Optimizing the method for generation of integration-free induced pluripotent stem cells from human peripheral blood

Background Generation of induced pluripotent stem cells (iPSCs) from human peripheral blood provides a convenient and low-invasive way to obtain patient-specific iPSCs. The episomal vector is one of the best approaches for reprogramming somatic cells to pluripotent status because of its simplicity and affordability. However, the efficiency of episomal vector reprogramming of adult peripheral blood cells is relatively low compared with cord blood and bone marrow cells. Methods In the present study, integration-free human iPSCs derived from peripheral blood were established via episomal technology. We optimized mononuclear cell isolation and cultivation, episomal vector promoters, and a combination of transcriptional factors to improve reprogramming efficiency. Results Here, we improved the generation efficiency of integration-free iPSCs from human peripheral blood mononuclear cells by optimizing the method of isolating mononuclear cells from peripheral blood, by modifying the integration of culture medium, and by adjusting the duration of culture time and the combination of different episomal vectors. Conclusions With this optimized protocol, a valuable asset for banking patient-specific iPSCs has been established. Electronic supplementary material The online version of this article (10.1186/s13287-018-0908-z) contains supplementary material, which is available to authorized users.


Background
Induced pluripotent stem cells (iPSCs) are a type of pluripotent stem cell resembling embryonic stem cells (ESCs) that can be directly generated from somatic cells by transcription factors [1]. The first common source for human iPSC derivation was skin dermal fibroblasts [2]. Since that discovery, a variety of somatic tissue cells have been reprogrammed to pluripotency [3][4][5]. Mononuclear cells (MNCs) from peripheral blood (PB) have been widely accepted as a more convenient and almost unlimited source of cells for reprogramming [6][7][8][9][10].
The original method using retroviral or lentiviral vectors expressing Oct4 (officially known as Pou5f1), Sox2, Klf4, and c-Myc (known as Myc) has high reprogramming efficiency, but neither type of viral vector (retroviral and lentiviral) is ideal for clinical application. Viral vectors carry a risk for insertion mutation, which can result in tumorigenicity and genomic instability of iPSCs [11]. To make iPSC-based therapies safer, great efforts have been exerted to establish the cells without integration of an exogenous sequence into the cellular genomes. These techniques include using recombinant proteins or mRNA as an alternative to exogenous DNA [12,13], Sendai virus methods [14], and episomal methods [15,16]. Although episomal vectors are the most practical and efficient of these options, the reprogramming efficiency of this strategy needs to be improved [17,18].
In this study, we optimized isolation of MNCs, modified the supplementation of culture medium, and adjusted the duration of culture time and the combination of different episomal vectors to improve the reprogramming technology of using human PB cells as donor cells. These optimizations have important implications for the clinical applications of iPSCs.

Cell culture
Primary murine embryonic fibroblasts (MEFs) were obtained from 13.5-day CD-1 IGS mouse embryos and cultured in standard DMEM containing 10% FBS (Hyclone, Logan, UT, USA) and 2 mM L-glutamine. The MEF cells (passage 3) were irradiated at 60 Gy and then plated on gelatinized plates. Irradiated MEFs (2 × 10 5 cells) were coated onto six-well plates to support the culture for iPSC generation.
iPSCs were usually maintained in a feeder-free culture system. Briefly, we precoated the well plates with Matrigel (BD Biosciences), and then seeded the iPSCs and cultured them with E8 medium. When iPSCs reached 30-60% confluence, they could be passaged routinely with EDTA (0.5 M/L).

Isolation and preparation of MNCs from peripheral blood
All human whole blood samples were obtained from volunteers at the Institute of Hematology and Blood Diseases Hospital. Each PB sample was divided equally into four parts. MNCs were isolated from part 1 using standard Ficoll procedures by loading 35 ml of diluted blood (blood:PBS = 1:2) onto a 15-ml layer of Ficoll-Paque PREMIUM (p = 1.077 g/ml; Sigma Aldrich) in a 50-ml conical tube. MNCs were isolated from part 2 using red cell lysis buffer procedures, with the addition of 4-fold of ACK buffer to the blood and centrifugation after 20 min of incubation in a 37°C water bath. Hydroxyethyl starch (HES; Sigma Aldrich) of 60-kDa molecular mass was added to PB from parts 3 and 4 in a 1:5 ratio. The supernatants collected after the sample remained stationary for 40 min at room temperature and were processed to yield MNCs either by the Ficoll method for part 3 or by the ACK method for part 4. In total, we used two isolating methods for enrichment of PB MNCs, including Ficoll and HES-Ficoll, and two isolating methods without enrichment, including ACK and HES-ACK. Except for MNCs, the other types of white blood cells would die quickly after being cultured in vitro, so we designated the isolated cells PB MNCs.

Picking iPSC colonies
When the colonies became visible to the naked eye, we started to pick them manually. We gently scratched the colonies using a 100-μl tip and transferred one colony to one well of the 24-well plates precoated with Matrigel and E8 medium. We typically picked 10-20 colonies for each donor.

Teratoma formation assay and histological analysis
Human iPSCs were suspended at 1 × 10 8 cells/ml in PBS, and 100 μl of the cell suspension (1 × 10 7 cells) was injected subcutaneously into the dorsal flank of SCID mice (five mice per cell line). One month after the injection, tumors were surgically dissected from the mice. Teratomas were weighted, fixed in PBS containing 4% formaldehyde, and embedded in paraffin. Sections were stained with hematoxylin and eosin.
Karyotyping and G-banding G-banding chromosome analysis of the iPSC line was performed following the protocol published by Li et al. [19]. Data were interpreted by a certified cytogenetic technologist.

Propidium iodide staining of live/dead cells
We used a flow cytometry assay to determine the ratio of live/dead cells. Briefly, harvested cells were washed with PBS, and then the cell pellet was suspended in PBS with 1 μg/ml propidium iodide and samples maintained in that solution at 4°C protected from light before analysis on a flow cytometer.

Statistical analysis
Data are presented as mean ± SEM. Two-tailed Student t tests were performed, and P < 0.05 was considered statistically significant.

Results
Isolating MNCs from human peripheral blood by different methods PB MNCs are ideal for reprogramming iPSCs and have the potential to expedite advances in iPSC-based therapies [20]. To improve the generation efficiency of integration-free iPSCs from human PB MNCs, we optimized the method of generating them from human PB (Fig. 1a). In the first step, we used two isolating methods for enrichment of PB MNCs, Ficoll and HES-Ficoll, and two isolating methods without enrichment, ACK and HES-ACK. The total number of cells isolated from 1 ml of PB with the four methods changed significantly. Yields with ACK and HES-ACK were significantly greater than with Ficoll or HES-Ficoll (Fig. 1b, Additional file 1: Figure S1A). In theory, the number of MNCs per milliliter of blood was almost the same in different groups from the same donor. Nonetheless, after 8 days of in-vitro culture, the number of live cells in the HES-Ficoll group was significantly greater than in the other groups (Fig. 1c, Additional file 1: Figure S1A). This result suggested that the HES-Ficoll group that yielded relatively purified MNCs initially was beneficial to cell culture.
We then generated the iPSCs from these cultured MNCs with a combination of reprogramming factors consisting of OCT4, SOX2, KLF4, and C-MYC. The ESC-like and TRA-1-60-positive colonies began to emerge at 7 days after nucleofection. Compared with the other three groups, the HES-Ficoll group generated significantly more TRA-1-60-positive colonies in every 1 × 10 6 live cells or every 1 ml of PB than the other three groups (Fig. 1d, e). This finding reveals that the different MNC isolation methods can affect iPSC generation.
The effect of culture medium and culture time on the generation of integration-free iPSCs CD34 + cells in PB MNCs would be expended and differentiated after culture in vitro. Different culture conditions can affect the efficiency of generating integration-free iPSCs from human PB [21]. ECM was used to expand and culture the PB MNCs and improve their reprogramming efficiency (Fig. 2a). Compared with ECM medium alone, adding StemRegenin1 (SR1, the inhibitor of the aryl hydrocarbon receptor) or G-CSF to the ECM medium did not improve the reprogramming (Fig. 2b). This result indicated that the nucleated erythrocyte cells may be reprogrammable cells with high efficiency, except for CD34 + cells.
To confirm our hypothesis, we selected PB cells from patients with polycythemia vera (PRV) disease, which is an uncommon neoplasm in which the bone marrow makes The results showed that the reprogramming efficiency of MNCs from a PRV patient's PB was significantly increased (Fig. 2c). After nucleofection, PB MNCs were cultured under normoxia or hypoxic conditions, respectively. Three weeks later, the number of TRA-1-60-positive colonies formed under hypoxic conditions (3%) was four to six times higher than that in normoxic conditions (Fig. 2d).
To identify the effect of age on reprogramming, we selected healthy volunteers of different ages (10-20 years, five donors; 20-30 years, five donors; 30-40 years, four donors; 40-50 years, three donors; 50-60 years, three donors). The reprogramming efficiency was not affected by the age of the donors (Fig. 2e). In addition to change the culture conditions, separated PB MNCs were expanded in vitro for 8-10 days, as reported previously [17]. With the longer culture time, the total number of living cells gradually decreased (Fig. 2f, Additional file 1: Figure S1B), while the percentage of living cells remained unchanged (Fig. 2g, Additional file 1: Figure S1B). This result indicated that there was a certain rate of cells dead every day. To confirm the optimal culture time, we transfected the cells that were cultured respectively at days 4, 6, 8, and 10. At 3 weeks after nucleofection, the number of TRA-1-60-positive colonies was greatest at day 6 ( Fig. 2h), and this result was confirmed by the AP staining method (Fig. 2i).

The effect of vectors on the generation of iPSCs from peripheral blood
We have noted that the reprogramming efficiency varied with different combinations of episomal vectors (pEB (C5 + Tg), pEV (OS+MK)), which may be associated with different promoters of these vectors [22]. MNCs from human PB were transfected with the pEV episomal vectors CAG, EF1, or SFFV, which had different promoters. At 48 h, the expression of pluripotent genes (Fig. 3a) did not differ among the different promoters. Three weeks later, the number of TRA-1-60-positive colonies was assessed, and the SFFV promoter looked propitious for the reprogramming of the human PB cells (Fig. 3b). We then compared the combination of transcription vectors and found that the different combinations of episomal vectors and transcription factors had different effects on the formation of iPSC colonies. OSMK and BCL-XL represented the best or the more efficient combination [18,22] (Fig. 3c, d).

Characterization of iPSC colonies generated from the human peripheral blood cells
We have established that iPSCs generated from PB MNCs using the optimized methods (Table 1) are indistinguishable in their behavior in culture and colony morphology from those of ESCs (Fig. 4a). Three iPSC lines were picked from the PB-iPSCs, and the expression of the pluripotency genes Oct4 and Sox2 in these three iPSCs were coincident with the H1 ESCs by real-time PCR (Fig. 4b). By immunostaining a b c d Data presented as mean ± SEM (n = 6) *P < 0.05, **P < 0.01, ***P < 0.001 assay, we found that clones of iPSCs established from human PB retained typical characteristics of pluripotent stem cells such as the expression of embryonic stem cell markers (e.g., Oct4, NANOG, TRA-1-60, and SSEA4) (Fig. 4c). PB-iPSCs could form teratomas and differentiate into the three embryonic germ layers in immunodeficient mice (Fig. 4d). Cytogenetic analysis of all PB iPSC colonies showed a normal karyotype (Fig. 4e). All of these data demonstrated the pluripotency of these iPSCs. Ultimately, according to previous reports [23,24], we passaged the iPSCs beyond 10 passages, and PCR-based detection of the vector sequence (EBNA1 and OSW) was not found in the expanded iPSCs after 10 passages (Fig. 4f). When we established iPSC lines, we also observed a certain proportion of clones undergoing differentiation (Additional file 2: Figure S2) and death in the same well derived from the same PB sample, which may indicate that there are differences between the different clones obtained from the same PB sample using the same method of reprogramming and cultivation.

Discussion
In the present study, we optimized the episomal method to generate integration-free iPSCs from PB MNCs to iPSCs. First, we found that much purer MNCs can be obtained from 1 ml of PB using the HES-Ficoll method compared to the other three options. After 6 days of in vitro culture, the most iPSC clones were acquired after transfection. ACK lysis buffer was used for lysis of the red blood cells. During this process, the polymorphonuclear cells were left in the ACK and HES-ACK procedures, which are not useful for MNC culture. On the other hand, Ficoll could not completely separate MNCs from red blood cells, while with the combination of HES and Ficoll most of the red blood cells could be iPSC induced pluripotent stem cell, JMML juvenile myelomonocytic leukemia, PB peripheral blood, PRV polycythemia vera a iPSC lines listed were identified by ESC characterization. We did not include iPSC lines without identification in the analysis precipitated and removed. MNCs could then be separated from the remaining cells with the least damage to themselves. CD34 + cells respond well to the cytokine cocktail and are reprogrammable with high efficiency [6,[25][26][27]. In our study, we found that the erythroid culture medium improved reprogramming efficiencies, favoring the expansion of erythroblasts instead of lymphocytes [17]. Therefore, adding granulocyte growth factors such as SR1 or G-CSF to ECM did not change the efficiencies, indicating that erythroblasts are the most important donor cell source except for CD34 + cells and can be reprogrammed with high efficiency.
MNCs from PRV patient PB cells had a high induction efficiency in forming iPSCs (Fig. 2c). The possible reason for this is that the erythroblasts are in specific epigenetic states that are more easily reprogrammed [23]. The reported PBMC reprogramming experiment recommends that PB MNCs are expanded over the course of 8-14 days in the culture medium [17]. We generated iPSCs from PB MNCs that had been cultured for different time periods and confirmed that the optimal culture time is on day 6, based on comparing the number of TRA-1-60-positive and AP-positive colonies formed.
The virus encapsulated with SFFV as a vector can transfect human hematopoietic cells more efficiently and d e f c a b Fig. 4 Characterization of integration-free iPSCs from PB MNCs. a Representative TRA-1-60 staining photograph of integration-free iPSC colony from PB MNCs. b Expression level of pluripotency genes of iPSCs compared with H1 by real-time PCR. c PB iPSCs expressed pluripotency markers OCT4, NANOG, TRA-1-60, and SSEA4. Representative images captured using Leica confocal microscope. d PB iPSCs formed teratoma in immunodeficient mice. H&E staining of representative teratoma from PB iPSCs with derivatives of three embryonic germ layers: cartilage (mesoderm), glands (endoderm), and neurotubules (ectoderm). e Representative karyotype of iPSC clone. All analyzed PB iPSC clones showed normal karyotype. f Vector sequence (EBNA1 and OSW) not found based on PCR-based detection in expanded iPSCs after 10 passages. MNC mononuclear cell, P passage be expressed for a long time [28]. In our data, the expression of transcriptional genes did not increase significantly in the SFFV group at 48 h after transfection compared to the other promoters. We suggest that the persistence of expression may be the key reason for the high efficiency of reprogramming. Our results show that when the promoter of the episomal vector is SFFV, the reprogramming efficiency is most optimal (Fig. 3b).
Thus far, many studies have proved that different combinations of transcription factors can be applied successfully to cell reprogramming [20,26]. BCL-XL is well known for acting as an antiapoptotic protein [29], which is beneficial [30]; in addition, OSMK with BCL-XL has the most positive effect on the formation of iPSC colonies [22] (Fig. 3c, d).
Earlier studies have reported that hypoxia can improve survival of neural spine cells [31] and hematopoietic stem cells [32] and can inhibit the differentiation of ESCs [33]. Our study also confirmed that hypoxic conditions can improve the reprogramming efficiency of PB MNCs after nucleofection.

Conclusions
In the present study, we sought to improve the episomal method for generating iPSCs from PB MNCs and to lay some foundation for individualized iPSCs for future clinical application. With this optimized protocol, we improved the generation efficiency of integration-free iPSCs from human peripheral blood mononuclear cells, and a valuable asset for banking patient-specific iPSCs has been established.

Additional files
Additional file 1: Figure S1. Availability of data and materials Please contact author for data requests.
Authors' contributions HG carried out the cell culture studies and drafted the manuscript. XH carried out the immunoassays and performed the statistical analysis. JX participated in the cell culture and animal experiments. LS drafted the manuscript. SL carried out the cell culture studies. WY and XBZ participated in the design of the study. YL conceived of the study and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.
Ethics approval and consent to participate Written approval for human tissue collection and subsequent iPSC generation and genome/gene analyses performed in this study was obtained from the Ethics Committee for Human Genome/Gene Analysis Research at the Institute of Hematology and Blood Diseases Hospital, and written informed consent was obtained from each individual volunteer. All animal protocols were approved by the Institutional Animal Care and Use Committee, Institute of Hematology and Blood Diseases Hospital, CAMS/ PUMC. All surgery was performed under sodium pentobarbital anesthesia, and all efforts were made to minimize animal suffering.

Competing interests
The authors declare that they have no competing interests.

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