Characterization and cardiac lineage differentiation potential of cardiogenic mesenchymal-like cells from human adults

doi:10.3877/cma.j.issn.2095-1221.2021.01.003

Abstract

Abstract:

Objective

To investigate the molecular phenotype of cardiac derived mesenchymal-like cells (CDMCs) and explore the differentiation potential into cardiac lineage.

Methods

The experiment was divided into: different culture time of CDMCs (passage 3, 5, 7) , and umbilical cord mesenchymal stem cells (UCMSCs) were used as control. Molecular characterization and cardiac lineage differentiation were carried out in each group. Cell morphological changes were observed by microscopy. Growth doubling time and population growth curves were generated. Surface marker antigen expressions were analyzed by flow cytometry. Real-time quantitative PCR and Western blot were used to detect the mRNA and protein expressions of pluripotent and tissue-specific molecules. Statistical analysis included repeated measures ANOVA, one-way analysis of variance and paired t-test.

Results

CDMCs had morphological characteristics and proliferative ability of UCMSCs, and there was no significant difference in the proliferation rate among four groups (P > 0.05) . Compared with UCMSCs, surface marker antigen (CD90) expression decreased in passage 3, 5, 7 CDMCs (before cryopreservation: 97.13﹪± 2.00﹪vs 59.87﹪± 34.14 ﹪, 38.83﹪±11.04 ﹪, 34.77﹪±14.78 ﹪; after cryopreservation: 99.83﹪±0.17 ﹪ vs 56.00﹪±19.47 ﹪, 47.48﹪±11.88 ﹪, 41.15﹪± 8.68 ﹪, P < 0.05) . The expressions of Rex1 (0.00±0.00 vs 0.68±0.50, 0.29±0.17, 0.38±0.50) , Oct3/4 (1.00±0.02 vs 5.28±0.78, 3.88±0.95, 3.63±0.34) and Nanog (1.00±0.16 vs 7.57±4.69, 5.40±3.58, 5.34±0.76) were significantly increased in CDMCs at different culture time compared with UCMSCs (P < 0.05) . Compared with UCMSCs, Nkx2.5 (1.00±0.12 vs 30.60±22.43, 19.69±9.65, 8.82±4.94) and Gata4 (1.00±0.85 vs 60467±25266, 44350±25800, 35067±23113) were highly expressed in each culture times of CDMCs (P < 0.05) . Compared with pre-differentiation, the expression of cTnT protein in each groups of CDMCs (0.40±0.13 vs 0.98±0.16, 0.38±0.18 vs 0.69±0.15, 0.17±0.11 vs 0.70±0.17) was significantly higher after 15 days myocardial differentiation (P < 0.05) .

Conclusion

CDMCs exhibited partial stem cell and mesenchymal features. In addition, it possessed cardiac tissue specificity. CDMCs could differentiate into cardiac lineage, and the differentiation potential of cardiac myocytes may be superior to UCMSCs.

Key words: Adult cardiac derived mesenchymal-like cells, Phenotype characteristics, Cardiac lineage, Differentiation induction

Yanyan Gao, Yanyan Pan, Yifan Jiang, Jun Ji, Li Zhang. Characterization and cardiac lineage differentiation potential of cardiogenic mesenchymal-like cells from human adults[J]. Chinese Journal of Cell and Stem Cell(Electronic Edition), 2021, 11(01): 16-24.

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URL: https://zhxbygxbzz.cma-cmc.com.cn/EN/10.3877/cma.j.issn.2095-1221.2021.01.003

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Figures/Tables 12

References 29

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基因	序列（5^'→3^'）	预期扩增产物长度（bp）
Rex1	上游GAGGCTGGAGCCTGTGTGAA	122
	下游ATATATCAACCACCTCCAGGCAGTA
Sox2	上游CCAAGATGCACAACTCGGAGA	143
	下游CCGGTATTTATAATCCGGGTGCT
Oct3/4	上游GACAGGGGGAGGGGAGGAGCTAGG	144
	下游CTTCCCTCCAACCAGTTGCCCCAAAC
Nanog	上游TCCAACATCCTGAACCTCAGCTA	186
	下游AGTCGGGTTCACCAGGCATC
Nkx2.5	上游GAGAAGGGGCTCCCAACAT	126
	下游TAATCGCCGCCACAAACTC
Gata4	上游AATGCCTGCGGCCTCTACA	108
	下游AGATTTATTCAGGTTCTTGGGCTTC
cTnT	上游GCTGTGGCAGAGCATCTATAACTTG	152
	下游GCCCGGTGACTTTAGCCTTC
α-SMA	上游ATTGCCGACCGAATGCAGA	110
	下游ATGGAGCCACCGATCCAGAC
β-actin	上游TGGCACCCAGCACAATGAA	186
	下游CTAAGTCATAGTCCGCCTAGAAGCA

基因	序列（5^'→3^'）	预期扩增产物长度（bp）
Rex1	上游GAGGCTGGAGCCTGTGTGAA	122
	下游ATATATCAACCACCTCCAGGCAGTA
Sox2	上游CCAAGATGCACAACTCGGAGA	143
	下游CCGGTATTTATAATCCGGGTGCT
Oct3/4	上游GACAGGGGGAGGGGAGGAGCTAGG	144
	下游CTTCCCTCCAACCAGTTGCCCCAAAC
Nanog	上游TCCAACATCCTGAACCTCAGCTA	186
	下游AGTCGGGTTCACCAGGCATC
Nkx2.5	上游GAGAAGGGGCTCCCAACAT	126
	下游TAATCGCCGCCACAAACTC
Gata4	上游AATGCCTGCGGCCTCTACA	108
	下游AGATTTATTCAGGTTCTTGGGCTTC
cTnT	上游GCTGTGGCAGAGCATCTATAACTTG	152
	下游GCCCGGTGACTTTAGCCTTC
α-SMA	上游ATTGCCGACCGAATGCAGA	110
	下游ATGGAGCCACCGATCCAGAC
β-actin	上游TGGCACCCAGCACAATGAA	186
	下游CTAAGTCATAGTCCGCCTAGAAGCA

细胞	实验次数	时间（d）
细胞	实验次数	1	2	3	4	5	6	7
UCMSCs	3	0.18±0.02	0.22±0.01	0.49±0.07	1.24±0.10	1.97±0.02	2.67±0.01	2.79±0.02
P3 CDMCs	3	0.18±0.01	0.24±0.01	0.55±0.03	1.24±0.11	2.05±0.02	2.64±0.03	2.85±0.03
P5 CDMCs	3	0.18±0.00	0.24±0.01	0.52±0.02	1.24±0.05	1.98±0.11	2.64±0.02	2.79±0.03
P7 CDMCs	3	0.18±0.00	0.24±0.00	0.53±0.01	1.21±0.07	2.00±0.05	2.73±0.03	2.81±0.02

细胞	实验次数	时间（d）
细胞	实验次数	1	2	3	4	5	6	7
UCMSCs	3	0.18±0.02	0.22±0.01	0.49±0.07	1.24±0.10	1.97±0.02	2.67±0.01	2.79±0.02
P3 CDMCs	3	0.18±0.01	0.24±0.01	0.55±0.03	1.24±0.11	2.05±0.02	2.64±0.03	2.85±0.03
P5 CDMCs	3	0.18±0.00	0.24±0.01	0.52±0.02	1.24±0.05	1.98±0.11	2.64±0.02	2.79±0.03
P7 CDMCs	3	0.18±0.00	0.24±0.00	0.53±0.01	1.21±0.07	2.00±0.05	2.73±0.03	2.81±0.02

细胞	实验次数	CD105		CD29		CD90
细胞	实验次数	冻存前	冻存后	冻存前	冻存后	冻存前	冻存后
UCMSCs	3	95.04±3.23	99.86±0.10	99.37±0.56	99.23±0.93	97.13± 2.00	99.83± 0.17
P3 CDMCs	3	96.17±5.06	96.47±5.28	97.17±2.44	99.47±0.40	59.87±34.14	56.00±19.47^b
P5 CDMCs	3	90.82±5.67	95.19±6.80	97.91±2.46	99.57±0.25	38.83±11.04^a	47.48±11.88^b
P7 CDMCs	3	93.12±3.78	95.21±2.48	98.08±1.70	99.30±0.40	34.77±14.78^a	41.15± 8.68^b
F值		0.799	0.725	0.660	0.227	6.466	14.170
P值		0.528	0.565	0.599	0.875	0.016	0.001
细胞	实验次数	CD45		CD34		HLA-DR
细胞	实验次数	冻存前	冻存后	冻存前	冻存后	冻存前	冻存后
UCMSCs	3	9.76±1.82	5.28±1.05	5.35± 1.32	2.31±0.72	0.48±0.40	0.49±0.27
P3 CDMCs	3	9.13±7.30	8.71±6.51	15.74±14.67	11.24±3.63^a	0.36±0.40	0.38±0.15
P5 CDMCs	3	3.43±1.85	8.92±5.40	7.04± 6.29	9.88±4.80	0.10±0.05	0.61±0.08
P7 CDMCs	3	5.21±5.08	7.43±4.75	6.08± 2.19	7.15±2.83	0.16±0.10	0.26±0.05
F值		1.307	0.352	1.075	4.176	1.120	2.758
P值		0.337	0.789	0.413	0.047	0.397	0.112

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