Butyrate greatly enhances derivation of human induced pluripotent stem cells by promoting epigenetic remodeling and the expression of pluripotency-associated genes - PubMed (original) (raw)

Bin-Kuan Chou, Jonathan Yen, Zhaohui Ye, Jizhong Zou, Sarah Dowey, Robert A Brodsky, Joyce E Ohm, Wayne Yu, Stephen B Baylin, Kosuke Yusa, Allan Bradley, David J Meyers, Chandrani Mukherjee, Philip A Cole, Linzhao Cheng

Affiliations

Butyrate greatly enhances derivation of human induced pluripotent stem cells by promoting epigenetic remodeling and the expression of pluripotency-associated genes

Prashant Mali et al. Stem Cells. 2010 Apr.

Abstract

We report here that butyrate, a naturally occurring fatty acid commonly used as a nutritional supplement and differentiation agent, greatly enhances the efficiency of induced pluripotent stem (iPS) cell derivation from human adult or fetal fibroblasts. After transient butyrate treatment, the iPS cell derivation efficiency is enhanced by 15- to 51-fold using either retroviral or piggyBac transposon vectors expressing 4 to 5 reprogramming genes. Butyrate stimulation is more remarkable (>100- to 200-fold) on reprogramming in the absence of either KLF4 or MYC transgene. Butyrate treatment did not negatively affect properties of iPS cell lines established by either 3 or 4 retroviral vectors or a single piggyBac DNA transposon vector. These characterized iPS cell lines, including those derived from an adult patient with sickle cell disease by either the piggyBac or retroviral vectors, show normal karyotypes and pluripotency. To gain insights into the underlying mechanisms of butyrate stimulation, we conducted genome-wide gene expression and promoter DNA methylation microarrays and other epigenetic analyses on established iPS cells and cells from intermediate stages of the reprogramming process. By days 6 to 12 during reprogramming, butyrate treatment enhanced histone H3 acetylation, promoter DNA demethylation, and the expression of endogenous pluripotency-associated genes, including DPPA2, whose overexpression partially substitutes for butyrate stimulation. Thus, butyrate as a cell permeable small molecule provides a simple tool to further investigate molecular mechanisms of cellular reprogramming. Moreover, butyrate stimulation provides an efficient method for reprogramming various human adult somatic cells, including cells from patients that are more refractory to reprogramming.

PubMed Disclaimer

Conflict of interest statement

Disclosure of Potential Conflicts of Interest: The authors indicate no potential conflicts of interest.

Figures

Figure 1

Figure 1

Butyrate stimulation of reprogramming of human fibroblasts. (A): IMR90 fibroblasts transduced by the standard four reprogramming factors, Oct4, Sox2, Klf4, and c-Myc (collectively called OSKM), were treated by individual small-molecule chromatin modifiers which were added in the cell culture in three different ways as shown: at an early stage (#1), a late stage (#2), or throughout (#3). The relative fold stimulation of TRA-1-60+ hES-like colonies compared with plain OSKM reprogramming (defined as one) was enumerated on day 16 after live staining (mean ± SEM, n = 3). Concentrations of the small molecules used in these experiments were: RG108: 5 _μ_M; BIX01294: 0.5 _μ_M; valproic acid: 1 mM; and sodium butyrate: 0.5 mM. (B): Flow cytometric analysis of TRA-1-60 expression in cells undergoing reprogramming. Cells at day 12 after OSKM transduction (basal recipe), or further treated with butyrate (N2 or N3), were harvested and stained. Abbreviations: B, BIX01294; hES, human embryonic stem; n, number; N, sodium butyrate; R, RG108; V, valproic acid.

Figure 2

Figure 2

Probing the mechanisms of butyrate stimulation using various inhibitors affecting protein acetylation. (A): Protein acetylation is critical to successful reprogramming. BASC fibroblasts were reprogrammed in the absence or presence of butyrate (0.25 mM). A HAT inhibitor (C646) or a control compound (C37) was added at increasing concentrations. HAT inhibition at high concentrations prevented reprogramming and also significantly diminished reprogramming efficiency, even in the presence of butyrate (from 27-fold to 4-fold). (B): A diagram of protein acetylation and deacetylation mediated by HATs and HDACs (and their inhibitors) using histone H3 lysine nine (H3K9) as a substrate. The compounds that promote protein acetylation are marked in red. (C): Detection of H3K9 acetylation by antibody staining in BASC fibroblasts with or without OSKM transduction and in the presence or absence of butyrate (day 6). (D): Butyrate stimulated reprogramming in the presence of fewer reprogramming factors. IMR90 cells were transduced by OSK or OSM and cultured in the presence or absence of sodium butyrate (N) in combination with BIX01294 (B, 0.5 _μ_M). The level of TRA-1-60+, human embryonic stem cell (hES)-like colonies at day 21 under each condition (mean ± SEM, n = 3) was compared with that by the optimal OSKM reprogramming. Abbreviations: HAT, histone acetyltransferase; N, sodium butyrate; OSKM, Oct4, Sox2, Klf4, and c-Myc collectively; OSK, Oct4, Sox2, Klf4; OSM, Oct4, Sox2, c-Myc.

Figure 3

Figure 3

Butyrate stimulated the expression of a group of pluripotency-associated genes during reprogramming. (A): IMR90 cells after OSKM transduction and treated by NaB or VPA (for 4 days) were harvested at day 6 (D6) or day 12 (D12). The genome-wide gene expression at these intermediate stages of reprogramming was analyzed in comparison with the parental IMR90 cells. The relative expression levels of the top genes such as POU5F1/OCT4 with the greatest stimulation by NaB under condition #1 (days 2 to 6) are shown [9]. By day 12, elevation of pluripotency-associated genes upon small molecule stimulation was overt, generally more so by NaB than by VPA. (B): Reverse transcription polymerase chain reaction validated the stimulation of endogenous genes such as DPPA2, DPPA3, DPPA4, DPPA5, ZFP42/REX1, and NANOG (but not KLF4). However, the expression from the transgenes used for reprogramming was not significantly affected by either treatment. Abbreviations: NaB, sodium butyrate; OSKM, Oct4, Sox2, Klf4, and c-Myc collectively; VPA, valproic acid.

Figure 4

Figure 4

Butyrate enhanced promoter DNA demethylation of pluripotency-associated genes. (A): We used Infinium HumanMethylation27 Bead-Chip to analyze the DNA methylome before and after successfully reprogramming. The level of DNA methylation (from 0 to 1) at gene promoters containing a CpG island (white) or without (black) is illustrated by color from blue to red. Loci are grouped as four clusters based on loss (#2), gain (#3), or unchanged (#1 remaining high and #4 remaining low) of DNA methylation. We used the K-means clustering algorithm to analyze the methylation data from three fibroblast lines (IMR90, MSC1640, and BASC), eight successfully reprogrammed iPS cells, and two ES lines (H1 and H9). The eight validated iPS cells showed a pattern highly similar to hES cells versus their parental fibroblasts. (B): Multidimensional scaling analysis of the relationship among different cell types based on DNA methylation profiles of 1,174 loci in cluster #2 was performed. Data along the first two components are plotted here. Two types of mesenchymal stem cells (MSCs) (MSC1640 and BASC), derived iPS cell lines, and intermediate cells during MSC reprogramming at day 6 (D6, condition #1) or day 12 (D12, condition #2) with or without sodium butyrate (N) were used. Duplicate intermediate MSC samples are referred by s1 and s2. It is evident that the butyrate-treated samples at day 12 (D12N2, red arrow) cluster away from MSCs and move closer to the iPS/ES group. (C): Detection of CpG DNA methylation by the microarray (numbers for 0 to 1.00) and by direct sequencing after bisulfite conversion (filled circles) of the promoter regions of POU5F1/OCT4 and DPPA2 genes. Reduction of DNA methylation in multiple CpG sites at both promoters is evident from parental MSCs to reprogramming intermediate cells at 12 (especially after butyrate treatment) and ultimately in successfully reprogrammed iPS cells. (D): DPPA2 transgene expression partially substitutes butyrate and further enhances reprogramming by the four reprogramming factors OSKM. BASC or IMR90 cells were transduced by a retroviral vector expressing either DPPA2 or GFP (as a control) together with OSKM, and fold change in TRA-1-60+ colonies was enumerated (mean ± SEM, n = 4). Abbreviations: BASC, Bone marrow Aspirate derived marrow Stromal Cells from patient with Sickle Cell Disease; GFP, green fluorescent protein; ES, ES, embryonic stem; hES, human embryonic stem; iPS, induced pluripotent stem; n, number; N, sodium butyrate; OSKM, Oct4, Sox2, Klf4, and c-Myc collectively.

Figure 5

Figure 5

Butyrate similarly stimulates human iPS cell derivation when piggyBac transposition is used for reprogramming. (A): Serially-passaged BASCs (passage 3 through passage 7), the MSCs from an adult female patient with sickle cell disease, were reprogrammed using piggyBac transposition. Butyrate significantly stimulated the formation of TRA-1-60+ colonies under conditions N2 and N3 for MSCs at each passage (passage 3 through passage 7), although later passage MSCs generated less TRA-1-60+ colonies (5- to 10-fold lower). Data from MSCs of five different passages were combined (n = 10) and plotted. Under the conditions #2 and #3 (N2 and N3), butyrate increased the reprogramming efficiency by 23- to 25-fold. (B): Five iPS lines (s1, s2, and s3) or pools (passage 3 and passage 4) were derived by picking TRA-1-60+ colonies at day 24 after piggyBac transposition. DNA fingerprinting was used to confirm the origin of the BASC line. (C): One of the five established lines, s1, was confirmed to have the homozygous HbS mutation (A to T) and a polymorphic C (*) found in the patient with sickle cell disease compared with T in the general population. (D): The line shows a normal (female) karyotype. (E): It expresses the characteristic pluripotency markers (left panels), readily differentiates in suspension culture to form cystic embryoid bodies with elements of all three germ layers (middle), and forms teratomas in nude mice comprising of various cell types derived from all of the three embryonic germ layers (right). Abbreviations: iPS, induced pluripotent stem; MSC, mesenchymal stem cells; BASC, Bone marrow Aspirate derived marrow Stromal Cells from patient with Sickle Cell Disease.

Similar articles

Cited by

References

    1. Hochedlinger K, Plath K. Epigenetic reprogramming and induced pluripotency. Development. 2009;136:509–523. - PMC - PubMed
    1. Huangfu D, Osafune K, Maehr R, et al. Induction of pluripotent stem cells from primary human fibroblasts with only Oct4 and Sox2. Nat Biotechnol. 2008;26:1269–1275. - PubMed
    1. Mikkelsen TS, Hanna J, Zhang X, et al. Dissecting direct reprogramming through integrative genomic analysis. Nature. 2008;454:49–55. - PMC - PubMed
    1. Shi Y, Desponts C, Do JT, et al. Induction of pluripotent stem cells from mouse embryonic fibroblasts by Oct4 and Klf4 with small-molecule compounds. Cell Stem Cell. 2008;3:568–574. - PubMed
    1. Lin T, Ambasudhan R, Yuan X, et al. A chemical platform for improved induction of human iPSCs. Nat Methods. 2009;6:805–808. - PMC - PubMed

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources