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Targeting JNK pathway promotes human hematopoietic stem cell expansion

Xiao, Xiong, Lai, Weifeng, Xie, Huangfan, Liu, Yang, Guo, Weijie, Liu, Yifang, Li, Yu, Li, Yuanjun, Zhang, Jingliang, Chen, Wenhan, Shi, Minhui, Shang, Lijun, Yin, Ming, Wang, Chengyan, Deng, Hongkui·2019·Cell Discovery
DOI 10.1038/s41421-018-0072-8PMC PMC6323118
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Abstract

The limited number of human hematopoietic stem cells (HSCs) has restrained their widespread clinical application. Despite great efforts in recent years, the in vitro expansion of HSCs remains a challenge due to incomplete understanding of the signaling networks underlying HSC self-renewal. Here, we show that culturing human cord blood (CB) CD34 + cells with JNK-IN-8, an inhibitor of the JNK signaling pathway, can enhance the self-renewal of HSCs with a 3.88-fold increase in cell number. These cultured CD34 + cells repopulated recipient mice for 21 weeks and can form secondary engraftment that lasted for more than 21 weeks. Knockdown of c-Jun , a major downstream target in the JNK pathway, promoted the expansion of hematopoietic stem and progenitor cells (HSPCs). Our findings demonstrate a critical role of the JNK pathway in regulating HSC expansion, provide new insights into HSC self-renewal mechanism, and may lead to improved clinical application of HSCs.

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Figures

Fig. 1 JNK-IN-8 promotes HSPC expansion in vitro. a Experimental schematic for the evaluation of JNK inhibitors on HSC expansion. CD34+ cells were cultured with StemSpan SFEM II medium supplemented with SCF, TPO, FLT3L in the presence of JNK-related molecules for 7 days, then the total cell expansion and CD34+CD45RA- cell frequency was determined. b Frequency of CD34+CD45RA- cell subsets in 7-day cultures of CD34+ cells supplemented with two representative JNK inhibitors including JNK-IN-8 (2 μM) and SP600125 (5 μM) (n = 3 experiments). c Chemical structure for JNK-IN-8 (hereafter called J8). d The increasing fold of CD34+CD45RA- subset frequency compared to DMSO after 10-day culture of CD34+ cells with different concentration of J8 (This data was drawn by R). See also to Supplementary Table S1. All data shown as mean values ± SD. Statistical significance was assessed using unpaired t test, where ***p < 0.001. See also Supplementary Fig. S1
Fig. 1 JNK-IN-8 promotes HSPC expansion in vitro. a Experimental schematic for the evaluation of JNK inhibitors on HSC expansion. CD34+ cells were cultured with StemSpan SFEM II medium supplemented with SCF, TPO, FLT3L in the presence of JNK-related molecules for 7 days, then the total cell expansion and CD34+CD45RA- cell frequency was determined. b Frequency of CD34+CD45RA- cell subsets in 7-day cultures of CD34+ cells supplemented with two representative JNK inhibitors including JNK-IN-8 (2 μM) and SP600125 (5 μM) (n = 3 experiments). c Chemical structure for JNK-IN-8 (hereafter called J8). d The increasing fold of CD34+CD45RA- subset frequency compared to DMSO after 10-day culture of CD34+ cells with different concentration of J8 (This data was drawn by R). See also to Supplementary Table S1. All data shown as mean values ± SD. Statistical significance was assessed using unpaired t test, where ***p < 0.001. See also Supplementary Fig. S1CC BY · PubMed Central
Fig. 2 The effects of JNK-IN-8 on cell expansion. a Representative flow plots of phenotypically defined cell subsets after 10-day culture of 10,000 fresh CD34+ cells supplemented with DMSO or J8 (2 μM) (n = 3 experiments). b Fold expansion of indicated phenotypically defined cell population after 10-day culture of 10,000 fresh CD34+ cells supplemented with DMSO or J8 (2 μM) (n = 3 experiments). TCC, total cell count. c CFU number of progenies from 1,000 day 0 CD34+ cells after 10-day culture supplemented with DMSO or J8 (2 μM) (n = 3 experiments). G, CFU-granulocyte; M, CFU-macrophage; GM, CFU-granulocyte and macrophage; CFU-E CFU-erythrocyte; BFU-E, erythroid burst-forming unit; GEMM, CFU-granulocyte, erythroid, macrophage, and megakaryocyte. All data shown as mean values ± SD. Statistical significance was assessed using unpaired t test, where *p < 0.05, **p < 0.01, and ****p < 0.0001; ns, not significant. See also Supplementary Fig. S1 and Table S2
Fig. 2 The effects of JNK-IN-8 on cell expansion. a Representative flow plots of phenotypically defined cell subsets after 10-day culture of 10,000 fresh CD34+ cells supplemented with DMSO or J8 (2 μM) (n = 3 experiments). b Fold expansion of indicated phenotypically defined cell population after 10-day culture of 10,000 fresh CD34+ cells supplemented with DMSO or J8 (2 μM) (n = 3 experiments). TCC, total cell count. c CFU number of progenies from 1,000 day 0 CD34+ cells after 10-day culture supplemented with DMSO or J8 (2 μM) (n = 3 experiments). G, CFU-granulocyte; M, CFU-macrophage; GM, CFU-granulocyte and macrophage; CFU-E CFU-erythrocyte; BFU-E, erythroid burst-forming unit; GEMM, CFU-granulocyte, erythroid, macrophage, and megakaryocyte. All data shown as mean values ± SD. Statistical significance was assessed using unpaired t test, where *p < 0.05, **p < 0.01, and ****p < 0.0001; ns, not significant. See also Supplementary Fig. S1 and Table S2CC BY · PubMed Central
Fig. 3 JNK-IN-8 promotes expansion of HSCs in primary recipients. a Human CD45+ engraftment level in the PB of recipients transplanted with DMSO or J8-expanded 10,000 day 0 CD34+ cells on 5, 9, 12, 16 weeks posttransplantation (n = 2 independent experiments). Statistics significance between DMSO and J8 group on 5, 9, 12, 16 weeks was assessed by multiple t test respectively, where *p < 0.05, **p < 0.01. b Representative flow plots of engraftment of J8-expanded and DMSO-expanded CD34+ cells in mouse recipients’ PB 16 weeks after transplantation. Progenies of 10,000 day 0 CD34+ cells after 10-day culture with DMSO or J8 were transplanted per mouse. c HSC frequencies of fresh CD34+ cells, J8, and DMSO cultured cells determined by LDA, calculated by ELDA software. As for overall test for differences in stem cell frequencies between any of the groups, p = 0.0055. The frequency of more than 1% human chimerism (human CD45 compared with all CD45) in the BM was regarded as positive response. d HSC frequencies presented as 1/fresh CD34+ cells equivalent for each condition from (c), the required confidence interval was 95%. See Supplementary Table S3A. All data shown as mean values ± SD. Statistical significance was assessed using unpaired t test, where *p < 0.05, and **p < 0.01. See also Supplementary Fig. S2
Fig. 3 JNK-IN-8 promotes expansion of HSCs in primary recipients. a Human CD45+ engraftment level in the PB of recipients transplanted with DMSO or J8-expanded 10,000 day 0 CD34+ cells on 5, 9, 12, 16 weeks posttransplantation (n = 2 independent experiments). Statistics significance between DMSO and J8 group on 5, 9, 12, 16 weeks was assessed by multiple t test respectively, where *p < 0.05, **p < 0.01. b Representative flow plots of engraftment of J8-expanded and DMSO-expanded CD34+ cells in mouse recipients’ PB 16 weeks after transplantation. Progenies of 10,000 day 0 CD34+ cells after 10-day culture with DMSO or J8 were transplanted per mouse. c HSC frequencies of fresh CD34+ cells, J8, and DMSO cultured cells determined by LDA, calculated by ELDA software. As for overall test for differences in stem cell frequencies between any of the groups, p = 0.0055. The frequency of more than 1% human chimerism (human CD45 compared with all CD45) in the BM was regarded as positive response. d HSC frequencies presented as 1/fresh CD34+ cells equivalent for each condition from (c), the required confidence interval was 95%. See Supplementary Table S3A. All data shown as mean values ± SD. Statistical significance was assessed using unpaired t test, where *p < 0.05, and **p < 0.01. See also Supplementary Fig. S2CC BY · PubMed Central
Fig. 4 JNK-IN-8-expanded HSCs repopulate secondary recipients. a Secondary engraftment in mouse recipients’ PB 21 weeks after transplantation of 1 × 107 cells from BM of the primary recipients injected with DMSO or J8-expanded 10,000 day 0 CD34+ cells 21 weeks after transplantation. (n = 10 mouse from two independent experiments per group, *p = 0.0418 by two-tailed unpaired t test.) b HSC frequency in secondary recipient of J8 or DMSO-expanded cells calculated by ELDA software. More than 1% human CD45 engraftment in the BM was regarded as positive. As for overall test for differences in stem cell frequencies between any of the groups, p = 0.0251. c HSC frequencies presented as 1/fresh CD34+ cells equivalent for each condition from (b), the required confidence interval was 95%. More than 1% human CD45 engraftment in the BM was regarded as positive. See also Supplementary Table S3B. All data shown as mean values ± SD. Statistical significance was assessed using unpaired t test, where *p < 0.05
Fig. 4 JNK-IN-8-expanded HSCs repopulate secondary recipients. a Secondary engraftment in mouse recipients’ PB 21 weeks after transplantation of 1 × 107 cells from BM of the primary recipients injected with DMSO or J8-expanded 10,000 day 0 CD34+ cells 21 weeks after transplantation. (n = 10 mouse from two independent experiments per group, *p = 0.0418 by two-tailed unpaired t test.) b HSC frequency in secondary recipient of J8 or DMSO-expanded cells calculated by ELDA software. More than 1% human CD45 engraftment in the BM was regarded as positive. As for overall test for differences in stem cell frequencies between any of the groups, p = 0.0251. c HSC frequencies presented as 1/fresh CD34+ cells equivalent for each condition from (b), the required confidence interval was 95%. More than 1% human CD45 engraftment in the BM was regarded as positive. See also Supplementary Table S3B. All data shown as mean values ± SD. Statistical significance was assessed using unpaired t test, where *p < 0.05CC BY · PubMed Central
Fig. 5 JNK-IN-8-induced CD34+ cell expansion acts by inhibiting c-Jun. a Relative mRNA expression of indicated JNK-related genes on day 5, CD34+ cells cultured with DMSO or J8 (n = 3 experiments). b Western blot analysis of inhibition of phosphorylated c-Jun for DMSO and J8-treated CD34+ cells following serum stimulation for 30 min. c Representative flow cytometry profiles of CD34+ cells 5 days after transduction with lentivirus expressing shRNAs targeting c-Jun or scrambled shRNAs (n = 3 experiments). d Total cell number of CD34+CD45RA- cell population in indicated cultured CD34+ cells on day 5 posttransfection (n = 3 experiments). e CFU numbers of 5, 000 cells on day 5 after transduction with lentivirus expressing shRNAs targeting c-Jun or scrambled shRNA (n = 3 experiments). G, CFU-granulocyte; M, CFU-macrophage; GM, CFU-granulocyte and macrophage; CFU-E, CFU-erythrocyte; BFU-E, erythroid burst-forming units; GEMM, CFU-granulocyte, erythroid, macrophage, and megakaryocyte. See also Table S4A. Sh-ctrl, CD34+ cells transfected with scrambled shRNA; Sh-J1, CD34+ cells transfected with 1# c-Jun shRNA; Sh-J2, CD34+ cells transfected with 2# c-Jun shRNA. All data shown as mean values ± SD. Statistical significance was assessed using unpaired t-test, where *p < 0.05; **p < 0.01, and ***p < 0.001; ND, not detected. See also Supplementary Fig. S3
Fig. 5 JNK-IN-8-induced CD34+ cell expansion acts by inhibiting c-Jun. a Relative mRNA expression of indicated JNK-related genes on day 5, CD34+ cells cultured with DMSO or J8 (n = 3 experiments). b Western blot analysis of inhibition of phosphorylated c-Jun for DMSO and J8-treated CD34+ cells following serum stimulation for 30 min. c Representative flow cytometry profiles of CD34+ cells 5 days after transduction with lentivirus expressing shRNAs targeting c-Jun or scrambled shRNAs (n = 3 experiments). d Total cell number of CD34+CD45RA- cell population in indicated cultured CD34+ cells on day 5 posttransfection (n = 3 experiments). e CFU numbers of 5, 000 cells on day 5 after transduction with lentivirus expressing shRNAs targeting c-Jun or scrambled shRNA (n = 3 experiments). G, CFU-granulocyte; M, CFU-macrophage; GM, CFU-granulocyte and macrophage; CFU-E, CFU-erythrocyte; BFU-E, erythroid burst-forming units; GEMM, CFU-granulocyte, erythroid, macrophage, and megakaryocyte. See also Table S4A. Sh-ctrl, CD34+ cells transfected with scrambled shRNA; Sh-J1, CD34+ cells transfected with 1# c-Jun shRNA; Sh-J2, CD34+ cells transfected with 2# c-Jun shRNA. All data shown as mean values ± SD. Statistical significance was assessed using unpaired t-test, where *p < 0.05; **p < 0.01, and ***p < 0.001; ND, not detected. See also Supplementary Fig. S3CC BY · PubMed Central

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Metadata

Authors
Xiao, X., Lai, W., Xie, H., Liu, Y., Guo, W., Liu, Y., Li, Y., Li, Y., Zhang, J., Chen, W., Shi, M., Shang, L., Yin, M., Wang, C., & Deng, H.
Venue
Cell Discovery
Publisher
Springer Science and Business Media LLC
Published
2019-01-08
Volume
5
Issue
1
ISSN
2056-5968
DOI
10.1038/s41421-018-0072-8
PMC
PMC6323118
Cited by
51
References
5
Language
en
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