Two Histone Variants TH2A and TH2B Enhance Human Induced Pluripotent Stem Cell Generation

Abstract

There are two major methods of reprogramming: generation of induced pluripotent stem cells (iPSCs) by overexpressing embryonic stem cell-specific transcription factors (OCT4, SOX2, KLF4, and c-MYC) and somatic cell nuclear transfer by oocyte-specific factors. Previously, we reported oocyte-enriched histone variants TH2A, TH2B, and the histone chaperone nucleoplasmin (NPM2) enhance the reprogramming by OSKM in mice by inducing open chromatin structure. In this study, we showed that human TH2A, TH2B, and NPM2 enhance the OSKM-induced reprogramming of adult and neonatal human dermal fibroblasts and umbilical vein endothelial cells. Pluripotency of iPSCs generated by coexpressing OSKM, TH2A, TH2B, and NPM2 was shown by in vitro and in vivo differentiation assays. These iPSCs gave rise to highly differentiated teratomas compared to iPSCs induced by OSKM alone. Genome-wide analysis suggests a possibility that TH2A, TH2B, and NPM2 might regulate genes that are involved in naïve stem cell stage. Thus, TH2A, TH2B, and NPM2 enhance reprogramming of human somatic cells and improve the quality of human iPSCs.

Introduction

Induced pluripotent stem cell (iPSC) generated by overexpressing defined transcription factors (OCT4, SOX2, KLF4, and c-MYC; OSKM) enriched in embryonic stem cells (ESCs) has a large impact in regenerative medicine [1,2]. However, the reprogramming process by OSKM is still slow and stochastic and resulting iPSCs often show the difficulty to differentiate into desired cell types [3]. Another technique for reprogramming is somatic cell nuclear transfer (SCNT) into oocytes [4 –6]. Recent studies showed that ESCs derived from SCNT embryo have less epigenetic abnormalities compared with iPSCs, likely due to orchestrated reprogramming events [3,7]. However, the generation of patient-specific ESCs by SCNT technique is still a challenge due to ethical reasons and scarcity of human oocytes.

Mouse histone variants TH2A and TH2B, which differ from canonical H2A and H2B by 15 and 16 amino acids, respectively, were first identified in mammalian testis [8,9]. We recently reported that mouse TH2A and TH2B are highly expressed in oocyte and zygote and gradually decreased as differentiation proceeds to blastocyst stage [10]. TH2A/TH2B double mutant oocytes showed significantly reduced ability to develop into blastocyst following fertilization compared with wild-type oocytes due to the defect in paternal genome activation in zygotes [10]. Since these results indicated that TH2A and TH2B are maternal effect factors, we tested whether TH2A and TH2B enhance somatic cell reprogramming like SCNT by oocyte-specific reprogramming factor(s). The OSKM-induced iPSC generations was enhanced ninefold by coexpression of TH2A and TH2B, although there was no effect when either TH2A or TH2B was expressed separately. Nucleoplasmin (Npm) is a histone chaperone, which is highly expressed in oocytes and is heavily phosphorylated during fertilization [11]. Therefore, we speculated that phosphorylation-mimic form of Npm (P-Npm), in which 11 predicted phosphorylation sites were changed to Asp, might further enhance the activity of TH2A and TH2B. As expected, coexpression of TH2B, TH2A, and P-Npm (so-called BAN) enhanced the OSKM-induced iPSC generation 18-fold. Interestingly, TH2A/TH2B more efficiently enhanced the iPSC generation from Xist mutant mouse embryonic fibroblasts (MEFs) compared with wild-type MEFs [10]. A previous study showed the cloning efficiency of Xist mutant nuclei by SCNT was much higher than that of wild-type nuclei [12]. Thus, addition of TH2A/TH2B to the somatic cell reprogramming process leads to greater similarity to oocyte-based reprogramming and SCNT.

TH2A and TH2B are also highly expressed during spermatogenesis, and they are essential for chromatin structure change during spermatogenesis [13]. Crystal structure of the nucleosomes containing canonical histones or TH2A/TH2B indicated that the number of strong hydrogen bond in the nucleosome containing TH2A/TH2B are less than that in the nucleosome containing canonical histones [14], suggesting that TH2A/TH2B induce open chromatin structure.

Human TH2A and TH2B have high homology with mouse TH2A and TH2B, respectively (Fig. 1). TH2B is highly expressed during human spermatogenesis [15] and forms unstable octamer, as demonstrated in mouse [10,16]. Human NPM2 is also highly enriched in oocyte [17] and has 66% identity with mouse Npm2. Human ESCs and iPSCs have similar properties with epiblast stem cells derived from the mouse epiblast [18,19]. Thus, they have a primed state of pluripotency that is different from the naïve pluripotent ground state of mouse ESCs and iPSCs [20]. Recently, some conditions to generate or enrich naïve human ESCs were demonstrated [21 –24]. Therefore, human TH2A, TH2B, and P-NPM2 may enhance the generation of human iPSCs with naïve ground state.

FIG. 1.

Gene structure and amino acid sequences of human histone variants TH2A and TH2B. (Upper) Structure of human TH2A and TH2B genes. The direction of transcription is shown by arrow, and the putative binding sites for transcription factors are indicated. (Lower) Amino acid sequences of canonical histone H2A (NP_066390.1), H2B (NP_003509.1), and histone variants TH2A (NP_734466.1) and TH2B (NP_733759.1) were aligned. Different amino acids are shown in red.

Here, we show that human TH2A, TH2B, and P-NPM2 enhance reprogramming of human somatic cells and improve the quality of human iPSCs.

Materials and Methods

Plasmid construction

TH2A (HIST1H2AA, NM_170745.3) and TH2B (HIST1H2BA, NM_170610.2) were amplified by polymerase chain reaction (PCR) and inserted into pLenti6.3 vector (Life Technologies). Putative phosphorylation sites of nucleoplasmin (NPM2) were deduced based on the method of Huang et al. [25]. Primer sequences used to generate theses vectors are listed in Supplementary Table S1 (Supplementary Data are available online at www.liebertpub.com/scd). The cDNA encoding phosphorylation-mimic nucleoplasmin (P-NPM2) was custom synthesized by Operon Biotechnologies, Inc., in which S4, S5, S7, S8, T9, S86, S99, S191, S196, and T204 of 10 putative phosphorylation sites were substituted to Asp. Nucleoplasmin 2 (NM_001286680.1) were used as the original sequence. We will deposit those plasmids to Addgene together with the maps and sequences.

Generation and maintenance of human iPSC

Adult human dermal fibroblasts (HDFs) and human umbilical vein endothelial cells (HUVECs) were purchased from Life Technologies, maintained in fibroblast medium (Medium M106 with LSGS supplement; Life Technologies) and EGM-2 BulletKit (Life Technologies), respectively. Human neonatal dermal fibroblasts (CC-2509) we obtained from Lonza-Life Technologies, and maintained in FGM-2 BulletKit fibroblast medium (Life Technologies). Lentivirus system was used to generate iPSCs. Plasmids pSIN4-EF2-O2S encoding human OCT4 and SOX2 and pSIN4-CMV-K2M encoding KLF4 and c-MYC were obtained from Addgene (#21162 and #21164, respectively) [26]. Lentiviruses were generated by cotransfection of pLenti6.3 vector with pLP1, pLP2, and pLP/VSVG (Life Technologies) or by cotransfection of pSIN4 vector with psPAX2, pMD2.G (Addgene). Virus-containing supernatant was concentrated by LentiX concentrator, and suspended into 1/10 of original volume using medium. Adult and neonatal HDFs and HUVECs (5 × 10⁴ cells) were infected with a mixture of each 50 μL lentiviral solution for 24 h and 3 h, respectively. Viral solution prepared using empty vector was used to adjust the volume. Virus-containing medium was changed to fibroblast medium or EGM medium, and cells were cultured for 48 h. Infected cells were passage 1/4 into 6-well plate containing mitomycin C-treated MEFs as feeders. Cells were maintained in human ES medium (20% Knockout serum replacement (Life Technologies), 10 ng/mL bFGF, 1 mM glutamine, 100 μM nonessential amino acid, 100 μM 2-mercaptoethanol, and 50 unit/mL streptomycin/penicillin in Knockout DMEM). In some case, adult HDFs were infected with virus to express BAN or empty vector for 24 h. Infected cells were then cultured in fibroblast medium and passaged once. After 5 days infection, cells were infected with virus to express OSKM as described.

Cell proliferation assay

At day 0, adult human fibroblasts were seeded at indicated number. Same amount of virus-containing medium as mentioned above were used to infect cells for 24 h. Infected cells were counted at day 5 and 8 postinfection by Countess II Automated Cell Counter (Life Technologies).

Immunofluorescence staining

Live cell staining was performed with anti-SSEA4 (BD Biosciences) and anti-TRA-1-60 antibodies (BD Biosciences) as described in Chan et al. [27]. For staining of OCT4 and NANOG, cells were fixed by 2% paraformaldehyde (PFA) in phosphate-buffered saline (PBS), permeabilized with 1% Triton-X100 in PBS, and then incubated with diluted (1/500) anti-OCT4 (Santa Cruz) and anti-NANOG (Abcam) antibodies for 2 h at room temperature. Secondary antibodies (Life Technologies) were diluted 1/1000 in 1% bovine serum albumin in PBS for 2 h at room temperature. Nuclei were stained by DAPI. For immunohistochemistry, tissue sections were stained with following antibodies: anti-SMA, anti-TUJ1, and anti-AFP (Invitrogen).

Alkaline phosphatase staining

Alkaline phosphatase staining was performed according to the instruction of Leukocyte Alkaline Phosphatase Kit (Sigma-Aldrich).

In vitro differentiation

Confluent iPSCs in one well of 12-well-plate were collected and suspended in 1 mL of human ES medium without bFGF. Cells were transferred into 12-well nonadherent plate and cultured for 3 days to form embryoid bodies. Embryoid bodies were transferred into adherent plate for spontaneous differentiation. Six days after transfer, differentiated cells were fixed and stained with antibodies against SMA, TUJ1, and AFP (Invitrogen).

Teratoma formation

Teratoma formation was performed as described by Gropp et al. [28]. Briefly 5 × 10⁵ iPSCs were mixed with 5 × 10⁵ mitomycin C-treated MEFs and suspended in 50 μL of PBS, then combined with 50 μL Matrigel (BD Biosciences) immediately before transplantation, and then injected subcutaneously into the dorsal flanks of NOD/ShiJic-scidJcl (NOD/SCID) mice (Clea Japan). Teratomas were dissected 6–9 weeks after injection. To analyze the area occupied by epithelial-like structures, images of nonconsecutive hematoxylin and eosin (H&E) sections (n = 10–12) of each teratoma were acquired and processed by Zeiss Axio Vision 4.8.2. For each teratoma, the areas of 100–120 epithelial-like structures were randomly selected and their sizes were measured. Experiments were conducted in accordance with the guidelines of the Animal Care and Use Committee of RIKEN Institute.

H&E staining

Tissues were fixed in 4% PFA in PBS at 4°C for 3 h, dehydrated by ethanol and butanol, and finally embedded in paraffin. Fixed tissues were sectioned into 5 μm thickness. Sections were rehydrated and stained with Mayer's hematoxylin (Wako) for 10 min, then rinsed with tap water for 15 min, and finally stained with 1% eosin for 15 min. Stained sections were dehydrated and mounted in Entellan neu (Merck). For immunohistochemistry, following antibodies were used: anti-SMA, anti-TUJ1, and anti-AFP (Invitrogen).

Quantitative reverse transcription-polymerase chain reaction

RNAs were extracted by Qiagen Rneasy Mini Kit (Qiagen). Total RNA (100 ng) was used for quantitative reverse transcription-polymerase chain reaction (qRT-PCR) reaction by RNA direct SYBR Green real-time PCR master mix (Toyobo). Sequence-specific primers were listed in Supplementary Table S2.

Microarray analysis

Total RNAs were extracted by ISOGEN (Nippon Gene) from three independent OSKM-induced iPSC clones and three independent OSKMBAN-induced iPSC clones. Total RNAs were amplified by Ambion WT Expression Kit (Life Technologies) and hybridized to Human Gene 1.0 ST Array (Affymetrix).

Data processing

Microarray data were processed with R package Oligo (www.r-project.org). RMA algorithm was used to normalize all the expression data at the same time. GEO dataset for human fibroblasts and human ESCs were retrieved from GSE23034 [29]. Gene set enrichment analysis (GSEA) was performed by default setting, except that the minimum size and maximum of gene set were set to 30 and 400 respectively. For GSEA of naïve stem cells, fourfold up- or downregulated transcripts by naïve stem cells compared to primed stem cells were used for up- or downregulated naïve stem cells transcripts gene sets [21]. GSEA analysis was performed by default setting.

Results/Discussion

We first examined whether efficiency of the OSKM-induced iPSC generation from HUVECs is stimulated by overexpression of BAN (TH2B, TH2A, and P-NPM2). After 11 days postinfection of lentiviruses to express the proteins, BAN enhanced the generation of OSKM-induced TRA-1-60-positive colonies 7.6-fold (Fig. 2A). At day 16, higher number of alkaline phosphatase-positive colony was observed in cells induced by OSKMBAN compared with OSKM alone (Fig. 2B). Number of SSEA4-positive colony was also increased by the coexpression of BAN (Fig. 2C). For application in regenerative medicine, adult HDFs are better sources, which can be collected from patients with a specific disease or disorder with an acceptable damage. BAN also enhanced the reprogramming of HDFs by 2.2-fold (Fig. 2D). When we used neonatal HDFs, BAN also enhanced the reprogramming by 2.2-fold (Fig. 2E). We also tested whether prolonged BAN expression modulates reprogramming efficiency of adult HDFs, because adult HDFs are better cell sources compared with neonatal HDFs for application purpose. Cells were infected with virus to express BAN or empty vector for 24 h. Infected cells were cultured in fibroblast medium and passaged once. After 5 days infection, cells were infected with virus to express OSKM. At day 16 after OSKM infection, the number of SSEA4-positive colony was enhanced 10.7-fold by prolonged BAN expression (Fig. 2F). This observation suggests that histone variant TH2A/TH2B need time to be incooporated into chromatin before OSKM expression. Colonies generated by OSKMBAN from HDFs expressed NANOG and OCT4 with the typical homogeneous nuclear staining pattern and showed flat morphology similar to human ESCs or iPSCs (Fig. 2G). Of note, overexpressing of BAN did not stimulate the proliferation rate of human fibroblasts (Fig. 2H), indicating that the appearance of more colonies by OSKMBAN compared with OSKM is not due to stimulation of cellular proliferation by BAN.

FIG. 2.

Histone variants TH2A and TH2B and histone chaperone P-NPM2 enhanced human iPSC generation. (A) The number of TRA-1-60-positive colonies induced by OSKM or OSKM plus TH2A/TH2B/P-NPM2 (OSKMBAN; BAN for TH2B, TH2A, and P-NPM-2). Human umbilical vein endothelial cells (HUVECs) were infected with lentiviruses to express indicated proteins, and stained with anti-TRA-1-60 antibody at day 11 postinfection (n = 3, mean ± standard deviation [SD]). Immunostaining of a colony induced by OSKMBAN were shown in the right panel. (B) Representative alkaline phosphatase staining of colonies at day 16 postinfection. (C) The number of SSEA4-positive colony from HUVECs induced by OSKM or OSKMBAN at day 11 postinfection (n = 4, mean ± SD). (D) The number of SSEA4-positive colony from adult human dermal fibroblasts (adult HDFs) induced by OSKM or OSKMBAN at day 16 postinfection (n = 4, mean ± SD). (E) The number of SSEA4-positive colony from neonatal HDFs induced by OSKM or OSKMBAN at day 16 postinfection (n = 4, mean ± SD). (F) Adult HDFs were infected with virus to express BAN or empty vector 5 days before infection with OSKM viruses. The number of SSEA4-positive colony at day 16 postOSKM virus infection is shown (n = 3, mean ± SD). (G) Immunofluorescence staining of pluripotency markers in iPSCs from HDFs generated by OSKMBAN. iPSCs are stained with anti-NANOG (red) and anti-OCT4 (green) antibodies, and DAPI (blue). (H) Cell proliferation rate of OSKM, OSKMBAN, BAN, and empty vector overexpressing human fibroblasts was monitored at indicated days. *P < 0.05 and **P < 0.001.

To judge the pluripotency of human iPSCs generated by OSKMBAN, we examined the in vitro and in vivo differentiation capacity. After 9 days of culture in differentiation medium, clones of the OSKMBAN-induced iPSCs expressed the markers for three germ layers: beta-III tubulin (TUJ1) for ectoderm, smooth muscle actin (SMA) for mesoderm, and alpha-fetoprotein (AFP) for endoderm (Fig. 3A). Subcutaneous injection of iPSC clones into NOD/SCID mice generated teratomas 4–7 weeks later (Supplementary Fig. S1). H&E staining of teratomas showed morphology of chondrocytes (mesoderm), neural rosette (ectoderm), and epithelial-like structure (endoderm) (Fig. 3B, Supplementary Fig. S2), and they were further confirmed by immunostaining of the three germ layer markers (Fig. 3C, Supplementary Fig. S3). Teratomas derived from the OSKMBAN-induced iPSCs contained more diverse structure and higher density of epithelium-like area than that from OSKM iPSCs (Fig. 3D, Supplementary Fig. S4, and Supplementary Data 1). These results suggest that OSKMBAN-induced iPSCs have a better differentiation capacity than OSKM-induced iPSCs in vivo. Similar higher degree of development in teratomas generated by naïve human ESCs compared with those by primed ESCs was recently reported [22].

FIG. 3.

OSKMBAN-induced iPSCs showed higher differentiation capacity than OSKM-induced iPSCs. (A) In vitro differentiation of iPSCs. OSKMBAN-induced iPSCs were cultured in differentiation medium. Immunostaining of beta-III tubulin (TUJ1) for ectoderm, smooth muscle actin (SMA) for mesoderm, and alpha-fetoprotein (AFP) for endoderm are shown. Scale bar: 100 μm. (B) Hematoxylin and eosin staining of teratoma sections. OSKMBAN-induced iPSCs generated well-differentiated teratomas containing all three germ layers. Typical morphologies of chondrocyte (mesoderm), neural rosette (ectoderm), and epithelial-like structure (endoderm) are shown. Scale bar: 100 μm. (C) Teratomas were positive for differentiation markers for three germ layers: SMA, TUJ1, and AFP. Scale bar: 100 μm. (D) OSKMBAN-induced iPSCs generated teratomas with larger differentiated area than OSKM-induced iPSCs. The area of epithelial-like structure in each three OSKM- and OSKMBAN-teratomas was measured, and shown in the boxplot. Total number of epithelial-like structures analyzed in OSKM- and OSKMBAN-teratomas are 320 and 318, respectively. Data are presented as boxplots. The middle line indicates the median value, and the upper and lower edges of the boxplot are 75 and 25th percentiles. The upper and lower whisker denote the value of the most extreme data point, which is not more than 1.5 times the interquartile range of the box. Data points that fall outside the whiskers are flagged as outliers and plotted as circles for visualization. **indicates P < 0.001. Analysis was performed using all the data including outliers. The raw data and the data in each teratoma are shown in Supplementary Data 1 and Supplementary Fig. S4, respectively.

To understand how BAN affects the characteristics of iPSCs at molecular level, we analyzed the whole transcript expression. OSKMBAN-induced iPSC clones were clustered in pluripotent stem cell group containing human ESCs and OSKM-induced iPSCs, indicating that OSKMBAN-induced iPSCs possess similar properties with human ESCs at gene expression level (Fig. 4A). The scatter plot of average gene expression levels of three OSKMBAN-induced iPSC clones and fibroblasts indicates that OSKMBAN-induced iPSCs are distinct from fibroblasts (Fig. 4B). The pluripotent markers (OCT4, SOX2, NANOG, ZFP42, and GDF3) shown in red dots were highly expressed in OSKMBAN-induced iPSCs than fibroblasts. Expression patterns of OSKMBAN-induced iPSCs showed a higher similarity to OSKM-induced iPSCs (r² = 0.99) than that of human ESCs (r ² = 0.96). The qRT-PCR data indicated that expression levels of endogenous OCT4, NANOG, and GDF3 were similar in OSKMBAN-induced iPSCs and OSKM iPSCs compared to human fibroblasts (Fig. 4C and Supplementary Fig. S5).

FIG. 4.

Gene expression profile of OSKMBAN-induced iPSCs. (A) Hierarchical cluster analysis of microarray expression data from three ES cell lines, each of three clones of OSKM-induced iPSCs and OSKMBAN-induced iPSCs, and three fibroblasts. Microarray data for human embryonic stem cells and human fibroblasts were obtained from Ohi et al. (2011). (B) Scatterplots of global gene expression patterns, comparing the indicated types of cells. Average expression levels of three clones were used. The expression levels of some pluripotent markers (OCT4, SOX2, NANOG, ZFP42, and GDF3) are shown in red. (C) Expression of endogenous OCT4, NANOG, and GDF3 mRNAs in OSKMBAN-induced iPSC lines was compared to that in three OSKM-induced iPSC lines or HDF. Data shown as mean ± SD (n = 3). NS, no significant difference. The data of individual iPSC clone are shown in Supplementary Fig. S5. (D) Gene set enrichment analysis (GSEA) of OSKM- and OSKMBAN-induced iPSCs. Upregulated genes in OSKMBAN-induced iPSCs compared to OSKM-induced iPSCs are shown (q < 0.1). (E) GSEA analysis for OSKMBAN- and OSKM-induced iPSCs probe sets with up- and downregulated transcripts of naïve pluripotent stem cells compared to primed pluripotent stem cells. Fourfold up- or downregulated transcripts were retrieved from Theunissen et al. (2014). P values are shown. The modified parts in the present revision are shown in blue.

One of the characteristics of pluripotent stem cells is the suppression of retroviral and lentiviral transgenes, which depend on the type of promoters. We evaluated expression levels of c-MYC and SOX2 from lentiviral vectors in iPSC clones. Exogenous c-MYC expression driven by CMV promoter was significantly downregulated in all iPSC clones (Supplementary Fig. S6A). On the other hand, SOX2 was persistently expressed at the same level in all iPSC clones under the control of EF1 promoter (Supplementary Fig. S6B), suggesting that EF1 promoter is relatively resistant to suppression in pluripotent cells in agreement with previous study [30]. However, it should be noted that levels of SOX2 expression in OSKMBAN-induced iPSCs were similar to those in OSKM-induced iPSCs, suggesting that it is unlikely that retained expression of Sox2 affects the quality of OSKMBAN- and OSKM-induced iPSCs.

To evaluate the difference in expression profile between OSKMBAN- and OSKM-induced iPSCs we performed the GSEA [31]. Upregulated genes in OSKMBAN-induced iPSCs compared to OSKM-induced iPSCs were involved in pathways including glycolysis and JAK-STAT pathway (Fig. 4D). We also examined whether up- or downregulated genes in OSKMBAN-induced iPSCs compared to OSKM-induced iPSCs resemble those in human naïve ESCs to primed ESCs. The upregulated genes in OSKMBAN-induced iPSCs were significantly enriched in the upregulated gene set of naïve ESCs (Fig. 4E, left). On the other hand, genes upregulated in OSKM-induced iPSCs were significantly enriched in the gene set downregulated in naïve stem cells (Fig. 4E, right). These results suggest the possibility that use of BAN may stimulate generation of naïve human iPSCs. Naïve state cells have a high cellular potency due to the genome-wide low methylation levels and low expression of lineage-specific genes [21]. Although the mechanism by which naïve state is established is still unclear, formation of open chromatin by TH2A and TH2B may help to set up the ground state of pluripotency, because they contribute to melting out the paternal genome in zygotes [8]. How BAN is involved in setting naïve state is an interesting topic for further research.

Footnotes

Acknowledgments

We are grateful to the staff of the Research Resources Center of the RIKEN Brain Science Institute for microarray analysis. This research was supported in part by the Mochida Memorial Foundation for Medical and Pharmaceutical Research.

Author Disclosure Statement

No competing financial interests exist.

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