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Multicolor Staining of Globin Subtypes Reveals Impaired Globin Switching During Erythropoiesis in Human Pluripotent Stem Cells

Adult hemoglobin composed of α‐ and β‐globin reflects a change from expression of embryonic ε‐ and fetal γ‐globin to adult β‐globin in human erythroid cells, so‐called globin switching. Human pluripotent stem cells (hPSCs) are a potential source for in vitro erythrocyte production, but they show prominent expression of γ‐globin with little β‐globin expression, which indicates incomplete globin switching. To examine the mechanism of this impaired globin switching, we optimized multicolor flow cytometry to simultaneously follow expression of different globin subtypes using different immunofluorescent probes. This enabled us to detect upregulation of β‐globin and the corresponding silencing of γ‐globin at the single‐cell level during cord blood CD34+ cell‐derived erythropoiesis, examined as an endogenous control. Using this approach, we initially characterized the heterogeneous β‐globin expression in erythroblasts from several hPSC clones and confirmed the predominant expression of γ‐globin. These hPSC‐derived erythroid cells also displayed reduced expression of BCL11A‐L. However, doxycycline‐induced overexpression of BCL11A‐L in selected hPSCs promoted γ‐globin silencing. These results strongly suggest that impaired γ‐globin silencing is associated with downregulated BCL11A‐L in hPSC‐derived erythroblasts and that multicolor staining of globin subtypes is an effective approach to studying globin switching in vitro.


INTRODUCTION
Blood transfusion is an indispensable tool in medicine today. At present, however, the availability of blood for transfusion is entirely dependent on blood collected from donors, the numbers of whom have been declining in most industrialized countries. This makes the safety and stability of the blood supply a significant and continuing concern. To overcome this problem, much effort has gone into developing methods for in vitro generation of human erythrocytes [1,2], including the use of human pluripotent stem cells (hPSCs; i.e., embryonic stem cells [hESCs] or induced pluripotent stem cells [hiPSCs]) [3,4]. hiPSCs are particularly advantageous, as blood group type O Rh-negativederived hiPSCs could potentially serve as a universal source of red blood cell transfusion. The hiPSC technology could also contribute to production of rare types of erythrocytes-for example, D2, Fy(a2b2), Di(b2), and Jr(a2) [5]. However, it is well known that hiPSC clones are heterogeneous with respect to their differentiation potential [6]. Consequently, the ability to distinguish clones suitable for in vitro erythrocyte production from among identical donor-derived hiPSC clones is crucial.
Adult hemoglobin is composed of two a-globin and two b-globin subunits (HbA), whereas fetal hemoglobin is composed of two a-globin and two g-globin subunits (HbF). The fetal protein binds oxygen with greater affinity than the adult form, giving the developing fetus better access to oxygen from the mother's bloodstream [7]. Moreover, changes in erythropoiesis, and thus the globin expression profiles, are hallmarks of the developmental stages of erythroblasts [7]. During erythropoiesis from hESCs and hiPSCs, the major globin forms are embryonic « and fetal g; little adult b-globin is expressed [7], indicating incomplete globin switching. Why the switching is not accomplished remains unclear, however [8][9][10][11][12][13][14][15][16][17][18].
In the present study, we combined multicolor immunostaining with an improved fixation technique to follow expression of individual globin subtypes during in vitro erythropoiesis from hPSCs and human cord blood (CB) CD34 + cells. This provided the time frame of globin switching during erythropoiesis and enabled selection of suitable clones from among a heterogeneous population of hPSC clones. In addition, we also sought to validate our tracing method by examining the effect of BCL11A-L on gand b-globin expression [7,19]. Our findings indicate that even in

Expansion and Differentiation of Erythroblasts From Human Cord Blood CD34 + Cells
To expand and differentiate human CB CD34 + cells, they were applied to C3H10T1/2 cells (8.0 3 10 5 cells per 100-mm dish) and cultured in hematopoietic differentiation medium supplemented with 50 ng/ml human SCF, 50 ng/ml human TPO, and 5 IU/ml human EPO for 6 days, after which they were cultured with 5 IU/ml human EPO alone for another 12 days. Throughout the culture period, the medium was replaced every 3 days, and nonadherent cells were collected and analyzed on days 3, 6, 12, and 18.
Samples were then washed with PBS and analyzed using flow cytometry.

Real-Time Reverse Transcription-Polymerase Chain Reaction
Total RNA was extracted from cells using an RNeasy Micro kit (Qiagen, Hilden, Germany, http://www.qiagen.com), after which complementary DNAs were generated using a reverse transcription-polymerase chain reaction (RT-PCR) system (Thermo Ochi, Takayama, Hirose et al. Fisher Scientific, Waltham, MA, http://www.thermofisher.com) with oligo(dT) primers (Invitrogen). RT-PCR was performed using a kit (SYBR Premix DimerEraser; Takara Bio, Shiga, Japan, http:// www.takara-bio.com) according to the manufacturer's instructions. Signals were detected using an ABI7900HT Real-Time PCR system (Life Technologies, Rockville, MD, http://www.lifetech. com). GAPDH was used as an internal control. Fold changes were calculated using the DDC T method, with day 18 human CB CD34 + cell-derived erythroblasts serving as a calibrator. The primer sets are listed in supplemental online Table 1.

Doxycycline-Inducible BCL11A Lentiviral Vector and Transfection Methods
Human BCL11A-L gene-inducible lentiviral vector was based on an all-in-one inducible lentiviral vector (Ai-LV) [24] from Dr. T. Yamaguchi (University of Tokyo). Using PCR, human BCL11A was cloned from human CB-derived CD34 + erythroblasts and used to replace the mOKS cassette in the lentiviral vector, thereby enabling doxycycline (DOX)-dependent induction of BCL11A-L. Viral supernatant was generated as described previously [25]. Virus-transfected hES/iPSCs were cloned and maintained as previously described.

Image Analysis
Phase-contrast and fluorescence images were captured using a BZ-9000 microscope (Keyence, Osaka, Japan, http://www. keyence.com), after which the images were analyzed using BZ-Analyzer software (Keyence).

Statistical Analysis
All experiments were performed in triplicate and repeated at least twice. Figures show the results from one representative experiment. All data are presented as mean 6 SD. Values of p , .05 were considered significant.

Optimization of Cell Fixation for Tracing Expression of Individual Globins During Erythropoiesis From hPSCs and CB-Derived CD34 + Hematopoietic Progenitors
We have developed a coculture system with which human ESCs or iPSCs can be differentiated into multipotent hematopoietic progenitors capable of yielding megakaryocytes, erythroblasts, or lymphocytes [21][22][23]27]. Using this culture system, we first sought to generate erythroblasts from the H1 and KhES-3 hESC lines using the protocol diagrammed in Figure 1A and from human CB-CD34 + cells using the protocol diagrammed in Figure 1B. Thereafter, we used flow cytometry to characterize several cell surface markers (e.g., CD235a, CD43, and CD71), which revealed the differentiation capabilities and time frame of the in vitro differentiation from the respective sources. We found that we were able to differentiate hESC H1 and CB-CD34 + cells into CD235a + CD71 + and CD235a + CD71 2 erythroid cells (Fig. 1C).
To examine globin switching during erythropoiesis in several hPSC and CB clones, we compared the mRNA levels for globin subtypes encoded in the b-globin locus (HBE1, HBG1/HBG2, or HBB). Because epigenetic memory must be taken into consideration [28,29], we generated eight different hiPSC clones from different sources using a Sendai-viral system in which nonintegration of transgenes was validated (hiPSCs 1-8). Of these, hiPSCs 1 and 2 were generated from human neonatal dermal fibroblasts, hiPSCs 3-6 from human CB CD34 + cells, and hiPSCs 7 and 8 from human adult peripheral blood cells ( Fig. 2A). As shown in Figure 2B, blood-derived hiPSC clones (i.e., hiPSCs 5, 6, 7, and 8) expressed higher levels of HBB mRNA than fibroblast-derived clones, but lower levels than were expressed by human CB CD34 + cellderived erythroblasts on day 18 of culture. Because hESC H1and hiPSC 8-derived erythroid cells exhibited similar upregulation of HBB, which was stronger than that seen in hESC KhES-3-or hiPSC 1-4-derived cells (Fig. 2C), we mainly used these clones in subsequent analyses of globin expression.
We next sought to optimize the fixation conditions so that they would enable us to trace the expression patterns of individual globin subtypes during development of hPSC-derived erythroblasts and determine the levels of each protein. As shown in Figure 3A, initial fixation with 4% PFA, followed by 100% methanol and permeabilization using 0.5% saponin were most suitable for simultaneous flow cytometric detection of individual globin subtypes as well as erythroblast surface markers (e.g., CD235a). The different intracellular globin subtypes were then labeled using FITC-, TRI-COLOR-, and PE-conjugated anti-human globin subtype antibodies (Fig. 3B). For example, cell populations detectable as side scatter (SSC) and forward scatter (FSC) on flow cytometry (P1) were subsequently selected as Pacific Blue-conjugated CD235a + cells (P3), which were also evaluated based on the anti-human globin immunofluorescence intensities (Fig. 3C). With this methodology, we were able to distinguish different anti-human globin immunofluorescence intensities, as evaluated based on mean fluorescence intensity. We ruled out nonspecific antibody binding using Jurkat cells, hESC H1-derived erythroblasts on day 6, human CB CD34 + cell-derived erythroblasts on day 6, and fresh red blood cells (RBCs) (supplemental online Fig. 1). hESC H1-derived erythroblasts showed positivity for human «-globin along with g-globin or b-globin (Fig. 3D), and CBerythroblasts (Fig. 3D) and fresh RBCs (supplemental online Fig.  1) showed profiles consistent with earlier reports [7,30]. The results from repeated experiments using this approach are summarized in Figure 3E. Collectively, these findings indicate that on day 18 of culture most hESC H1-and hiPSC 8-derived erythroblasts simultaneously express «-, g-, and b-globin. However, a small population of «-globin-negative cells was also observed, whereas few or no g-globin-negative cells were seen (Fig. 3E). This indicates that hPSC-derived erythroblasts coexpress fetal gand adult b-globin. However, this experiment does not reveal why globin switching from fetal gto adult b-globin is impaired despite selection of good hPSC clones.

Reduced BCL11A-Mediated Silencing of g-Globin Expression in PSC-Derived Erythroid Cells May Be Associated With Impaired Globin Switching
It has been postulated that BCL11A facilitates g-globin silencing [19,31]. In that regard, we found that the mRNA level for one BCL11A isoform, BCL11A-L, was significantly lower in hESC H1and hiPSC 8-derived erythroid cells than in the CB-derived cells (Fig. 4). By contrast, mRNA expression of KLF1, an accelerator for b-globin upregulation [32], and its upstream molecules in hPSC-derived cells, GATA1 and cMYB, was comparable to or higher than in CB-erythroid cells (Fig. 4) [33]. In addition, we noticed that expression of SOX6, which is essential for formation of the BCL11A signaling complex [33,34], was also lower in hPSC-derived erythroid cells (Fig. 4). With those findings in mind, we hypothesized that downregulated BCL11A-L expression was associated with the impaired g-globin switching seen in hPSC-derived erythroid cells.
To test that idea, we used a lentiviral vector harboring a DOXinducible overexpression system (Fig. 5A) to establish hPSC clones (hESC H1-BCL11A-L-GFP and hiPSC 8-BCL11A-L-GFP) expressing BCL11A-L plus GFP as a marker, and the pluripotent state was indicated by SSEA-4 positivity (Fig. 5B, 5C). With this system, the DOX-OFF and DOX-ON states showed no expression and overexpression of BCL11A-L plus GFP, respectively. We then tested the three protocols depicted in Figure 6A (protocols i, ii, and iii). Flow cytometric analysis showed that differentiation phasedependent changes in BCL11A-L levels were associated with reductions in the expression of both g-globin mRNA (Fig. 6B) and protein (Fig. 6C) in hESC H1-BCL11A-L-GFP-and hiPSC Ochi, Takayama, Hirose et al. 8-BCL11A-L-GFP-derived erythroid cells. Interestingly, protocol iii induced substantial silencing of g-globin expression without affecting b-globin expression (Fig. 6C, 6D, day 18 of culture), which is consistent with earlier reports [19,31].
These results strongly suggest that downregulated expression of BCL11A-L and its signaling complex is associated with impaired g-globin silencing, whereas b-globin expression is unaffected. We also conducted CE-HPLC studies to detect different hemoglobin chain types. Sorted hiPSC 8-BCL11A-L-GFP-derived CD235a + GFP + cells (2.0 3 10 7 cells) generated using protocol i (DOX-OFF) or protocol iii (DOX-ON from day 0 to day 18) were evaluated on day 18 of culture. The majority of the hemoglobin in cells generated using protocol i was HbF (a2g2) and HbE (a2«2), although small amounts of HbA (a2b2) were also present. Sustained overexpression of BCL11A-L in protocol iii altered the hemoglobin expression pattern, as exemplified by the reduction in HbF (a2g2) and the increase in HbA (a2b2). This profile was similar to that of CB-derived erythroblasts, except that HbE (a2«2) was also retained in hiPSC 8-derived erythroid cells (Fig. 6E).

DISCUSSION
The globin expression profile is a hallmark of the developmental stages of erythroblasts [7,33]. In adults, human hemoglobin is composed of aand b-globins. The human b-globin gene cluster contains five functional genes that are expressed in the order of their arrangement within the locus («-Gg-Ag-d-b) on chromosome 11. In humans, these genes undergo two major transitions in expression during ontogeny; the first is the transition from embryonic («) to fetal (Gg, Ag) globins, and the second is from fetal to adult (d, b) globins [7]. The human system does not always correspond to the murine one, however [35,36]; consequently, tracing globin switching using hPSCderived erythroblasts could be an effective approach to characterizing human hemoglobin ontogeny. We therefore sought to use hPSC clones (i.e., hESCs and hiPSCs) to trace the changes in the expression of b-globin during in vitro differentiation toward erythrocytes. To accomplish this, we optimized multicolor flow cytometric analysis to simultaneously follow the expression of individual globin subtypes using several immunofluorescent probes. Initially, we used human CB CD34 + cell-derived and hPSC-derived erythroid cells to determine the optimal methodology for cell fixation and the most suitable probes for detection of the protein expression of individual globin subtypes (Figs. 1-3). Most of the hES/iPSC clone-derived erythroblasts showed prominent expression of «and g-globin, but little expression of b-globin, likely reflecting primitive erythropoiesis. By contrast, clones established from human blood cells (CB CD34 + cells or peripheral blood cells) were strongly positive for b-globin expression (Fig. 3D, 3E). This suggests that "epigenetic memory"-based mechanisms may influence definitive erythropoiesis, keeping it at a primitive stage [28].
We also found that the heterogeneous b-globin expression among hESC-and hiPSC-derived erythroid cells might reflect differences in the endogenous expression of BCL11A-L, the fulllength form of BCL11A (Fig. 4). BCL11A is a zinc-finger transcription factor that acts as a regulator of HbF expression [31]. It is also suspected that BCL11A levels correlate with developmental stage such that high levels of g-globin are evident in the absence of the full-length forms of BCL11A [7,19,30]. Abundant expression of BCL11A-L is developmentally restricted to adult erythroid cells (i.e., primary BM-erythroblasts), strongly indicating that BCL11A-L acts to repress g-globin gene expression [19]. In the present study, g-globin expression was silenced in human CB CD34 + cell-derived erythroid cells, but that silencing was incomplete in hES/iPSC clone-derived cells (Fig. 3E). Furthermore, overexpressed BCL11A-L in hPSC-derived erythroid cells appeared to downregulate g-globin expression at both the mRNA (Fig. 6B) and protein (Fig. 6C, 6D) levels without affecting b-globin (Fig. 6D, left). Most interesting was the observation that continuous overexpression of BCL11A-L in hPSC-derived CD34 + CD43 + multipotent progenitors selectively silenced g-globin expression (Fig. 6D).
CE-HPLC analysis confirmed that ectopic expression of BCL11A-L in hiPSC-derived erythroid cells upregulated HbA and downregulated HbF, as compared with control cells without overexpression of BCL11A-L. The relative upregulation of HbA observed in the CE-HPLC analysis is thought to result from the downregulation of HbF. These hemoglobin composition patterns are similar to those of human CB CD34 + cell-derived erythroid cells (Fig. 6E), confirming the flow cytometry results.
This suggests that BCL11A-L must be upregulated from the CD34 + CD43 + multipotent progenitor stage, as is seen in CB CD34 + cell-derived cells (Fig. 4). Based on these findings, we suggest that future identification of the mechanism underlying the reduced expression of BCL11A-L along with reduced formation of the BCL11A signaling complex [33,34] in hPSC-derived cells will be a valuable step toward in vitro production of functional erythrocytes.

CONCLUSION
Our multicolor staining method used with flow cytometry enabled us to shed new light on the mechanism underlying the impaired g-globin silencing in hPSC-derived erythroid cells. Moreover, hPSCderived erythroid cells have the potential to provide an infinite supply of erythrocytes by using immortalized erythroblast cell lines from hPSCs [26], thereby replacing donor-dependent transfusion therapy, and our method could be useful for choosing the best clone for generating adult type erythrocytes after solution of globin switching.