Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2016 Oct 24;215(2):187-202.
doi: 10.1083/jcb.201601061. Epub 2016 Oct 17.

The Mitochondrial Protein CHCHD2 Primes the Differentiation Potential of Human Induced Pluripotent Stem Cells to Neuroectodermal Lineages

Affiliations
Free PMC article

The Mitochondrial Protein CHCHD2 Primes the Differentiation Potential of Human Induced Pluripotent Stem Cells to Neuroectodermal Lineages

Lili Zhu et al. J Cell Biol. .
Free PMC article

Abstract

Human induced pluripotent stem cell (hiPSC) utility is limited by variations in the ability of these cells to undergo lineage-specific differentiation. We have undertaken a transcriptional comparison of human embryonic stem cell (hESC) lines and hiPSC lines and have shown that hiPSCs are inferior in their ability to undergo neuroectodermal differentiation. Among the differentially expressed candidates between hESCs and hiPSCs, we identified a mitochondrial protein, CHCHD2, whose expression seems to correlate with neuroectodermal differentiation potential of pluripotent stem cells. We provide evidence that hiPSC variability with respect to CHCHD2 expression and differentiation potential is caused by clonal variation during the reprogramming process and that CHCHD2 primes neuroectodermal differentiation of hESCs and hiPSCs by binding and sequestering SMAD4 to the mitochondria, resulting in suppression of the activity of the TGFβ signaling pathway. Using CHCHD2 as a marker for assessing and comparing the hiPSC clonal and/or line differentiation potential provides a tool for large scale differentiation and hiPSC banking studies.

Figures

Figure 1.
Figure 1.
Variations in the ability of hiPSCs to undergo neuroectodermal differentiation. (A) Phase-contrast images of hESCs and hiPSCs used in this study. Bars, 100 µm. (B) Representative flow cytometric analysis indicating a high expression level of the pluripotency markers TRA1-60 and NANOG. (C) All human pluripotent stem cells formed EBs in suspension culture. Bars, 100 µm. (D) Graph representation of flow cytometric analysis for PAX6 expression at day 8 of neural induction process. Data are shown as mean ± SEM (n = 3). **, P < 0.005. (E) Immunofluorescence with SOX1 antibody at day 15 of neural induction process (nuclei were labeled with blue-fluorescent DAPI). Bars, 100 µm.
Figure 2.
Figure 2.
Transcriptional profiling demonstrating differences between hiPSCs and hESCs and NSCs derived therefrom. (A) Unsupervised hierarchical clustering of global gene expression data in hESCs and hiPSCs. (B) Global view of gene expression comparison between hESCs and hiPSCs. The array data were filtered using P < 0.05 and fold change >1.5. (C) Gene Ontology analysis of genes with different expression levels in hESCs and hiPSCs. The Gene Ontology terms are noted on the y axis and the log10(p-value) on the x axis. (D) Venn diagram analysis visualizing the overlap between the genes differently expressed in hESCs versus hiPSCs as well as NSCs derived from both sources.
Figure 3.
Figure 3.
CHCHD2 expression in human pluripotent stem cells and NSCs derived therefrom. (A) Quantitative RT-PCR results indicate that CHCHD2 expression is significantly lower in hiPSCs than in hESCs, and this difference is maintained in NSCs. Data are shown as mean ± SEM (n = 3). **, P < 0.005. (B) Western blot analysis of CHCHD2 in human pluripotent stem cells and NSCs derived therefrom. (C) Immunofluorescence with CHCHD2 antibody and MitoTracker red or CHCHD2 and mtTFA antibodies showing CHCHD2 expression in H9 and NSC-H9, but not in 19-9-7T or NSC-7T. Please note that CHCHD2 expression is only localized to the mitochondria of a subset of hESCs and all NSCS derived therefrom (nuclei were labeled with blue-fluorescent DAPI). Bars, 10 µm. (D) Schematic chart of colocalization coefficients between CHCHD2 and MitoTracker red or CHCHD2 and mtTFA in hiPSCs and NSCs derived therefrom. Data are shown as mean ± SEM (n ≥ 4).
Figure 4.
Figure 4.
CHCHD2 and its impact on the spontaneous differentiation of human pluripotent stem cells. (A) Quantitative RT-PCR results with markers of neuroectoderm (PAX6, SOX1, SOX2, and NESTIN), mesoderm (MIXL1 and T), endoderm (FOXA2 and GATA4), and trophectoderm (CDX2, EMOES, and HAND1) at day 6 of differentiation. Data are shown as mean ± SEM (n = 3). *, P < 0.05; **, P < 0.005. (B) Phase-contrast images of 19-9-7T cells stably transfected with vector or CHCHD2 construct. Bars, 200 µm. (C) Representative flow cytometric analysis indicating a high percentage of TRA1-60 and NANOG expression. (D) Quantitative RT-PCR analysis to assess the overexpression of CHCHD2 in CHCHD2 stable overexpression 19-9-7T. Data are shown as mean ± SEM (n = 3). ** P < 0.005. (E) Western blot analysis to assess the overexpression of CHCHD2 in CHCHD2 stable overexpression 19-9-7T. (F) Double staining of CHCHD2 and MitoTracker red or CHCHD2 and mtTFA indicates that CHCHD2 expression is localized to the mitochondria in 19-9-7T-CHCHD2 cells (nuclei were labeled with blue-fluorescent DAPI). Bars, 10 µm. (G) Schematic chart of colocalization coefficient between CHCHD2 and MitoTracker red or CHCHD2 and mtTFA. Data are shown as mean ± SEM (n ≥ 4). (H) Quantitative RT-PCR analysis indicating increased expression of neuroectodermal markers at the expense of other germ lineage markers upon stable overexpression of CHCHD2 in 19-9-7T. Data are shown as mean ± SEM (n = 3). **, P < 0.005. (I) Flow cytometric analysis at day 8 of neural induction indicating increased commitment to neuroectodermal lineages upon overexpression of CHCHD2 in 19-9-7T. Data are shown as mean ± SEM (n = 3). *, P < 0.05.
Figure 5.
Figure 5.
CHCHD2 expression correlates with the neural differentiation potential of hiPSC derived and characterized in our laboratory as well as others. (A) Large-scale transcriptional data from eight hESCs and 18 hiPSCs reported by Koyanagi-Aoi et al. (2013) to have different potentials to differentiate into neural lineages were analyzed for CHCHD2 expression. Data are shown as mean ± SEM (n = 3). (B) Relative CHCHD2 mRNA levels in H9, neonatal (Neo-1) fibroblast, adult (AD3) fibroblast, and reprogrammed hiPSC clones derived from them (Neo1-1# to 7# and AD3-1# to 9#). Data are shown as mean ± SEM (n = 3). (C) Phase contrast images of hiPSC lines Ad2 CL1 and Ad3 CL1. Bars, 100 µm. (D) Representative FACS analyses showing a high expression of TRA1-60 and NANOG in Ad2 CL1 and Ad3 CL1. (E) Relative expression of CHCHD2 in 19-9-7T, Ad2 CL1, and Ad3 CL1. Data are shown as mean ± SEM (n = 3). **, P < 0.005. (F) Western blot analysis of CHCHD2 in 19-9-7T, Ad2 CL1, and Ad3 CL1 lines. (G) Representative FACS analysis at day 8 of neural induction showing variable proportion of PAX6-positive cells from hiPSCs. Data are shown as mean ± SEM (n = 3). **, P < 0.005.
Figure 6.
Figure 6.
Variable expression of CHCHD2 in hiPSC clones and regulation of its expression during the reprogramming process. (A) Relative CHCHD2, OCT4, SOX2, KLF4, and c-MYC mRNA levels in AD3-CL7#, AD3-CL8#, AD3-CL9#. Data are shown as mean ± SEM (n = 3). (B) Western blot analysis of CHCHD2 in AD3-CL7#, AD3-CL8#, and AD3-CL9#. (C) Relative CHCHD2 mRNA levels in AD3 fibroblasts and AD3 fibroblasts transduced with Sendai-EGFP, Sendai-OCT4, Sendai-SOX2, Sendai-KLF4, and Sendai-c-MYC for 3 d. Data are shown as mean ± SEM (n = 3). **, P < 0.005. (D) Schematic presentation of CHCHD2 promoter showing the location of the putative OCT4 and SOX2 binding sites, as well as the location of primers used for ChIP. OCT4 and SOX2 binding sites are highlighted in blue and red. NS, nonspecific primers for region without potential OCT4, SOX2 binding sites; P1, P2 specific primers for OCT4, SOX2 potential binding sites. (E) ChIP assays demonstrating the capacity of OCT4 and SOX2 to bind to the CHCHD2 upstream fragment in H9 cells.
Figure 7.
Figure 7.
CHCHD2 represses TGFβ signaling activity. (A) CHCHD2 interacts with SMAD4. CHCHD2 and SMAD4 were immunoprecipitated (IP) and analyzed for the presence of SMAD4 and CHCHD2 by Western blot from the protein lysates of whole cells, as well as nuclear and mitochondrial fractions. (B) CHCHD2 regulates Smad4 intracellular localization. SMAD4 is predominantly expressed in the nucleus; however, upon CHCHD2 overexpression, less SMAD4 is found in the nucleus and more in mitochondria. Upon CHCHD2 knockdown, more SMAD4 is found in the nucleus and less in mitochondria. Nuclear and mitochondrial fractions were isolated from 19-9-7T overexpressed with vector (OE-Vector) or CHCHD2 (OE-CHCHD2) and H9 transfected with si-NS (KD-Control) or si-CHCHD2 (KD-CHCHD2). Histone H3 and VDCA1 were used as nuclear and mitochondrial markers, respectively. (C) SMADs transcriptional activity is repressed by CHCHD2. Cells were cotransfected with the reporter plasmid with the vector or CHCHD2 construct (left) or cotransfected with the reporter plasmid with si-NS or si-CHCHD2 (right). Cells that were transfected with the reporter and incubated with 10 µM SB431542 were used as controls. Data are shown as mean ± SEM (n = 3). **, P < 0.005. (D) Western blot analysis of phosphorylated-SMAD2 (p-SMAD2), total-SMAD2, phosphorylated-SMAD3 (p-SMAD3), total-SMAD3, SMAD4, and CHCHD2 in the cells as indicated. (E) Relative protein expression levels in indicated cells. Data are shown as mean ± SEM (n = 3). *, P < 0.05; **, P < 0.005. (F) Quantitative RT-PCR results of TGFβ-related genes in the cells as indicated. Data are shown as mean ± SEM (n = 3). *, P < 0.05; **, P < 0.005. (G) Quantitative RT-PCR results of markers of neuroectoderm (PAX6), mesoderm (MIXL1), endoderm (GATA4), and trophectoderm (CDX2, HAND1) at day 6 of spontaneous differentiation of H9 cells transfected with si-NS or si-CHCHD2 or si-CHCHD2 with TGFβ inhibitors. Data are shown as mean ± SEM (n = 3). *, P < 0.05; **, P < 0.005. (H) Graph representation of flow cytometric analysis for PAX6 expression at day 8 of the monolayer differentiation process. Data are shown as mean ± SEM (n = 3). **, P < 0.005.

Similar articles

See all similar articles

Cited by 10 articles

See all "Cited by" articles

References

    1. Aras S., Pak O., Sommer N., Finley R. Jr., Hüttemann M., Weissmann N., and Grossman L.I. 2013. Oxygen-dependent expression of cytochrome c oxidase subunit 4-2 gene expression is mediated by transcription factors RBPJ, CXXC5 and CHCHD2. Nucleic Acids Res. 41:2255–2266. 10.1093/nar/gks1454 - DOI - PMC - PubMed
    1. Aras S., Bai M., Lee I., Springett R., Hüttemann M., and Grossman L.I. 2015. MNRR1 (formerly CHCHD2) is a bi-organellar regulator of mitochondrial metabolism. Mitochondrion. 20:43–51. 10.1016/j.mito.2014.10.003 - DOI - PubMed
    1. Armstrong L., Tilgner K., Saretzki G., Atkinson S.P., Stojkovic M., Moreno R., Przyborski S., and Lako M. 2010. Human induced pluripotent stem cell lines show stress defense mechanisms and mitochondrial regulation similar to those of human embryonic stem cells. Stem Cells. 28:661–673. 10.1002/stem.307 - DOI - PubMed
    1. Balakrishnan S.K., Witcher M., Berggren T.W., and Emerson B.M. 2012. Functional and molecular characterization of the role of CTCF in human embryonic stem cell biology. PLoS One. 7:e42424 10.1371/journal.pone.0042424 - DOI - PMC - PubMed
    1. Birket M.J., Orr A.L., Gerencser A.A., Madden D.T., Vitelli C., Swistowski A., Brand M.D., and Zeng X. 2011. A reduction in ATP demand and mitochondrial activity with neural differentiation of human embryonic stem cells. J. Cell Sci. 124:348–358. 10.1242/jcs.072272 - DOI - PMC - PubMed

MeSH terms

Feedback