Enhanced Generation of Human Induced Pluripotent Stem Cells from Peripheral Blood and Using Their Mesoderm Differentiation Ability to Regenerate Infarcted Myocardium

Тhe most pressing issue in generating induced pluripotent stem cells (iPSCs) in clinical practice is the cell source. Compared to human dermal fibroblasts (HDFs), which have been widely used, human peripheral blood could be a more easily obtainable alternative. However, iPSCs generated from fresh peripheral blood require inconvenient specific methods including isolation. Recently, we succeeded in isolating and culturing human heart-derived circulating cells called circulating multipotent stem (CiMS) cells. Here, we investigated the generation efficiency of CiMS-derived iPSCs (CiMS-iPSCs) and tested their differentiation potential into mesodermal lineages and cardiovascular cells. We isolated and cultured CiMS cells from peripheral mononuclear cells with a high efficiency. Moreover, our method succeeded in reprogramming the CiMS cells and generating iPSCs with higher efficiency compared to when HDFs were used. Compared to HDF-iPSCs or human embryonic stem cells (hESCs), CiMS-iPSCs showed high differentiation potential into mesodermal lineage cells and subsequently into endothelial cells, vascular smooth muscle cells, and cardiomyocytes. Further, we checked the epigenetic status of each cell type. While methylation of the CpG site of the brachyury T promoter did not differ between cell types, the histone H3 lysine 4 trimethylation level in the brachyury T promoter region was enhanced in CiMS-iPSCs, compared to that in other cell types. In contrast, histone H3 lysine 9 acetylation was downregulated during the differentiation process of the CiMS-iPSCs. In the myocardial infarction model, the CiMS-iPSCs group showed more therapeutic potential in regenerating the myocardium than other cell types. Our study showed a new method to isolate human heart-derived stem cells from human peripheral blood and to generate iPSCs efficiently. Due to epigenetic memory, these CiMS-iPSCs easily differentiated into cardiovascular lineage cells, resulting in improved efficiency in vivo. These results suggest that our new method using CiMS cells has therapeutic potential in regenerative medicine using cell therapy.


Introduction
In 2006, Takahashi and Yamanaka [1] found that ectopic expression of Oct3/4, Sox2, Klf4, and c-Myc using viral (retroviral) gene transfer can transform murine somatic cells into induced pluripotent stem cells (iPSCs). One year later, human iPSCs from fibroblasts were independently generated by two research groups [2,3]. Since iPSCs have similar plu-ripotent potential to embryonic stem (ES) cells, they can differentiate into almost every somatic cell type. Furthermore, they can be produced from autologous sources without ethical concerns and the problem of immune rejection, unlike that of ES cells. Therefore, they have been considered ideal for patient-and disease-specific regenerative therapy.
In humans, fibroblasts are typically used for reprogramming. However, because of the invasive preparation method and the need for a long culture period for application in reprogramming [2], several new candidates have been proposed. Since then, human terminally differentiated circulating T cells, which are highly accessible and can be obtained noninvasively, have been isolated and used for reprogramming [3]. However, these cells showed low virus transduction efficiency and T cell receptor gene rearrangement patterns in the original patient T cell clone.
Recently, we succeeded in culturing a new type of adult stem cells isolated from human peripheral blood samples for reprogramming of donor cells. These are multipotent stem cells derived from the heart endocardium called circulating multipotent stem (CiMS) cells [4]. CiMS cells are advantageous for iPSC production, because they divide actively enough to store extra cells and have high transduction efficiency. In addition, CiMS can be obtained from only 10 ml of blood, which can be obtained in the outpatient department.
Selecting donor cells for reprogramming is also important, because some epigenetic characteristics of the original cell can remain and form the different characteristics of multiple iPSCs [5,6]. Recent studies described that some epigenetic status of original cell can be remained and make differentiation potentials distinguishable among iPSCs [6,7]. CiMS cells are multipotent stem cells derived from the heart endocardium. Moreover, we observed high expression of GATA4, an early cardiomyocyte (CMC) differentiation marker, and SOX17, which is involved in heart development, in CiMS cells [8]. Thus, we presumed that CiMS-derived iPSCs (CiMS-iPSCs) could show higher potency in differentiating into cardiovascular cells.
Loosely compacted chromatin is epigenetically more accessible. Typically, DNA methylation and histone modification can alter chromatin compaction level and genetic activity. DNA methylation at CpG islands makes chromatin more compact [9][10][11]. In general, histone H3 lysine 4 trimethylation (H3K4me3) is observed at gene promoters and is considered a genetically active signal [12]. In contrast, H3K27me3 and H3K9ac act as genetically repressive signals [13]. We presumed that CiMS could remain in an euchromatic state in the mesodermal gene [14,15]. In this study, we examined the generation efficiency of CiMS-iPSCs and tested their differentiation potential into cardiovascular cells, compared to other cell types. and Culture of CiMS. This study was approved by the Insti-tutional Review Board of the Seoul National University Hospital (IRB Number). Human peripheral blood samples (10 cc) were obtained from donors after informed consent. PBMCs were isolated from the blood samples using Ficoll-Paque PLUS (GE Healthcare, NJ, USA) according to the manufacturer's recommendations. Freshly isolated PBMCs were suspended in EGM-2MV BulletKit™ (Lonza, Basel, Switzerland) and seeded on the fibronectin-coated (Sigma-Aldrich, MO, USA) six-well plate at 6 × 10 6 cells per well. The media were changed every single day for up to 2 weeks after plating. Adherent CiMS cells were observed from as early as five days after the start of culture and gradually formed colonies. CiMS were passaged using 0.05% Trypsin-EDTA solution.

Infection of Yamanaka's Reprogramming Factor
Retrovirus and Generation of Induced Pluripotent Stem Cells. Human embryonic kidney (HEK) 293T cells were plated and transfected with retroviral vectors containing human OCT3/ 4, SOX2, KLF4, c-Myc gene, and packaging vectors with PEI solution (Sigma-Aldrich). Forty-eight hours after transfection, the retrovirus-containing supernatant was harvested and concentrated using ultracentrifugation with 25,000 rpm for 1 hour 30 min in 4°C. CiMS cells were seeded at 2 × 10 5 cells per well in a 6-well plate before transduction. Concentrated retroviruses encoding the four reprogramming factors were added to CiMS cells with the 10 μg/ml Polybrene (Sigma-Aldrich). Twenty-four hours after transduction, the transduction medium was changed with new fresh EGM-2MV medium. Six days after transduction, transduced CiMS cells were harvested by trypsinization and replated at 2 × 10 5 cells onto new mitomycin C-(MMC-) treated STO feeder layers in a 6-well plate. Two days later, the medium was changed with human ES cell medium supplemented with 10 ng/ml bFGF (R&D systems, MN, USA) and the medium was replaced every single day. Fourteen days after transduction, iPS cell colonies were mechanically picked and transferred into new MMC-treated STO feeder layers.

Real-Time PCR.
Real-time PCR was performed using FastStart SYBR Green (Roche, Mannheim, Germany) and analyzed with Applied Biosystems 7500 Real-Time PCR System (Applied Biosystems). Gene expression level was normalized to the level of 18s rRNA and quantified using the 2 (-ΔCt) method. PCR primers are listed in Supplementary  Table S1 After washing away the PFA or methanol with 0.05% TBS-T three times, blocking/permeabilization process was performed with 1%BSA solution including 0.05% Triton X-100. Cells were incubated with primary antibodies overnight at 4°C. Goat anti-PECAM-1 (M-20) (Santa Cruz Biotechnology), mouse anti-SMA (1A4) (Abcam), and monoclonal anti-α sarcomeric actin (Sigma-Aldrich) antibodies were used as primary antibodies. Alexa Flour 488 donkey anti-mouse IgG, Alexa Flour 555 donkey anti-goat IgG, and Alexa Flour 555 donkey anti-mouse IgM (Invitrogen) were used as secondary antibody. Nuclei were stained with DAPI (4 ′ ,6-diamidino-2-phenylindole). Samples were observed using a confocal microscope (Carl Zeiss).
2.11. Chromatin Immunoprecipitation Assay. Chromatin immunoprecipitation (ChIP) assay was performed using chromatin immunoprecipitation (ChIP) assay kit (Merck) and followed manufacturer's recommendation. 1 × 10 6 cells were used for each immunoprecipitation reaction. Histone H3 tri methyl K4, Histone H3 acetyl K9, and Histone H3 tri methyl K27 antibodies from Abcam were used. For negative control of ChIP reaction, Rabbit IgG (Invitrogen) was used also included to allow for normalization. Primer information is listed in Table S1.

In Vivo Experiments.
For heart injection, the stem cells of each group were differentiated to mesoderm progenitor cells. The cells of each group were treated with 10 μM CHIR 99021 in RPMI1640 containing 1% B27 without insulin. After 24 hours, the medium containing CHIR99021 was changed to RPMI-B27 without insulin and incubated for additional 24 hours. Differentiated cells at this early mesodermal progenitor stage were harvested and prepared for injection into a mouse myocardial infarction (MI) model. For MI model, male nude mice (6-8 weeks old) were anesthetized by inhalation of 1.5% isoflurane. 5 μl PBS containing a half million cells was injected into the myocardium of the mice with or without surgical manipulation such as 3 Stem Cells International the ligation of the coronary artery. The measurement of heart function with echocardiography was performed at baseline and after two weeks after myocardial infarction. Mouse heart tissues were harvested and fixed in 4% PFA overnight in room temperature. The tissues were embedded in paraffin and cut into 3 μm thick sections. After deparaffination, all slides were boiled in retrieval solution (Dako). Goat anti-PECAM-1 (M-20) (Santa Cruz Biotechnology), mouse anti-SMA (1A4) (Abcam), and monoclonal anti-α sarcomeric actin (Sigma-Aldrich) antibodies were used as primary antibodies. Alexa Flour 488 donkey anti-mouse IgG, Alexa Flour 555 donkey anti-goat IgG, and Alexa Flour 555 donkey anti-mouse IgM (Invitrogen) were used as secondary antibodies. In each experiment, the controls were a medium-(PBS) injected group. The percentages of positive, a-SA-positive, SMA-positive, and CD31-positive cells versus total nucleated cells were quantified in 5 different sectors per tissue section in the peri-infarct zone at day 28 after the injection of cells.
2.13. Statistical Analysis. All data were calculated as mean ± SD. Group comparisons were performed by one-way ANOVA using GraphPad Prism 5 (GraphPad Software, San Diego, CA, USA), and the number of asterisks on top of each graphs means statistical significance; " * ," " * * ," and " * * * " mean that the p value range is 0.01 to 0.05, 0.001 to 0.01, and 0.001 to 0.01, respectively.

High Reprogramming Efficiency of CiMS Cells from
Human Peripheral Blood. We isolated peripheral blood mononuclear cells and obtained CiMS cells using EGM-2MV with 95.6% efficiency within 2 weeks (Figure 1(a)). Then, 2 × 10 5 CiMS cells were transduced with retroviral virus containing the genes described by Yamanaka (Oct3/4, Sox2, KLF4, and c-Myc) for 18 h. Five days after transduction, morphologically transformed cells started forming colonies ( Figure 1(b)). Eight days after transduction, these transformed colonies were mechanically picked and passaged on the STO feeder layer (Figures 1(a) and 1(b)). CiMS-iPSCs were positive for ALP staining and expressed pluripotency markers such as Oct3/4, Nanog, and TRA-1-81 (Figure 1(c)). When we tested transduction efficiency with GFP retrovirus using 9:4 × 10 4 TU virus, the transduction efficiency of CiMS cells was 93.75%, more than double that of human dermal fibroblasts (HDFs, 49.44%). The transduction efficiency was even higher than that of 293T cells (76.44%) (Figure 1(d)). We compared the efficiency of ALP-positive colony formation on a feeder layer after introducing a reprogramming factor into CiMS cells and HDFs. CiMS cells displayed approximately 1.47 times higher ALPpositive colony formation efficiency than HDF cells (Figure 1(e)). These results indicate that CiMS cells have higher potential for reprogramming into pluripotent stem cells than HDFs [16].
Reverse transcription PCR data showed the expression of pluripotent ES cell markers in CiMS-iPSCs. Various markers of human pluripotent ES cells (hESCs) were detected in all CiMS-iPSC clones at levels similar to those of hESCs. However, they did not appear in the parental CiMS cells (Figure 1(f)). To compare the global transcript profiles, cDNA of CiMS cells, hESCs, and CiMS-iPSCs were examined using DNA microarrays. Gene patterns upregulated in hESCs and CiMS-iPSCs appeared similar, but not with exactly the same pattern, and gene expression patterns in CiMS cells were very different from the previous two patterns. A total of 764,885 genes were analyzed by microarray in three different cell lines. Expression differences between CiMS cells and CiMS-iPSCs and between CiMS cells and hESCs showed large gaps, with 3,665 genes and 4,025 genes showing more than 2-fold difference in expression, respectively. In contrast, very few differences in expression appeared between CiMS-iPSCs and hESCs, with only 427 genes showing >2-fold difference (Figure 1(g)).

High Potential for CiMS-iPSC Differentiation into
Cardiovascular Lineage Cells. There have been reports that the differentiation efficiency of iPSCs depends on their origin [6]. Since CiMS cells are cells found in the endocardium of the heart, we predicted that CiMS-iPSCs would be superior to iPSCs of other origins in differentiation into cardiovascular lineage cells. In order to observe the differentiation efficiency according to the epigenetic difference, iPSCs prepared from HDFs and CiMS cells and hESCs were compared. We treated iPSCs and hESCs with CHIR99021, a GSK3β inhibitor for mesodermal     Stem Cells International Oct3    11 Stem Cells International differentiation [18], and estimated the expression of mesodermal genes by real-time PCR. The pluripotency markers OCT3/4 and NANOG were downregulated in all iPSCs and hESCs upon initiation of differentiation (Figure 2(a)). As expected, CiMS-iPSCs showed increased expression of early mesoderm markers, compared to HDF-iPSCs or hESCs (Figure 2(a)). No such trend was observed in the differentiation of other germ layers, such as the endoderm or ectoderm (Figure 2(a)).
Next, we observed epigenetic differences in the differentiation into cardiovascular lineage cells, such as CMCs, endothelial cells (ECs), and vascular smooth muscle cells   14 Stem Cells International (VSMCs). When we differentiated each group cell into CMCs, ECs, and VSMCs, it was observed that each marker was strongly increased in CiMS-iPSCs, compared to HDF-iPSCs or hESCs (Figure 2(b)). Comparably, immunofluorescent staining for endothelial lineage markers, such as CD31; VSMC lineage markers, such as SMA; and CMC markers, such as alpha sarcomeric actin, showed that CiMS-iPSCs had higher potential to differentiate into those cardiovascular lineage cells than other cell types (Figures 2(c) and 2(d)).

High Mesodermal Differentiation Potential due to
Different Epigenetic Status. Epigenetic differences are mainly induced by methylation of gene promoters and modifications of histone proteins that cause changes in chromatin structure. To determine the cause of the tendency of CiMS-iPSCs to differentiate into cardiovascular cells, methylation of the CpG site of the brachyury T promoter, a mesoderm marker, was observed. When two CpG islands of the brachyury T promoter were examined using bisulfite PCR, there was little difference in CpG methylation between cells (Figures 3(a) and 3(b)). Therefore, we found that CpG methylation was not the cause of the differentiation tendency and observed histone modification of brachyury T through a chromatin immunoprecipitation (ChIP) assay. During mesoderm differentiation, H3K4me3 in the brachyury T promoter region was clearly increased in CiMS-iPSCs, and a tendency to decrease in H3K9ac levels in the brachyury T promoter region was observed. There was little difference in H3K27me3 in the brachyury T promoter region (Figure 4(a)). However, when iPSCs were differentiated into the endoderm (GSC) and ectoderm lineage (Sox1), there were no significant differences in histone modification levels between each cell group (Figures 4(b) and 4(c)). A reverse experiment was conducted using the H3K4 transferase (KMT) inhibitor, MM102, and the H3K9 KMT inhibitor, chaetocin, in order to identify the role of histone modification more accurately. At the mesoderm differentiation stage, treatment with 50 μM MM102 strongly suppressed H3K4me3 in the brachyury T promoter region of CiMS-iPSCs (Figure 4(d)) and reduced the expression of the brachyury T gene in CiMS-iPSCs (Figure 4(f)). In HDF-iPSCs, MM102 treatment had a slight inhibitory effect on H3K4me3 in the brachyury T promoter region, leading to a slight decrease in brachyury T gene expression (Figures 4(d) and 4(f)). In hESCs, H3K4me3 was suppressed, but brachyury T gene expression was not affected (Figures 4(d) and 4(f)). In contrast, treatment with 100 nM chaetocin slightly increased H3K9ac of the brachyury T promoter region (statistically not significant) (Figure 4(e)  15 Stem Cells International ( Figure 4(g)). In HDF-iPSCs and hESCs, chaetocin treatment had no effect on H3K9ac in the brachyury T promoter region, but the expression of brachyury T was reduced (Figures 4(e) and 4(g)). We performed additional experiments measuring the expression of cardiovascular lineage markers (Nkx2.5 and GATA4) using H3K4 KMT inhibitor or H3K9 KMT inhibitor (Supplemental Figure S1). Both inhibitors suppressed the expression of cardiovascular lineage markers.

Regenerative Potential of CiMS-iPSCs in Myocardial
Infarction Model. To evaluate the in vivo therapeutic potential of CiMS-iPSCs, we employed a myocardial infarction model in nude mice. The group injected with CiMSderived cells showed significant improvement in infarction area reduction and in heart systolic function (mean ± SEM, HDF-iPSC vs. CiMS-iPSC vs. hESC, 21:57 ± 2:1% vs. 27:90 ± 1:56% vs. 18:21 ± 1:39%, p value < 0.001; n = 10 per group) (Figures 5(a) and 5(b)). We also found that CiMS-  Figure S2). We found that CiMS-iPSCs could differentiate into cardiomyocytes more efficiently than the other cells. These results suggest that the tendency of differentiation into cardiovascular lineage cells could contribute to therapeutic potential in regenerating damaged myocardium.

Discussion
In this study, we presented CiMS cells, adult stem cells from the heart endocardium, a new candidate cell type for establishing iPSCs. CiMS cells can be easily obtained with a small volume of peripheral blood (10 ml) ( Figure 6). This approach can prevent using invasive preparation methods for obtaining samples and long culture maintenance periods, which iPSC preparation from fibroblasts requires. CiMS cells showed a twofold higher transduction efficiency compared to fibroblasts (Figure 1(d)). These advantages make CiMS cells an ideal cell line for establishing iPSCs. After 14 days of culture, we introduced Yamanaka factors into CiMS cells and identified ES-like colonies without feeder cells (Figure 1(b)). These CiMS-iPSCs showed the essential features of iPSCs, including expression of pluripotent genes (Figures 1(f) and 1(g)), epigenetic status similar to hESCs (Figure 1(h)), in vitro differentiation ability into three germ layers (Figure 1(j)), and teratoma formation (Figure 1(k)).
This tendency of CiMS-iPSCs to differentiate into cardiovascular lineage cells is presumed to be due to the epigenetic memory of CiMS cells. There have been reports that epigenetic features of the original cell can remain after reprogramming, which can affect their differentiation potential, even when iPSCs acquire the molecular and functional characteristics of hESCs [6,7]. Recently, we confirmed that CiMS cells highly expressed the early CMC markers GATA4 and SOX17, which are involved in cardiac development [8]. Based on previous reports and our observations, we hypothesized that CiMS-iPSCs might be more potent in differentiating into cardiovascular cells than HDF-iPSCs and hESCs, as they present a more accessible epigenetic state than other iPSCs [8]. Genomic DNA methylation levels at the CpG site of brachyury T did not differ significantly among the three groups ( Figure 3). However, we observed that the H3K4me3 level of the brachyury T promoter region in CiMS-iPSCs was significantly increased, and the H3K9ac and H3K27me3 levels of the brachyury T promoter region were decreased significantly during mesodermal differentiation (Figure 4(a)), whereas differentiation into ectoderm and endoderm showed no significant difference between  Figure 6: Production of iPSCs using CiMS cells and its application in cardiovascular cell differentiation. CiMS cells separated from the endocardium of the heart can be easily isolated from the blood and have high transduction efficiency; therefore, reprogramming is efficient. These CiMS cells are specialized for differentiation into cardiovascular cells even when they become iPSCs because of the epigenetic memory of the endocardium. Therefore, the generation of CiMS-iPSCs shows great advantages in cardiovascular diseaserelated modeling and is an appropriate cell line for stem cell therapy in regenerative medicine.

Stem Cells International
PSCs (Figures 4(b) and 4(c)). Treatment with MM102, a H3K4 KMT inhibitor, strongly reversed H3K4me3 in the brachyury T promoter region of CiMS-iPSCs (Figure 4(d)) and reduced the expression of the brachyury T gene in CiMS-iPSCs (Figure 4(f)). In general, H3K4me3 is observed at gene promoters and functions as a genetically active signal [12]. In contrast, H3K27me3 and H3K9ac act as genetically repressive signals [13]. Therefore, the prediction that CiMS-iPSC could specialize in the differentiation of cardiovascular cells is appropriate when analyzing the results of histone methylation in the promoter region of brachyury T.
In conclusion, our study showed that the superior ability of CiMS-iPSCs to differentiate into cardiovascular lineage cells is due to their different epigenetics and histone modification status and not their DNA methylation status. CiMS cells are a feasible option for efficient iPSC generation. Moreover, our results suggest that their superior mesodermal differentiation ability could facilitate regenerative potential in various heart diseases, such as myocardial infarction.

Data Availability
The data used to support the findings of this study are available from the corresponding author upon request.

Ethical Approval
All human samples were obtained after the approval by the Institutional Review Board (IRB) of Seoul National University Hospital (Approval No. H-0908-036-290). All animal experiments were performed after receiving approval from the Institutional Animal Care and Use Committee (IACUC) of Clinical Research Institute in Seoul National University Hospital.

Consent
All human samples were obtained with written informed consent.