Histone deacetylases differentially regulate the ...

Stem Cell Research 17 (2016) 170–180

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Histone deacetylases differentially regulate the proliferative phenotype of mouse bone marrow stromal and hematopoietic stem/progenitor cells Neha R. Dhoke a,b, Evangelene Kalabathula a, Komal Kaushik a,b, Ramasatyaveni Geesala a,b, SravaniB. a, Amitava Das a,b,⁎ a b

Centre for Chemical Biology, CSIR-Indian Institute of Chemical Technology, Uppal Road, Hyderabad, 500 007, India Academy of Scientific & Innovative Research, 2 Rafi Marg, New Delhi 110 001, India

a r t i c l e

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Article history: Received 17 November 2015 Received in revised form 27 June 2016 Accepted 1 July 2016 Available online 02 July 2016 Keywords: BMSCs HSPCs Cyclin B1 Cyclin D2 HDAC2 HDAC3

a b s t r a c t Mouse bone marrow stromal stem/progenitor cells (BMSCs, also known as bone marrow-derived mesenchymal stem cells) and Hematopoietic Stem and Progenitor Cells (HSPCs) with differential proliferative potentials were investigated for identifying epigenetic signals that can modulate their growth. In the present study, immunodepletion of granulo-monocytic (CD11b) and erythroid (Ter119) population yielded CD11b−/Ter119− cells, capable of differentiating into chondrogenic, osteogenic and adipogenic cells. Enrichment of the CD11b+ population by positive selection of multipotent stem/progenitor marker (CD133) yielded CD11b+/CD133+ cells, efficiently differentiated into hematopoietic lineages. Molecular characterization revealed the expression of BMSC and HSPC markers in CD11b−/Ter119− and CD11b+/CD133+ sorted populations, respectively. Cell expansion studies depicted a higher growth rate and percentage of proliferating cells in G2/M phase of cell cycle in BMSCs (13.9 ± 2.9%) as compared with HSPCs (5.8 ± 0.8%). Analysis of the HDACs gene expression revealed a differential expression pattern in BMSCs and HSPCs that modulates the cell cycle genes. Trichostatin A (TSA)-mediated HDAC inhibition led to an increased level of AcH3 and AcH4 along with cyclins B1 and D2. Chromatin immunoprecipitation revealed alleviation of HDAC2 and HDAC3 binding by TSA on cyclins B1 and D2 promoter, thereby enhancing cell proliferation. This study identifies epigenetic modulation on the proliferative potential of BMSCs and HSPCs for stem cell transplantation therapies. © 2016 Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction Bone marrow stromal stem/progenitor cells (BMSCs, also known as bone marrow-derived mesenchymal stem cells), possess properties like self-renewal, multipotency and immuno-modulation that make them an attractive candidate for cell transplantation therapy. Friedenstein et al., pioneered the isolation of MSC populations that were adherent and depicted the self-renewal property (Bianco and Gehron Robey, 2000). Bone marrow contains heterogeneous cell populations, including BMSCs, HSPCs, endothelial progenitor cells (EPCs), etc.

Abbreviations: BMSCs, bone marrow stromal stem/progenitor cells; HSPCs, hematopoietic stem/progenitor cells; MACS, magnetic activated cell sorting; LSK, lineage, Sca-1, c-Kit; HDAC, histone deacetylases; CDKs, cyclin dependent kinases; CKIs, cyclin dependent kinase inhibitors; FBS, fetal bovine serum; MTT, 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; BrdU, bromodeoxy uridine; PI, propium iodide; G0/G1 Phase, gap 1 phase of the cell cycle; G2/M Phase, gap 2 phase and mitotic phase of the cell cycle; S Phase, DNA synthesis phase of the cell cycle; FITC, fluoroscein isothiocyanate; PE, phycoerythrin; TSA, trichostatin A; ChIP, chromatin immunoprecipitation. ⁎ Corresponding author. E-mail addresses: [email protected], [email protected] (A. Das).

(Kucia et al., 2005). Studies in the past have used plastic adherence property to isolate BMSCs from bone marrow (Friedenstein et al., 1982). However, this technique suffers with limitations as cells from hematopoietic origin and various fibroblastic cells often contaminate the cultures. To date, a number of putative markers have been employed for isolation of BMSCs (Sacchetti et al., 2007). Among these, CD146+ and STRO-1 expressing cells from human bone marrow cells (BMSC) exhibited self-renewal capability (Sacchetti et al., 2007). Recently various studies have been performed to identify murine bone marrow BMSCs using different cell surface markers including Nestin (Mendez-Ferrer et al., 2010), leptin receptor (Zhou et al., 2014), and Grem1 (Worthley et al., 2015) markers. Also studies by Chan et al. identified postnatal BMSCs from growth plate of femurs that differentially expressed markers such as CD45−/Ter-119−/Tie2−/AlphaV+/Thy−/6C3−/ CD105−/CD200+ with capabilities to undergo skeletogenesis (Chan et al., 2015). BMSCs have also been isolated by immunodepletion of either CD11b or CD34 or CD45 expressing cells. However isolation with a single surface marker resulted in contamination of hematopoietic cells (Xu et al., 2010). Recent studies showed CD34−/low populations are also comprised of long-term hematopoietic stem cells (HSCs) as well as reports of CD45low expressing BMSC (Donnelly et al., 1999; Osawa et al.,

http://dx.doi.org/10.1016/j.scr.2016.07.001 1873-5061/© 2016 Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

N.R. Dhoke et al. / Stem Cell Research 17 (2016) 170–180

1996; Yu et al., 2010). These studies suggest the use of a combination of markers for isolation of BMSCs. HSCs isolated by the set of markers which are Lin−/Sca-1+/C-kit+ (LSK) have been also reported to be heterogeneous (Osawa et al., 1996). Moreover, Sca-1 expression differs between mouse strains and therefore is not very effective to enrich HSCs (Spangrude and Brooks, 1993). Likewise, the SLAM family member CD150 is not useful for emerging and developing HSCs in embryos (McKinney-Freeman et al., 2009). Other markers such as CD11b/Mac1 is often expressed in the early HSPCs (Matsubara et al., 2005; Mikkola and Orkin, 2006). The inconsistencies of surface markers on BMSCs and HSPCs led us to identify alternative markers with greater specificity to sort the BMSC and HSPC populations from bone marrow. Maintenance of stemness of adult stem cells in their niches is governed by unique combinations of epigenetic regulators (Oh and Humphries, 2012). Epigenetic mechanisms, including Histone acetylation/deacetylation, play a crucial role in transcriptional regulation via remodelling of chromatin architecture (Huang et al., 2015; Oh and Humphries, 2012). Histone deacetylases (HDACs), classified into four classes, (Gregoretti et al., 2004; Telles and Seto, 2012; Yang and Seto, 2003) catalyzes a wide spectrum of physiological process including proliferation, differentiation, apoptosis and cell cycle regulation (Choudhary et al., 2009). Mammalian cell cycle progression through four distinct phases, G1/ S/G2/M are mediated via cyclins and cyclin dependent kinases (CDKs) (Nurse, 1994). These CDKs are successively regulated by Cdk inhibitors (CKIs) (Sherr and Roberts, 1999). HDACs deacetylate and regulate the activity of key cell cycle proteins (Glozak et al., 2005). In the past, studies have been performed to assess the role of HDACs in modulating the differentiation ability of stem cells, including Embryonic Stem Cells (ESCs) and adult stem cells (Qiao et al., 2015). However, studies are lacking in identification of HDACs regulation of adult stem cell proliferation. Often in the event of an injury, adult stem cells migrate from their niche to regenerate the damaged tissues, which is directly dependent on its proliferative capacity. Thus, understanding the molecular mechanisms involved during HDAC-mediated cell cycle gene regulation may provide a better insight into the proliferative potential of these adult stem/progenitor cells. In the present study, we have isolated and characterized both BMSCs and HSPCs from mouse bone marrow based on the presence and absence of CD11b, Ter119 and CD133 markers. The two resultant sorted cell populations, CD11b−/Ter119− and CD11b+/CD133+, were successfully differentiated into skeletal and hematopoietic lineages, respectively. CDK and CKI gene expression correlated well with the cell cycle analysis depicting a relatively higher percentage of BMSCs in G2/M phase, and HSPCs in the G0 phase of the cell cycle. The expression profile of HDACs in these populations suggests the plausible mechanism of differential proliferation. Finally, HDAC inhibition led to an increase in acetylated Histones and Cyclins B1/D2 levels in these cell populations. This study suggests the translational role of epigenetic modifiers in regulating the proliferative potential of these adult stem/progenitor cells. 2. Materials and methods 2.1. Isolation and culture of mouse BMSCs and HSPCs Mouse BMSCs were isolated according to a modified protocol (Kopen et al., 1999). Animal experimentation protocols were approved by the institutional Animal Ethics Committee. Briefly, bone marrow was flushed from the femurs and tibias of 6–8 week old male C57BL6/J mice, cells were counted and subjected to immunodepletion of granulomonocytic cells using CD11b (Kopen et al., 1999) and Ter119 (Morikawa et al., 2009) antibody coated magnetic microbeads (Miltenyi Biotech, USA) sequentially in a MACS system according to the manufacturer's protocol. Similarly, from the CD11b+ cell populations, selection of HSPCs were achieved using CD133 antibody microbeads

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(Hess et al., 2006). Two consecutive positive selections yielded CD11b+/CD133+ bone marrow cells which were separately plated for subsequent characterization and experiments. Detailed methods of cell sorting are in the supporting information. Purity check of these sorted cell populations was achieved using flow cytometry analysis. 2.2. Molecular characterization of BMSCs and HSPCs Sorted CD11b−/Ter119− and CD11b+/CD133+ cell populations were characterized for the expression of BMSC- and HSPC- genes and cell surface proteins, respectively using quantitative Real-time PCR (qRT-PCR) and flow cytometry approaches. Detailed methodology of semi-quantitative (Baddoo et al., 2003) and qRT-PCR (Das et al., 2006), flow cytometric analysis (Baddoo et al., 2003; D'Alessio et al., 2011), along with cell lineage differentiation assays (Shivdasani and Schulze, 2005; Zhu et al., 2010) are in the supporting information. 2.3. Growth curve analysis and cell proliferation assays Sorted cells were seeded at a cell density of 1 × 103 cells per well in at least triplicates. Cells were harvested at regular intervals of 48 h and counted microscopically to plot cell growth curves (Pendleton et al., 2013). Viability of the sorted cell populations were evaluated using MTT (Geesala et al., 2016) and BrdU (Ploenes et al., 2013) analysis as discussed in detail in the supporting information. 2.4. Cell cycle analysis DNA content in the sorted cell populations was also evaluated after staining with propidium iodide (PI, 0.5 mg/mL, Sigma-Aldrich, USA) using flow cytometry analysis as described previously (Peng et al., 2008). 2.4.1. Cell cycle and HDAC gene expression assays Mouse-specific forward and reverse primers of cyclins, CKIs, as well as genes belonging to different classes of HDACs were used for qRT-PCR analysis to identify their expression levels in the sorted CD11b−/ Ter119− and CD11b+/CD133+ cell populations. All target gene expression was normalized to eukaryotic 18S rRNA and shown as the – fold change relative to bone marrow cells as controls (Das et al., 2006). 2.4.2. Colony formation assays Colony formation efficiencies of sorted cells were performed as described earlier (Sacchetti et al., 2007). CD11b−/Ter119− cells were cultured in Mesencult medium (StemCell Technologies, Canada) with different treatments and colonies with 50 or more cells were scored under inverted microscope. Similarly, CD11b+/CD133+ cells were cultured in Methocult medium and colonies formed were evaluated according to standard criteria. 2.4.3. HDAC inhibition by trichostatin A Sorted CD11b−/Ter119− and CD11b+/CD133+ populations were treated with increasing concentrations (0, 1, 5 and 10 nM) of trichostatin A (TSA, Sigma-Aldrich, USA), an HDAC inhibitor, and evaluated for its effect on colony formation assays, growth curve analysis, cell proliferation using BrdU analysis and cell cycle gene expression analysis as described previously (Ploenes et al., 2013). 2.4.4. Immunofluorescent staining Cells were briefly fixed with 4% formaldehyde, permeabilized, and subjected to methanol fixation overnight. Subsequently, cells were washed, and either stained with anti-Ki67 rabbit antibody or antiBrdU antibody or stained with anti-CD11b, anti-Ter119, anti-Sca-1, anti-CD29, anti-CD133 antibodies (Biolegends, USA). Detailed steps are provided in the supporting information.

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2.5. Immunoblot analysis Cells treated with or without TSA were lysed, subjected to 10% SDS–PAGE and transferred onto a polyvinylidene difluoride membrane. Membranes were incubated with primary antibodies against Cyclins B1, D2, p27, (Cell Signaling Technologies, USA), HDACs 1, 2, 3 (Abcam, USA) and Acetylated Histones 3 (AcH3) and 4 (AcH4) (Merck Milipore, USA) at 4 °C for overnight. Detailed methodology is described in the supporting information (Ploenes et al., 2013).

2.5.1. Chromatin immunoprecipitation (ChIP) assays ChIP assays were performed as described previously (Kawamura et al., 2005). Briefly, after fixation of cells with formaldehyde for cross-linking, cell extracts were sonicated and subsequently immunoprecipitated separately with mouse anti-HDAC2 or antiHDAC3 antibody (Merck Millipore, USA), or with control goat IgG. Immunocomplexes were pulled down, washed and DNA was isolated to run qRT-PCR with primers specific for mouse cyclins B1 and D2 promoter sequences. Detailed steps of the assays are described in the supporting information.

2.6. Statistical analysis Results represent the mean ± standard error of the mean of at least three independent experiments, which were performed with a minimum of triplicate samples. Photomicrographs represent typical experiments reproduced at least three times with similar results. Statistical analyses were performed using a Student's t-test with values of p b 0.05 considered significant.

3. Results 3.1. Isolation of mouse BMSCs and HSPCs Freshly isolated mouse bone marrow cells were immunodepleted of granulo-monocytic (CD11b) as well as pro-erythroid (Ter119) cells in two consecutive rounds of negative selection yielding CD11b−/ Ter119− cell populations (Fig. 1A). Reports have suggested an existence of CD11b+/low cells in bone marrow that are capable of self-renewal and differentiation into hematopoietic lineages. Depletion of these cells often results in exclusion of important primitive repopulating cells (Pearce et al., 2004). Thus the CD11b+ cells were further subjected to a second round of MACS using a multipotent stem/progenitor cell marker, anti-CD133 (anti-Prominin-1) microbeads yielding CD11b+/ CD133+ sorted cell populations (Fig. 1A). To further establish the purity of these sorted populations, flow cytometric analysis was performed for CD11b, Ter119 and CD133 surface markers. CD11b−/Ter119− cells were negative for CD11b and Ter119 protein expression (Fig. 1B, upper panel) while, CD11b+/CD133+ cells were positive for both the surface proteins (Fig. 1B, lower panel), thus confirming the purity of these sorted cell populations. Both the sorted CD11b−/Ter119− and CD11b+/CD133+ cell populations were cultured for 48 h in their preferred medium and further evaluated for the expression of CD11b, Ter119 and CD133 using immunofluorescence cytometry. CD11b−/ Ter119− cells depicted an expression of only CD133 but lacked expression of CD11b and Ter119 whereas CD11b+/CD133+ cells expressed both CD11b and CD133 (Fig. 1C). CD11b−/Ter119− and CD11b+/ CD133+ cell populations when sub-cultured under optimal expansion conditions for 7 days depicted strikingly different morphologies. The phase-contrast microscopic images depict CD11b−/Ter119− cells with typical fibroblast morphology, whereas CD11b+/CD133+ cells have a cobble stone appearance (Fig. 1D).

3.1.1. Gene expression profile of CD11b−/Ter119− and CD11b+/CD133+ sorted cell populations We evaluated the expression of BMSC-, HSPC-, pluripotency-specific markers along with ligand and cell surface receptor gene expressions in both sorted populations relative to mouse bone marrow cells using mouse gene-specific designed primer setsi as described in Table S1 of the Supporting information. Further, we compared the gene expressions in these sorted cell populations with bona fide/established mouse bone marrow-derived BMSCs (Cyagen Biosciences Inc., USA) and mouse embryonic stem cell line, ES-E14TG2a (ATCC® CRL-1821™, USA). As shown in Fig. 2A, CD11b−/Ter119− cell populations expressed approximately 2–3 – fold higher BMSC-related genes such as Nestin, PodxL, CD73 and CD90.2 than the CD11b+/CD133+ sorted cell populations. However, CD49f and CD105 (endoglin) expression was comparable between these sorted populations. Expression of Nestin, CD90.2 and CD105 in CD11b−/Ter119− cells were comparable to mouse BMSCs (Fig. 2A). Similarly, CD11b+/CD133+ sorted cells expressed 2–8 – fold higher HSPC-specific genes such as CD117 (c-Kit), CD14 and Procr1 than CD11b−/Ter119− cell populations (Fig. 2B). The expression of BMSC-related genes in CD11b+/CD133+ cells and hematopoietic marker genes in CD11b−/Ter119− cells and mouse BMSCs were significantly lower. These gene expression profiles suggest the stromal-character of CD11b−/Ter119− and hematopoietic-character of CD11b+/CD133+ sorted cell populations. Interestingly, expression of pluripotency-specific genes such as Oct-4, Sox-2 and Nanog was observed to be 4–12 – fold higher in CD11b−/Ter119− cell populations than CD11b+/CD133+ sorted cells (Fig. 2C). The higher expression patterns of pluripotencyspecific genes in CD11b−/Ter119− cell populations depicted a similar high expression in bona fide mouse embryonic stem cell line, ESE14TG2a (Fig. 2C). However, the expression of these pluripotency-specific genes in ES-E14TG2a, except Sox-2, were several folds higher than our BMSCs. Expression of SDF-1 was approximately 3.75–fold higher in CD11b−/Ter119− cell populations than CD11b+/CD133+ sorted cells (Fig. 2D). However its cognate receptor, CXCR4 as well as other cell surface receptor expression, VEGFR2, PDGFR-β and TGFRβ -II was equally expressed in both the freshly sorted cell populations. VEGFR-1 expression was 6–fold high in CD11b+/CD133+ than CD11b−/Ter119− cell populations (Fig. 2D). A similar expression profile of these cell surface receptor genes was observed in bona fide mouse BMSCs and our sorted CD11b−/Ter119− cell populations (Fig. 2D). 3.2. Surface marker characterization Next, we performed immuno-phenotype analysis of surface marker expression in both the sorted cell populations. Flow cytometric analysis showed that both CD11b−/Ter119− and CD11b+/CD133+ cell populations were strongly positive for markers like CD29 and CD44. CD49f along with other markers such as CD106, Sca-1 and CD140a were expressed differentially by the sorted cell populations (Fig. 3A, B). Interestingly CD11b−/Ter119− cell populations were comparable with bona fide mouse BMSCs in expression of these surface markers (Fig. S1A). Our sorted CD11b−/Ter119− cell populations were very low for CD31 whereas CD11b+/CD133+ cell populations were strongly positive for CD31 (Fig. 3B) suggesting the stromal origin of the former, and hemangioblast origin, a common precursor of hematopoietic and endothelial cells, of the later sorted cell populations. 3.2.1. Cell lineage-specific differentiation of CD11b−/Ter119− and CD11b+/ CD133+ sorted cell populations The potency of these sorted populations was further characterized by subjecting to differentiation into various cell lineages. The CD11b−/Ter119− sorted cell populations were separately subjected to osteogenic, adipogenic, and chondrogenic induction medium. Differentiation of CD11b−/Ter119− sorted cell population into osteogenic cells was confirmed by staining with Von Kossa stain to evaluate the mineralization (calcium) deposits (Fig. S2A), whereas


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Fig. 1. Isolation and characterization of CD11b−/Ter119− and CD11b+/CD133+ cells (A) Flow chart of the MACS methodology adapted for sorting CD11b−/Ter119− and CD11b+/CD133+ cell population. (B) Purity check of the MACS sorted cell populations depicting CD11b−/Ter119− cells were negative for both CD11b and Ter119 expression whereas CD11b+/CD133+ cell population were positive for both CD11b and CD133. (C) Immunocytochemical analysis performed after 48 h of culture depicting the expression of these surface protein markers. (D) Phase-contrast microscopic images of different sorted populations after culturing for 7 days. Data are representative of more than six independent experiments, sorted and cultured at different timings.

adipocytes were confirmed by staining with oil-red-O stain to visualize the fat (oil) droplets formed in the cell (Fig. S2B), and chondrocytes by staining with toluidine blue for identification of cartilage matrix formation (Fig. S2C). Respective control cells did not stain for the above three differentiation lineage marker specific stains. Quantitation of these images depicted a significant increase in number of positively stained cells (Fig. S2D). These cytological data was further supported by semi-quantitative RT-PCR analysis depicting increased expression of osteoblast marker genes, Sparc (Osteonectin), Bglap2 (Osteocalcin), SPP1 (Osteopontin), Col1α1 and Cbfa1 (RunX2) in CD11b−/Ter119− sorted cells cultured in osteogenic induction medium (Fig. S2E). Adipsin, CEBP-α, Lpl and PPAR-γ expression was markedly high in CD11b−/Ter119− sorted cells cultured in adipocyte induction medium (Fig. S2F). Similarly, CD11b− /Ter119 − sorted cells cultured in chondrogenic medium depicted high expression of Col2α1, Col10α1 and Acan (Fig. 4G), chondrocyte-specific genes (Fig. S2G). Next, to identify the differentiation potential of CD11b+/CD133+ sorted stem cell populations of hematopoietic origin, we subjected these cells to colony forming cell assays for enumerating multipotent hematopoietic stem and progenitors. Colonies exhibiting burst forming unit-erythroid (BFU-E), colony forming units –erythroid (CFU-E), –macrophage (CFU-M), –granulocyte (CFU-G), –granulocyte, –macrophage (CFU-

GM) and –granulocyte, erythrocyte, macrophage, megakaryocyte (CFU-GEMM) appeared after 6–7 days of culture (Fig. S3A). Separately, CD11b +/CD133+ sorted cell populations cultured in megakaryocyte-specific differentiation medium and stained with Giemsa stain, depicted large cells with lobulated nuclei whereas control cells retained the undifferentiated hematopoietic morphology (Fig. S3B). Quantitation of images revealed a high number of positively stained cells (Fig. S3C). Also, megakaryocyte-specific gene expression (LGALS1, MUC1, Gp9) were observed to be higher as compared to CD11b +/CD133 + sorted cell populations when cultured in megakaryocyte induction medium (Fig. S3D). 18S rRNA or β-Actin expression was used as an internal control. Interestingly, both the sorted cell populations although expressed CD133, an endothelial progenitor marker; however, DiI-AcLDL uptake, a characteristic feature of endothelial cells, was negative (Fig. S4). When these cells were cultured in EGM-2, they stained positive for DiIAcLDL indicating the capability to differentiate into endothelial lineages as reported earlier (Fig. S4) (Janeczek Portalska et al., 2012). Together, these data suggests that CD11b−/Ter119− sorted cell population of bone marrow are BMSCs with tri-lineage differentiation capacity, whereas the CD11b+/CD133+ sorted population of bone marrow are HSPCs, with capability of differentiation into hematopoietic lineages.

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Fig. 2. Gene expression analysis: Expression of mRNA in these sorted stem cell population was analyzed by evaluating (A) BMSC markers, (B) Hematopoietic Stem Cell markers, (C) Pluripotency Stem Cell markers and (D) Surface receptor markers. Data depicted are the results of three independent experiments represented as mean ± sem of fold change relative to bone marrow cells as control (*p b 0.05). Bona fide mouse BMSCs and ESCs were used as positive controls.

3.3. Expansion characteristics To determine the differences in growth properties of BMSCs and HSPCs, the growth curve was analyzed in the sorted populations for a period of 10 days. HSPCs demonstrate a slower growth characteristic with a doubling time of approximately 142.57 h whereas BMSCs exhibited a relatively higher growth rate with a doubling time of approximately 93.2 h, based on the logarithmic growth curves (Fig. 4A). Next, the proliferation rate of these sorted CD11b−/Ter119− and CD11b+/ CD133+ cell populations, evaluated after 48 h using an MTT assay (Fig. S5A) and BrdU assay (Fig. S5B), depicted lower proliferative potential with the latter as compared to the former. These observations were confirmed by immunofluorescence imaging depicting higher BrdU nuclear staining in CD11b−/Ter119− cells (Fig. S5C) as compared with CD11b+/ CD133+ cells (Fig. S5D).

3.4. Differential proliferation of BMSCs and HSPCs To corroborate the expansion characteristics of isolated CD11b−/ Ter119− (BMSCs) and CD11b+/CD133+ (HSPCs), we analyzed the cell cycle status of these sorted populations. Our results exhibited a significant increase in frequency of HSPC populations in G0/G1 phase as compared to BMSCs (89.9 ± 0.6% vs 75.1 ± 3.8%; n = 6; p b 0.004). Incidentally, a significantly higher population of BMSCs were also observed to be in sub-G1 phase (11.46 ± 1.63 vs 4.40 ± 0.56%; n = 6; p b 0.002). In contrast, BMSCs showed significant increase in frequency

of cells in S-G2/M phase than HSPCs (13.9 ± 2.9 vs 5.8 ± 0.8%; n = 6; p b 0.05) (Fig. 4B). Next, we investigated the expression of cell cycle control genes in these two sorted populations relative to bone marrow cells. As shown in Fig. 6C, cyclin B1 and cyclin D2 expression were 5.78- and 2.3-fold higher in BMSCs as compared with HSPCs (cyclin B1: 2.6 ± 0.93 vs 0.45 ± 0.28; cyclin D2: 2.05 ± 0.95 vs 0.89 ± 0.35), respectively (Fig. 4C). Furthermore, BMSCs depicted a 3.07–fold higher expression of cyclin E1 than HSPCs (BMSCs: 4.67 ± 3.1 vs HSPCs: 1.52 ± 0.9) indicating accumulation at the G0/G1 phase (Fig. 4C). The low proliferative phenotype of HSPCs was further substantiated by a 4.01– and 4.28–fold increase in expression of CDK inhibitors such as p16 (HSPC: 4.25 ± 0.54 vs BMSC: 1.06 ± 0.16) and p27 (HSPC: 6.37 ± 0.75 vs BMSC: 1.49 ± 0.27), respectively (Fig. 4D). Although there was an insignificant differential expression of p21 in these two sorted populations, the increased expression of both p16 and p27 in HSPCs suggests that cells are mostly detained in the G0/G1 phase of the cell cycle. Interestingly, p53 expression was very low in both BMSCs (0.48 ± 0.05) and HSPCs (0.23 ± 0.05), indicating that cells are not undergoing apoptosis even though there were fractions of cell populations in both BMSCs and HSPCs in sub-G 1 phase of cell cycle (Fig. 4D). BMSCs in comparison with HSPCs not only depicted 3.04–fold higher expression of proliferating cell nuclear antigen (PCNA), but also 2– to 4–fold higher gene expression of E2F transcription factors and cdc20, which are involved in cell cycle progression and transition from G1 to S phase, indicating a higher capacity for proliferation of BMSCs in comparison with isolated HSPCs (Fig. 4E).


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Fig. 3. Cell-surface antigen expression analysis: (A) CD11b−/Ter119− sorted cells differentially expressed various cell surface antigens but were very low for CD31 indicating the mesenchymal origin. (B) CD11b+/CD133+ sorted cell populations were also positive for different cell surface antigens including the hematopoietic markers, CD31. Black histogram on the extreme left of each row represents negative control (FITC- or PE- labeled IgG1 isotype control). The data reported are the representative of three independent experiments each performed in duplicates.

3.4.1. HDACs are differentially expressed in BMSCs and HSPCs Different classes of HDACs modulate different cell cycle proteins. BMSCs showed a 5.3– and 7.5–fold increase in HDAC1 and HDAC2 expression as compared with HSPCs (HDAC1: 9.73 ± 5.49 vs 4.66 ± 2.13; HDAC2: 97.53 ± 40.36 vs 13.02 ± 4.18), thereby suggesting a HDAC1 and HDAC2–mediated repression of p21 expression, an inhibitor of cyclinD-CDK4/6 complexes (Fig. 5A). This correlates well with our observation of low expression of p21 and 2–fold higher expression of cyclin D2 in BMSCs, leading to maintenance of a higher proliferative state. A decreased p21 expression in our HSPC populations clearly suggests an involvement of other HDACs. Recent studies have shown that over-expression of HDAC3 in T cells leads to cell cycle arrest and increased p27 expression, an inhibitor of cyclin E-CDK2 and cyclin DCDK4 complexes (Hartwell et al., 1970). Similarly, our results depicted a 2.6–fold increase in expression of HDAC3 in HSPCs as compared with BMSCs (45.15 ± 1.16 vs 17.5 ± 1.44), suggesting an HDAC3-mediated increased expression of p27 rendering these cells into a slow proliferative phenotype (Fig. 5A). Up-regulation of HDAC8 has been reported to inhibit apoptosis and stimulate cellular proliferation (Evans et al., 1983). Our results also depicted an increased HDAC8 expression in BMSCs (25.73 ± 2.51 vs 0.84 ± 0.46), thereby correlating with the increased cell proliferation phenotype of these cells (Fig. 5A).

Further, the role of class II HDACs has been elucidated in proliferation and differentiation of stem cells (Grana and Reddy, 1995). Among the various class II type HDACs, HDAC4 and HDAC6 depicted a 4.11– and 2.65–fold higher expression in BMSCs than HSPCs (HDAC4: 48.31 ± 17.4 vs 30.96 ± 14.18; HDAC6: 6.90 ± 3.9 vs 2.55 ± 0.56) (Fig. 5B). These HDACs primarily function by repressing p21-mediated cell cycle arrest and up-regulating expression of cyclins-CDK complexes via indirect mechanisms. Similarly, Class III HDACs are also associated in a wide variety of cellular functions including cellular response to stress, cellular proliferation and longevity of cells (Michan and Sinclair, 2007). Our analysis of nuclear Sirtuins (SIRT) -SIRT1, SIRT2, SIRT3, SIRT4, SIRT6 and SIRT7 in BMSCs and HSPCs depicted a 5.5–6–fold increase in expression of SIRT6 (1.19 ± 0.69 vs 6.46 ± 3.75) and SIRT7 (0.35 ± 0.19 vs 2.12 ± 0.50) in HSPCs suggesting reduced cell proliferation (Fig. 5C). This finding is in concurrence with earlier reported over-expression of SIRT6 in HeLa cells resulting in a low population of cells in mitotic phase with reduced proliferation rate (Ardestani and Liang, 2012). Finally HDAC11 of class IV types of HDAC, whose role is poorly understood in cell cycle regulation did not depict a marked differential expression pattern in our sorted BMSC and HSPC populations (Fig. 5D).The differential expression of HDACs in these cells suggests a probable epigenetic modifications in cell proliferation and progression.

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Fig. 4. Cell cycle genes expression decides the cell growth status of BMSCs and HSPCs: (A) CD11b−/Ter119− and CD11b+/CD133+ showed differential expansion properties for a period of 10 days. HSPCs depicted a slower growth rate with a doubling period of approximately 142.57 h whereas BMSCs exhibited a relative higher growth rate with doubling period of approximately 93.2 h, based on the logarithmic growth curves. (B) Flow cytometry analysis of CD11b−/Ter119− and CD11b+/CD133+ sorted cell populations depicting a high peak of G0/G1 in the latter whereas a relatively high peak of G2/M in the former. Fractions of cell populations of CD11b−/Ter119− and CD11b+/CD133+ sorted cells in different phases of cell cycle (Inset). qRT-PCR analysis using mouse-gene specific primer sets depicted differential expression of (C) Cyclin genes, (D) cyclin-dependent kinase inhibitor (CKI) genes and (E) Cell cycle-associated genes. Data represented are from results of three independent experiments represented as fold change relative to bone marrow cells as control (*p b 0.05).

3.4.2. HDAC inhibition promotes BMSC and HSPC proliferation HDAC deregulation has been revealed in various malignancies. However, their role in adult stem cells is poorly understood. Among the various HDAC inhibitors, TSA has been reported to possess a higher affinity to class I HDACs, HDAC1, HDAC2 and HDAC3, and few Class II b HDACs including HDAC6 and HDAC10 (Bantscheff et al., 2011). TSA treatment at low nanomolar concentration depicted an increased number of colonies in both the sorted cell populations (Fig. 6A, BMSC (CD11b−/ Ter119−)-upper panel and HSPC (CD11b+/CD133+)-lower panel). BMSCs (CD11b−/Ter119−) depicted a further marked decrease in doubling period of cell populations in the presence of TSA (5 nM, 48 h) as compared with control (84 h) (Fig. 6B). Similarly, our sorted slowly proliferative HSPC (CD11b+/CD133+) population, when treated with TSA (5 nM), potentiated the cell growth with a considerable decrease in doubling time to 33.6 h as compared with control (139.2 h) (Fig. 6C). These data indicate that overall, the growth rate of both BMSCs and HSPCs are negatively modulated by HDACs. Next, a dose-dependent increase in cell proliferation was also observed in the presence of TSA (5 nM), as analyzed using the BrdU incorporation assay, further confirming the observed cell expansion and growth curve analysis (Fig. 6D). It was noted that TSA at 5 nM concentration depicted a maximum increase in cell proliferation and growth rate in both the analyses (Fig. 6B, C and D), which corroborates well with the literature (Han et al., 2013). We further evaluated the effect of TSA in these sorted cell populations using cell cycle analysis. TSA induced BMSCs (CD11b−/ Ter119−) to exit G0/G1 phase and enter into S phase (33.2 ± 0.6 vs 7.4 ± 1.1) as compared with control (Fig. 6E). Similarly, HSPCs (CD11b+/CD133+) also exited the G0/G1 phase and entered into the S

phase (18.3 ± 3.4 vs 2.8 ± 1.1) (Fig. 6F). Next, the immunostaining of these cells depicted a marked increase in Ki67 nuclear staining in TSA treated group as compared with their respective controls (Fig. 6E and F). Thus, TSA-mediated inhibition of HDACs induced the cell cycling capability of these sorted cell populations. 3.4.3. HDAC2 and 3 regulate the cyclins B1 and D2 expression by chromatin remodelling Gene expression studies of cyclins revealed a 3.13– and 4.87–fold increase in expression of cyclins B1 and D2 mRNA levels in HSPCs treated with TSA as compared to untreated cell populations. Similarly BMSCs with a higher growth rate also depicted a significant increase in cyclins B1 and D2 mRNA expression in the presence of TSA (Fig. 7A). At the protein level, TSA treated cells corroborated with mRNA expression studies depicting a higher expression of cyclin B1 and cyclin D2 in both BMSCs and HSPCs (Fig. 7B and C). There was also a marked decrease in p27 mRNA (Fig. 7A) and protein expression in both BMSCs and HSPCs treated with TSA (Fig. 7D and E) as compared with untreated control cell populations. These observations suggest a plausible mechanism of HDAC inhibition-mediated increased cyclins B1 and D2 expression positively regulates the cell proliferation and growth. TSA treatment of both BMSCs (CD11b−/Ter119−) and HSPCs (CD11b+/CD133+) depicted a large increase in acetylated histones H3 and H4 (Fig. 7F, S6A) with a concomitant decrease in HDAC (HDAC1, HDAC2 and HDAC3) expression (Fig. 7G, S6B). This further suggests that the hyper-acetylation of H3 and H4 proteins in the presence of HDAC-inhibition leads to downstream activation of cyclins B1 and D2 in both BMSCs and HSPCs. Finally, to confirm this phenomenon, ChIP analysis of cyclins B1 and D2 with


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Fig. 5. Differential expression of HDACs in CD11b−/Ter119− and CD11b+/CD133+ cell population: HDACs mRNA expression analyzed in these two sorted stem cell population depicted differential expression of (A) Class I HDACs (B) Class II HDACs (C) Class III HDACs (D) Class IV HDACs. Data depicted are the results of three independent experiments represented as mean ± sem of fold change relative to bone marrow cells as control (*p b 0.05).

anti-HDAC2 and anti-HDAC3 revealed a significant decrease in HDAC2 and 3 binding to cyclins B1 and D2 promoters in the presence of TSA. The amount of promoter precipitated with anti-HDAC2 and antiHDAC3 was significantly decreased in both the cell populations treated with TSA, thereby depicting a decrease in qRT-PCR amplification of cyclins B1 (Fig. 7H) and D2 (Fig. 7I) promoter. This observation suggests a negative regulation of HDAC2 and HDAC3 on the proliferative phenotype of these cells. 4. Discussion BMSCs and HSPCs are the most intensely studied adult stem cell types because of their great potential in the field of regenerative medicine. Isolation of murine bone marrow derived BMSCs has been performed by immuno-depletion of CD11b cells (Kopen et al., 1999). But, use of a single marker for isolation of BMSCs often leads to contamination with other cells of hematopoietic origin which lack CD11b. Moreover, reports of isolation of murine BMSCs using combinatorial markers have revealed these to be Ter119 negative (Morikawa et al., 2009). Therefore, in our experimental approach we combined double negative selection of Ter119, a marker of erythroid cells ranging from early pro-erythroblasts to mature erythrocytes, along with CD11b for enrichment of BMSCs. The CD11b−/Ter119− cell population thus obtained appeared morphologically similar to BMSCs in previously published literature (Kopen et al., 1999). CD11b+ cell fractions were earlier reported to contain short term HSPCs. We have therefore, further

enriched HSPCs using CD133 (prominin-1), a stem cell marker known to be expressed in primitive hematopoietic progenitor cells (Hess et al., 2006). Studies by Hess et al., demonstrated that CD133+/ALDHhigh/ lin− HSC population significantly engrafted in NOD/SCID mice as compared to the CD133− population. Our sorted CD11b+/CD133+ HSPC populations depicted morphological similarity with the aforementioned cell populations (Hess et al., 2006). The expression profile of mRNA and protein, examined in our sorted cell populations to gauge their biological activity, matched with expression profile of markers observed by Terskikh et al., in purified HSPCs and committed progenitors (Terskikh et al., 2003). The pluripotent marker expression on BMSCs has been studied in detail only in human BMSCs. Riekstina et al., have shown a differential expression pattern of embryonic stem cell markers including Oct-4, Nanog and SOX-2 in stromal cells derived from various sources such as bone marrow, adipose tissue, dermis and heart (Riekstina et al., 2009). Similarly, Palma et al. have also shown that enforced expression of Oct-4 in human BMSCs increases the expression of other pluripotent genes including KLF-4, SOX-2, FOXD3, NANOG and c-MYC (Palma et al., 2013). Our studies revealed a similar differential profile of pluripotency gene expression in murine bone marrow-derived BMSCs and HSPCs; although the levels are generally much lower than in ES cells. The sorted BMSC and HSPC populations were observed to exist in distinct states of the cell cycle, equally important for their functions as stem cells. The cell cycle gene expression studies further ascertained the cellular physiology of both the sorted cell populations. Our results

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Fig. 6. HDAC inhibition by TSA induces cellular proliferation: HDAC inhibition by TSA leads to increased proliferation in both the CD11b−/Ter119− and CD11b+/CD133+ cell population. (A) Image depicting increased number of colonies in cells treated with TSA (5 nM) in both the subsets of cells. Growth curve analysis showed an increased cell doubling time after TSA treatment as compared to control in (B) CD11b−/Ter119− and (C) CD11b+/CD133+ sorted cells. (D) BrdU proliferation assay illustrating a higher percent of proliferative cells in 5 nM TSA treated cells. Three independent set of experiments were performed and results are depicted as percent proliferation as compared to control (*p b 0.05). (E) CD11b−/Ter119− and (F) CD11b+/CD133+ cells were separately treated with 5 nM TSA and stained with PI and subjected to cell cycle analysis. TSA treatment increased the cells in S phase (lower panels) as compared to control (upper panels). The fraction of cells in G0/G1 and S phases of the cell cycle are represented as mean ± sem of three independent experiments. Immunofluorescence imaging of Ki67 positive cells depicting higher nuclear staining in TSA treated groups.

also coincide with studies reported by Zou et al., that both p57 and p27 cooperate with Hsp-70 to maintain HSPC quiescence (Zou et al., 2011). Similarly, Passague et al., demonstrated a sequential expression pattern of cyclins and CKIs during different stages of self-renewal and differentiation in HSPCs (Passegue et al., 2005). The literature suggested that overexpression of cyclin D2 also led to an increased proliferation in human BMSCs (Kono et al., 2013). It is pertinent to mention that although we demonstrated a high percentage of BMSCs expressing cyclins D2 and B1 in the proliferative phase, the data do not exclude the existence of a fraction of BMSCs in G0 phase. In the bone marrow stem cell niche, various molecular cues maintain the physiological states such as proliferation or differentiation of BMSCs and HSPCs. Emerging evidences suggest that stem cells undergo major epigenetic alterations during development and differentiation (Huang et al., 2015). Epigenetic regulation via histone acetylation/deacetylation is a dynamic process that is orchestrated by the interplay between histone acetyl transferases and HDACs. Studies in the past revealed an important role of HDACs in stem cell differentiation of both adult and embryonic stem cells. Small molecule HDAC inhibitors have been shown to enhance BMSC's differentiation into the adipocyte lineages by upregulating the expression of PPAR gamma (Yoo et al., 2006). Further, in human embryonic stem cells (hESC), TSA has been shown to induce cardiomyocyte differentiation by involving GATA-4 (Kawamura et al., 2005). Additionally, during neural differentiation, an inhibition of HDAC activity by TSA promoted pluripotency maintenance at the initial stage of hESC differentiation, but later inhibited differentiation during the neural commitment stage (Qiao et al., 2015). HDAC1 and HDAC2

deletion in mouse embryonic stem (mESC) cells did not affect ESC proliferation, but led to a marked decrease in ESC differentiation, i.e., embryoid body (EB) formation (Dovey et al., 2010). Further, HDAC1deficient EBs were significantly smaller, showed spontaneous rhythmic contraction, and increased expression of both cardiomyocyte and neuronal markers (Dovey et al., 2010). Although the role of HDAC inhibition on stem cell differentiation has been explored extensively, its role on stem cell proliferation is limited. Wilting et al., have revealed the overlapping roles of HDAC1 and HDAC2 in hematopoiesis and cell cycle regulation by dual inactivation of both HDAC1 and HDAC2, which led to apoptosis of megakaryocytes and thrombocytopenia (Wilting et al., 2010). Zupkowitz and colleagues demonstrated the essential requirement of HDAC1 for mouse development which is to regulate cellular proliferation and represses the CKI, p21 (Zupkovitz et al., 2010). In addition, studies also suggested that loss of HDAC1 in T cells led to increased proliferation, indicating that the role of HDACs on proliferation is cell type-dependent. These HDACs are known to regulate different sets of target genes that govern the outcome of cellular proliferation in a particular cell type. Our analysis also revealed a higher expression of both HDAC1 and HDAC2 in BMSCs as compared with HSPCs, suggesting their role in regulating the cell cycle. HDAC3, another class I HDAC, has been shown to control G1-S transition in resting T cells by repressing Skp2 transcription, leading to increased levels of p27 and consequently halting the cell cycle in the G0/G1 phase (Zhang et al., 2008). Similarly, HSPCs expressed a higher level of HDAC3 in our study, which implies its indirect role in regulating p27-mediated low proliferative state. Thus, HDACs participate in maintenance of wide physiological functions


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Fig. 7. TSA-mediated HDAC inhibition differentially regulated histone acetylation, cyclins and CKIs expression: TSA treatment induced acetylation of Histone proteins H3 and H4 and expression of cyclins at both mRNA and protein level in CD11b−/Ter119− and CD11b+/CD133+ cell population. (A) Graph representing increased mRNA expression of cyclin B1 and cyclin D2 in TSA treated cells. Three independent set of experiments were performed and results are depicted as difference in mRNA expression in TSA treated group as compared with respective untreated group (#p b 0.05). Image depicting (B) an increased protein expression of cyclin B1 and cyclin D2, (C) quantitation of band intensities in cyclins B1 and D2, (D) a decreased protein expression of p27 in TSA treated cells, and (E) densitometric analysis of the observed bands intensities from replicative experiments. Blots depicting (F) increased acetylation of histones H3 and H4, and (G) decreased protein expression of HDAC1, HDAC2 and HDAC3 in TSA treated cells. ChIP analysis of promoters (H) cyclin B1 and (I) cyclin D2 using anti-HDAC2 and anti-HDAC3 antibodies. Data representing significant differences in TSA treated group as compared with respective untreated group (#p b 0.05).

in the adult stem cells. Pharmacological inhibition of HDACs with small molecule inhibitors will provide further insight into the molecular functioning of this highly important class of enzymes. TSA, a HDAC inhibitor, has a differential affinity to bind various HDAC complexes (Bantscheff et al., 2011). In the present study, ChIP analysis revealed that HDAC2 and HDAC3 are bound with higher affinity to the Cyclin B1 promoter in BMSCs and the Cyclin D2 promoter in HSPCs, which inhibits the expression of these cyclins. However, inhibition of HDACs with TSA leads to prevention of HDACs from binding to its target gene promoter, thereby inducing its gene expression. This phenomenon was evident by a higher pull down of cyclin B1 promoter by anti-HDAC2 in control cells as compared with TSA treated cells. Similarly, an increased pull down of cyclin D2 promoter by anti-HDAC3 was observed in control cells as compared with TSA treated cells (graphical abstract). The present study thus suggests the role of specific epigenetic modulators that can induce stem cell growth and expansion. 5. Conclusion Our study demonstrates use of unique combinations of CD11b, Ter119 and CD133 markers to isolate enriched BMSCs and HSPCs from a heterogeneous bone marrow population. The sorted CD11b−/ Ter119− and CD11b+/CD133+ cell populations were successfully differentiated into skeletal and hematopoietic lineages, respectively and confirmed with BMSC and HSPC marker expression. Further, a differential proliferative potential of these BMSC and HSPC was observed due to

expression of specific HDACs which acts in concert to express cyclins and CKIs. Finally, TSA-mediated HDAC inhibition led to decreased binding of HDAC2 and 3 on the cyclins B1 and D2 promoters, thereby up-regulating the expression of these cyclins B1, D2, and subsequent lowering of the doubling time of these differentially proliferative adult stem cellsBMSCs and HSPCs. Acknowledgement AD acknowledges the funding provided by “CSIR-Mayo Clinic for Innovation and Translational Research, CKM-CMPP-07” and CSIR, Ministry of Science and Technology, Government of India, XIIth Five-year Plan Project # CSC-0111. Fellowships provided by ICMR and UGC are gratefully acknowledged by ND (ICMR-SRF), KK (UGC-JRF) and RG (UGC-SRF). We also thank the technical assistance provided by Mr. Suresh Y in assisting the Flow-cytometry analysis and confocal imaging. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.scr.2016.07.001. References Ardestani, P.M., Liang, F., 2012. Sub-cellular localization, expression and functions of Sirt6 during the cell cycle in HeLa cells. Nucleus 3, 442–451.

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