAP2 (22.50±3.54 vs 42.50±2.12) and PAX6 (5403.00±569.93 vs 38756.00±1068.44) decreased compared with H9 cells (all P < 0.05) . The capacity of Amp (1q) hESCs to form NPCs was significantly decreased, and the expression level of NPCs marker PAX6 in Amp (1q) hESC-derived cells was significantly lower than H9 cells (13.83±3.75 vs 88.33±1.53, all P < 0 05) .

Conclusion

Amp (1q) hESCs maintained the ability of self-renewal, while the neural differentiation ability was impaired due to the extra copy of chromosome 1q.

Key words: Chromosome 1q amplification, Human embryonic stem cells, Self-renewal, Neural differentiation

Jiayi Li, Meili Zhang, Yue Huang. Amplification of chromosome 1q impairs neural differentiation ability of human embryonic stem cells[J]. Chinese Journal of Cell and Stem Cell(Electronic Edition), 2022, 12(03): 129-137.

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Figures/Tables 14

References 20

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基因	引物序列（5'-3'）	预期扩增长度（bp）
GAPDH	上游TGATGACATCAAGAAGGTGGTGAAG	240
	下游TCCTTGGAGGCCATGTGGGCCAT
OCT4	上游CGACCATCTGCCGCTTTGAG	573
	下游CCCCCTGTCCCCCATTCCTA
SOX2	上游GGGAAATGGGAGGGGTGCAAAAGAGG	151
	下游TTGCGTGAGTGTGGATGGGATTGGTG
NANOG	上游CAGTCCTGGAGCAACCACTC	188
	下游ACATTAAGGCCTTCCCCAGC
REX1	上游ACATTTCCTGGTTCATACTGG	169
	下游ATTTGACTGAATAAACCTCCTG
MAP2	上游AGGAAGGGAAGAGCATGTAA	190
	下游ACACCACTTCCTCAACCACAG
PAX6	上游AACGATAACATACCAAGCGTGT	120
	下游GGTCTGCCCGTTCAACATC
HAND1	上游CCATGCTCCACGAACCCTTC	170
	下游CCTGGCGTCAGGACCATAG
T(Brachyury)	上游TATGAGCCTCGAATCCACATAGT	109
	下游CCTCGTTCTGATAAGCAGTCAC
SOX17	上游CTGCCACTTGAACAGTTTGG	138
	下游GAGGAAGCTGTTTTGGGACA
GATA4	上游GTGTCCCAGACGTTCTCAGTC	102
	下游GGGAGACGCATAGCCTTGT

基因	引物序列（5'-3'）	预期扩增长度（bp）
GAPDH	上游TGATGACATCAAGAAGGTGGTGAAG	240
	下游TCCTTGGAGGCCATGTGGGCCAT
OCT4	上游CGACCATCTGCCGCTTTGAG	573
	下游CCCCCTGTCCCCCATTCCTA
SOX2	上游GGGAAATGGGAGGGGTGCAAAAGAGG	151
	下游TTGCGTGAGTGTGGATGGGATTGGTG
NANOG	上游CAGTCCTGGAGCAACCACTC	188
	下游ACATTAAGGCCTTCCCCAGC
REX1	上游ACATTTCCTGGTTCATACTGG	169
	下游ATTTGACTGAATAAACCTCCTG
MAP2	上游AGGAAGGGAAGAGCATGTAA	190
	下游ACACCACTTCCTCAACCACAG
PAX6	上游AACGATAACATACCAAGCGTGT	120
	下游GGTCTGCCCGTTCAACATC
HAND1	上游CCATGCTCCACGAACCCTTC	170
	下游CCTGGCGTCAGGACCATAG
T(Brachyury)	上游TATGAGCCTCGAATCCACATAGT	109
	下游CCTCGTTCTGATAAGCAGTCAC
SOX17	上游CTGCCACTTGAACAGTTTGG	138
	下游GAGGAAGCTGTTTTGGGACA
GATA4	上游GTGTCCCAGACGTTCTCAGTC	102
	下游GGGAGACGCATAGCCTTGT

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