Human Induced Pluripotent Stem Cell-Derived B Lymphocytes Express sIgM and Can Be Generated via a Hemogenic Endothelium Intermediate

Abstract

The differentiation of human pluripotent stem cells to the B-cell lymphoid lineage has important clinical applications that include in vitro modeling of developmental lymphogenesis in health and disease. Here, we first demonstrate the capacity of human induced pluripotent stem cells (hiPSCs) to differentiate into CD144⁺CD73⁻CD43/CD235a⁻ cells, characterized as hemogenic endothelium, and show that this population is capable of differentiating to CD10⁺CD19⁺ B lymphocytes. We also demonstrate that B lymphocytes generated from hiPSCs are able to undergo full VDJ rearrangement and express surface IgM (sIgM⁺), thus representing an immature B-cell subset. Efficiency of sIgM expression on the hiPSC-derived B lymphocytes (∼5% of CD19⁺ cells) was comparable with B lymphocytes generated from human umbilical cord blood (UCB) hematopoietic progenitor cells. Importantly, when assessed by global transcriptional profiling, hiPSC-derived B-cells show a very high level of similarity when compared with their UCB-derived counterparts, such that from more than 47,000 different transcripts, only 45 were significantly different (with a criteria adjusted P value P<0.05, log FC >1.5 or 2.8-fold). This represents a unique in vitro model to delineate critical events during lymphogeneisis in development and lymphoid diseases such as acute lymphocytic leukemia.

Introduction

Hematopoietic progeny has routinely been derived from pluripotent stem cell (PSC) sources, yet the in vitro generation of hematopoietic stem cells (HSCs), as assessed by high-level, long-term multi-lineage reconstitution in immune-compromised mice continues to elude the field. The generation of myeloid and erythroid cells from various pluripotent sources has been described in mice and humans [1,2], as has the generation of T lymphocytes [3 –5]. Differentiation of B-cell lymphocytes from PSCs has, however, proved to be much more challenging. To date, B-cell differentiation from human embryonic stem cells has been described by Vodyanik et al. [6] and the generation of B-cells from human induced pluripotent stem cells (hiPSCs) has been reported by our own laboratory [7].

In hematopoietic ontogeny, the first wave of hematopoietic development, termed primitive hematopoiesis, occurs in the extra-embryonic yolk sac (YS) and, until recently, has been reported to be restricted to the generation of erythrocytes and macrophages (reviewed in McGrath and Palis [8]). HSCs, in contrast, are generated intra-embryonically within the aorta-gonad-mesonephros (AGM) region in the second wave of definitive hematopoiesis [9 –11]. Lymphocyte differentiation is an important readout of increased hematopoietic progenitor potential that arises from a transient wave of hematopoiesis, and precedes the production of HSCs [12 –14]. Furthermore, evidence that lymphocytes are generated in the extra-embryonic YS has recently been described [14 –16]. Analysis of the B-cell potential of hiPSC-derived hemogenic endothelium (HE) is important, as lymphoid potential is considered a distinguishing characteristic of definitive hematopoiesis.

Hematopoietic precursors from the transient and definitive waves of hematopoiesis are thought to arise from an endothelial intermediate with hematopoietic potential. Termed HE, this population of cells retains an endothelial phenotype, but undergoes an endothelial-to-hematopoietic transition (EHT) that results in the generation of hematopoietic progeny [17 –20]. In PSC cultures, populations that lack the expression of hematopoietic markers (typically CD45, CD41a, and CD43) but express CD144 are frequently classified as HE [21]. This phenotype, however, describes the entire endothelial fraction and therefore does not specifically identify HE. Recently, Choi et al. [22] have described the isolation of human HE from PSC/OP9 cell co-cultures using a CD235a/CD43⁻CD144⁺CD73⁻ cell surface profile that distinguishes this HE population from CD73⁺ (CD235a/CD43⁻CD144⁺) endothelial cells. Whether this population is capable of generating hematopoietic progenitors with T or B lymphoid potential has not been described.

In this study, we investigate the origins of hiPSC-derived B lymphocytes, and characterize them in relation to their cord blood-derived counterparts. First, we demonstrate that putative HE, defined as CD235a/CD43⁻CD144⁺CD73⁻ and produced on day 8 of hiPSC/OP9 cell co-culture, is capable of differentiating to yield CD19⁺ B lymphocytes. Second, we show that a subpopulation of the B-cell lymphocytes generated are able to undergo full VDJ recombination and express cell surface IgM (sIgM). Interestingly, ∼5% of the total CD19⁺ B-cell population expressed sIgM⁺, a proportion highly comparable to the production of sIgM⁺CD19⁺ cells from human umbilical cord blood hematopoietic progenitor cells (HPCs) when cultured in the same conditions. Finally, analysis of the transcriptome revealed a strikingly high level of similarity between hiPSC-derived B-cells and those generated from umbilical cord blood (UCB) HSC/HPCs using the same in vitro conditions, such that from more than 47,000 different transcripts, only 45 were significantly different (P<0.05, log1.5 FC or >2.83-fold difference). These included, among others, the developmentally significant genes PDGFRα and Lin28b.

Materials and Methods

Generation, maintenance, and characterization of hiPSCs

The c19 hiPSC line was derived in-house from normal human dermal fibroblasts (Lonza Biologics) and transduced with lentiviral vectors according to protocols first described by Takahashi et al. [23], and also in detail by us elsewhere [7]. These were used for the majority of the work described here. For transcriptome array profiling of arising B-cells, additional lines were employed; OPM2 and OC3 hiPSC lines were generated from human peripheral blood (PB) and UCB mononuclear cells (MNCs) and reprogrammed by episomal plasmids as described elsewhere [24]. OC3 was reprogrammed on mouse embryonic feeders (MEFs), in Dulbecco's modified Eagle's medium/F12 media (Life Technologies Corporation) with basic fibroblast growth factor (bFGF) and N2B27 (Life Technologies) supplementation as described by Yu et al., while OPM2 reprogramming was performed without feeder cells [25]. A fourth hiPSC line, designated as ‘PA’ hiPSCs, were kindly provided by Dr. Polyanna Goh (University College London, United Kingdom) and derived from human fibroblasts, again using episomal plasmids [26]. All hiPSC lines were characterized as described in Carpenter et al. [7] to demonstrate an embryonic stem (ES) cell-like phenotype and pluripotent potential. hiPSC lines formed teratomas in RAG^−/− mice, were negative for the expression of transgenes, and expressed the pluripotency associated markers SSEA4 and TRA-160. hiPSC lines were karyotypically normal as determined by G-banding.

MNC separation and isolation of CD133⁺ progenitors

Human UCB and adult PB were collected with written informed pre-consent and ethical approval from Oxford and Berkshire National Research Ethical Committees. Studies were conducted with approval of the NHSBT research committee. Cells were collected, processed, and stored under a Human Tissue Authority licence. MNCs were isolated by density gradient centrifugation using lymphocyte separation medium 1077 (GE Healthcare).

CD133⁺ or CD34⁺ cells from UCB or hiPSC sources, respectively, were isolated by magnetic activated cell separation (MACS) separation with anti-CD133 (for UCB) or CD34 beads (for hiPSC) (Miltenyi Biotec) as outlined in the manufacturer's protocols.

Hemato-endothelial and B-lymphocyte differentiation

hiPSC colonies were differentiated on OP9 stroma (kindly provided by Prof. Igor Slukvin, Department of Pathology and Laboratory Medicine, Wisconsin National Primate Research Center, University of Wisconsin, WI) but were originally from Dr. Nakano, Kyoto University, Japan, as previously described [6, 7]. Briefly, whole hiPSC colonies were harvested by collagenase type IV (Invitrogen, Inc.) pretreatment (at 1 mg/mL for up to min at 37°C), and seeded onto over-confluent OP9 stroma (low passage) on gelatinized 10 cm plates. Culture media consisted of alpha minimum essential media (αMEM), 20% (v/v) defined fetal calf serum (FCS) (Hyclone, Thermo Fisher Scientific), 1% (v/v) PenStrep, and 100 μM monothioglycerol (MTG) (Sigma-Aldrich Ltd.). hiPSC were seeded at a density of 1.0–1.5×10⁶ cells/10 cm dish, and after 24 h, a complete media change with warm media was performed to remove dead or unattached cells, with half-media changes then conducted on days 4, 6, and 8. After 10 days of co-culture with OP9 stroma, hematopoietic progenitors were harvested by 20 min digestion with collagenase type IV (1 mg/mL) and further trypsinization [0.05% (v/v) for 10–15 min at 37°C]. CD34⁺ cells were then isolated to 81.0%±3.5% purity from this suspension by magnetic activated cell separation using CD34 microbeads (Miltenyi Biotec) as outlined in the supplier's protocol.

To induce B-cell lymphopoiesis, MS-5 stroma (DSMZ) were maintained on gelatinized plates (as described earlier) in αMEM with 10% (v/v) FCS (Hyclone) and 1% (v/v) Penstrep (Invitrogen). Confluent monolayers were used for differentiation of CD34⁺ cells toward the B lymphoid lineage, using 25,000–50,000 hiPSC-derived CD34⁺ cells. These were seeded onto MS-5 stroma in differentiation media as described earlier, together with IL-7 (20 ng/mL), IL-3 (10 ng/mL), stem cell factor (SCF) (50 ng/mL), and Flt3L (50 ng/mL) (all from R and D Systems). Cultures are then fed with complete media changes weekly, with IL-7 (20 ng/mL), SCF (50 ng/mL), and Flt3L (50 ng/mL). Culture of these cells on mouse MS-5 stroma for 21 days gave rise to phase dark cobblestone-shaped cells that appeared underneath the stroma. These cells were then harvested by collagenase and trypsin digestion as described earlier, and CD45⁺ cells isolated by MACS separation (Miltenyi Biotec) as outlined in the manufacturer's protocol. Staining of cells with relevant conjugated antibodies [CD19-PerCP-Cy5.5 (HIB19) (eBioscience)/CD19-PE (LT19) (Miltenyi Biotec)/CD19-FITC (HIB19), CD45-APC (HI30), and CD10-PE (HI10a) (BD Biosciences)] performed before analysis by a fluorescence activated cell analyser (LSR II from BD Biosciences) was used to demonstrate double-positive populations for CD45⁺ and CD19⁺ and CD19⁺ and CD10⁺.

For extended B-cell cultures (day 45), CD45⁺ cells were harvested on day 21 from MS-5 stroma by 20 min digestion with collagenase type IV (1 mg/mL) and further trypsinization [0.05% (v/v) for 10 min at 37°C]. CD45⁺ cells were once again isolated using MACS separation as described earlier (96.1%±1.5% purity), with 5×10⁴ cells reseeded onto confluent MS-5 stroma as before.

Hematopoietic colony-forming assay

Hematopoietic colony-forming (erythomyeloid) potential was assayed using Methocult H4434 (Stem Cell Technologies). hiPSC-derived, FACS isolated cells were plated at 1×10³ cells/well by resuspending them in 50 μL phosphate-buffered saline and deposited into the methocult. Assays were maintained for 14 days before colonies were scored and counted, according to Stem Cell Technologies' guidelines.

Flow cytometry and fluorescent-activated cell sorting

The following monoclonal antibodies (mAb) were used for characterizing the arising B-cells: CD10-PE (Hl10a), CD144-FITC (55-7H1), CD235a-APC (HIR2), CD41aAPC/FITC (HIP8), CD45-APC/FITC (Hl30), CD73-PE (AD2), and IgM-PE (G20-127), all from BD Biosciences; CD19-PE (LT19), CD133-PE (293C3), and CD34-APC/FITC (AC136), all from Miltenyi Biotec; and CD144-PE (16B1), CD19-PerCP-Cy5.5 (HlB19), and CD43-APC/FITC (84-3C1), all from eBiosciences. Cells for flow cytometry analysis were incubated with the appropriate mAb in the dark, on ice, for 20 min. Cells were then washed and resuspended for analysis with the addition of 0.1 μg/mL 4′,6-diamidino-2-phenylindole DAPI (Life Technologies Corporation) as a viability stain. Analysis was performed on an LSR II flow cytometer (BD Biosciences) with gating for live cells; doublet exclusion was implemented based on forward scatter height (FSC-H) versus forward scatter area (FSC-A), and fluorescence minus one (FMO) was used to define negative and positive gates. For the analysis of B-cell maturation, cells were also gated on the CD19⁺ fraction.

For fluorescence activated cell sorting (FACS), cell populations were isolated using Aria (BD) or Becton Coulter cell sorters at the Nuffield Department of Medicine, Weatherall Institute for Molecular Medicine or Sir William Dunn School of Pathology, University of Oxford, by the trained FACS operator.

Polymerase chain reaction analysis of VDJ recombination

Genomic DNA was isolated from CD19⁺ cells on day 21 of MS-5 co-culture, separated by MACS isolation, using the QIAamp DNA Blood Mini Kit (Qiagen), and the IgH Gene Rearrangement Assay (InVivoScribe) was used to assess VDJ recombination status. Briefly, 5 μL of sample DNA (20–100 ng/μL) was added to Master Mix aliquots and reactions were run with the following program: 95°C 7 min with 37 cycles of 94°C for 30 s, 55°C for 30 s, 72°C for 60 s, followed by 95°C for 10 min. Oligo primer sequences are proprietary, but are designed toward the FR1 and JH region of the IgH locus. DNA amplicons were loaded onto a 2% (w/v) agarose (Sigma-Aldrich), TBE (Life Technologies) gel containing Gel Red Nucleic Acid Stain (Biotium). Hyperladder II (Bioline) was included as a molecular ladder. DNA fragments were separated in a TBE buffer by electrophoresis at 150 mV. Images were acquired using UVi Tec Gel Version 12.6 documentation (UVITEC) and image acquisition software.

Transcriptome profiling using gene array chips

RNA was isolated from FACS-sorted CD19⁺CD10⁺ cells from the following cell samples, each with four biological replicates per cell type: (1) day 21 hiPSC-CD34⁺/MS-5 co-cultures, (2) day 21 UCB-CD133/CD34⁺/MS-5 co-cultures, (3) uncultured UCB MNCs, (4) uncultured PB MNCs, and (5) undifferentiated hiPSCs, using the RNAeasy minikit (Qiagen). RNA purity was at least 1.8–2.0 (260/280 nm) with 50–100 ng used for hybridization. Gene arrays were performed by the High-Throughput Genomics Group, at The Wellcome Trust Center for Human Genetics, University of Oxford, using the Illumina Human-HT-12v4 expression BeadChip (Illumina). Quality controls were analyzed using the Bioanalyzer, and RNA was converted to biotin-labeled cRNA for hybridization. Chips were scanned using GenomeStudio (Illumina). Data were normalized using the lumi bioconductor package and analyzed using MeV software [27]. Data normalization and advice on bioinformatic methods were provided by the Computational Biology Research Group, WIMM, the United Kingdom.

Statistical test

The significance of differences between the mean values was determined using GraphPad Prism 6 and Student's t-test.

Results

CD144⁺CD73⁻CD43/CD235a⁻ putative HE is able to generate B lymphocytes

Reports have demonstrated that myeloid hematopoietic progeny from pluripotent sources are generated via a hemogenic endothelial intermediate [20, 22, 28, 29]. We therefore wanted to determine whether putative HE, a CD144⁺CD73⁻CD43/CD235a⁻ population as characterized by Slukvin et al., was capable of yielding B-cell lymphoid progeny. Initially, we assessed the co-expression of hematopoietic (CD41a, CD43, CD45) and hemato-endothelial (CD34, CD31, CD144) cell surface markers on differentiating hiPSCs in OP9 cell co-culture. On day 5 of co-culture, cells expressing CD144 were negative for hematopoietic markers (Fig. 1); however, by day 6, CD41 and CD43 were detected on a small fraction of CD144⁺ and CD34⁺ cells, respectively, and this expression continued over 12 days (Fig. 1B, C). By day 8, the fraction of cells co-expressing hematopoietic and endothelial markers (CD144 with CD41a, and CD34 with CD43) had increased substantially. By day 12, CD45 expression was also detected on a small proportion of cells and the expression of the hemato-endothelial cell surface marker CD144 had begun to decline (Fig. 1D). When B-cell potential of these fractions was probed using populations isolated from day 10 hiPSC/OP9 co-cultures and enriched for CD34⁺, CD43⁺, or CD34⁺CD43⁻ subsets by MACS (Fig. 2B), the capacity to generate lymphocytes as defined by CD19⁺CD10⁺ cells (Fig. 2C) was found exclusively in the hematopoietic population (Fig. 2D, E). With the description by Kennedy et al. [3], of a T-cell potential in the CD34⁺CD43⁻ on and before day 9, we conducted further studies to characterize B-cell potential of HE on day 8, focusing on this CD34⁺CD43⁻ fraction.

FIG. 1.

Emerging hematopoietic cells co-express endothelial-associated antigens. Hemato-endothelial cells were gated on forward scatter (FSC); viability and exclusion of doublets with specific antibody signature were assessed using fluorescence minus one (FMO) controls, as outlined in Supplementary Fig. S1. Here, we show typical staining for (A) isotype controls (B) CD144/CD41a, (C) CD34/CD43, and (D) CD144/CD45 which show double-positive populations that occur transiently during human induced pluripotent stem cell (hiPSC)/OP9 co-culture and peak around days 8–10. Generally, these populations mirror one another, where hemato-endothelial cells (double positive) appear before the hematopoietic population (CD41a⁺ or CD43⁺ alone) during hiPSC/OP9 co-culture. In (D), we show that CD45 is expressed after CD43 and CD41a in hiPSC/OP9 co-cultures. Representative dot-plots are shown, and mean±SD are included for all relevant quadrants (n=3 experiments).

FIG. 2.

B-cell potential on day 10 of iPS/OP9 co-culture is found exclusively in the hematopoietic fraction. In (A), we show a schematic of the experimental approach, with (B) flow cytometric analysis as dot plots which show CD34⁺, CD43⁺, and CD34⁺CD43⁻ fractions enriched after magnetic activated cell separation (MACS) from day 10 iPS/OP9 co-cultures. In (C), we show typical positive staining for B lymphocytes as indicated by a CD10 CD19 double-positive population, with specificity for staining confirmed with isotype (IgG1) controls. In (D), we show that CD34⁺ and CD43⁺ populations generated by MACS separation on day 10 (as in B), generate CD19⁺CD10⁺ cells on day 21 of MS-5 co-culture. The CD34⁺CD43⁻ population does not have B-cell potential. Values are mean±SD where n=3 independent experiments, and asterisk indicates statistical significance as determined by Student's t-test: *P≤0.05. In (E) CD34⁺ (i) and CD43⁺ (ii), populations generate cobblestone-like colonies (as indicated by arrows) in MS-5 co-culture that are absent in CD34⁺CD43⁻ cultures (iii) (magnification at ×20, where arrows indicate B-cell colonies observed in both (i) and (ii) by bright field microscopy).

Since Choi et al. [22] and Rafii et al. [29] have recently reported that the differential expression of CD73 could be used to distinguish HE from endothelium on day 8 of OP9 co-culture, we adopted this more stringent selection criteria to better characterize our cellular subsets. Cells from day 8 hiPSC/OP9 co-culture were first CD34 selected using immunomagnetic beads and then flow sorted based on the following cell surface profiles: CD43/CD235a⁺ hematopoietic (haem), CD43/CD235a⁻CD144⁺CD73⁻ HE, CD43/CD235a⁻CD144⁺CD73⁺ endothelium (endo), and CD43/CD235a⁻CD144⁻CD73⁻ negative (neg) cells as shown in Figure 3A. FMO controls and FACS profiles of sorted populations are shown in Supplementary Fig. S2 (Supplementary materials are available online at http://www.liebertpub.com/scd), which confirms specificity and purity of each fraction. The day 8 CD34⁺ fraction was shown to be 19.7%±3.4% haem, 9.1%±6.2% HE, and 32.3%±11.3% endo cells (mean±SD). FACS isolated populations were assayed subsequently for purity (consistently being more than 99% for HE and endo, and more than 90% for haem fractions) and seeded at 5,000 cells/mL into the MS-5 co-culture assay to promote B-cell differentiation, which was then evaluated after 21 days of culture. Figure 3B and C show that both HE and haem fractions were able to generate CD19⁺CD10⁺ B-cells, which were absent in the endo population. In addition, the endo fraction did not produce CD45⁺ cells, while as expected those from the HE and haem fractions did (Fig. 3D). Interestingly, when the total number of CD19⁺ B-cells generated from the HE fraction was compared with the number of B-cells generated from haem fraction, an 80-fold enrichment in this potential was revealed (Fig. 3C). Moreover, the HE population generated 6-fold more CD45⁺ cells than when an equivalent number of haem cells were seeded (Fig. 3E).

FIG. 3.

hiPSC-derived B-cells arise from a hemogenic endothelium (HE). CD43/CD235a⁻ CD144⁺ CD73⁺ endothelium and CD43/CD235a⁺ hematopoietic cells are indicated by plots shown in (A), where we also observe CD43/CD235a⁻ CD144⁺ CD73⁻ hemogenic endothelial cells, detected on day 8 of hiPSC/OP9 co-cultures. The CD73 versus CD144 plot shown is derived from the CD43/CD235⁻ fraction, as indicated by the arrow. Supplementary Fig. S2 demonstrates specificity of staining using FMOs controls. In (B), we show that HE, and to a much lesser extent hematopoietic (haem) populations, generates CD19⁺CD10⁺ B cells in the MS-5 co-culture; however, the sorted endothelial fraction (endo) does not contribute toward B-cell formation. Populations were FACS isolated and seeded at 5,000 cells/mL, onto MS-5 stroma with appropriate cytokines for B-cell differentiation. In (C), we show that B-cell potential is enriched by more than 80-fold, in the HE fraction more than that of the haem population (HE 94,748±30,998; Haem 1,141±567; mean±SD, n=3 independent experiments). We also show that hematopoietic (CD45⁺) progeny is generated from HE and haem fractions but not endo populations (D), where HE populations show a 6-fold greater enrichment for CD45⁺ cells than compared with haem populations (E) (HE 269,435±63,291; Haem 43,198±9,331; mean±SD for n=4 independent experiments). Asterisks indicate statistical significance as determined by Student's t-test: **P≤0.01. Cells were gated first on forward scatter (FSC) and side scatter (SSC) properties, but then only CD19⁺ or CD45⁺ cells counted by cytometry were included, as shown in (B) and (D), respectively. Absolute cell counts were determined by total events in each gate, where all cells were analyzed.

This work not only describes the hematopoietic potential of day 8 HE from pluripotent sources as previously reported by others [22] but also for the first time identifies HE with B lymphoid potential, which is also in agreement with Kennedy et al. [3], who describe T-cell potential of CD34⁺CD43⁻ cells before and upto day 9. Indeed, demonstration specifically of the transition of B-cell potential from endothelial to hematopoietic fraction between days 8 and 10 has not been previously described.

hiPSC-derived B-cells mature to undergo full V(D)J recombination and express cell sIgM

Since we were able to generate pre-B-cells lacking IgM expression in 21 day cultures [7], we hypothesized that maturation of hiPSC-derived B-cells to express sIgM might occur with extended culture time, with the experimental approach outlined in Figure 4A. Cells from day 21 hiPSC-CD34⁺/MS-5 co-cultures were harvested and selected to enrich for the B-cell population (CD19⁺) or for the pan-hematopoietic marker CD45. Cells were then re-plated onto a fresh MS-5 stromal layer and cultured for a further 21 days. When CD19⁺ cells were enriched directly by immuno-magnetic bead separation, there was a dramatic reduction in the number of B-cells observed after 21 days of culture (Fig. 4B). B-cell numbers were maintained, and indeed increased 4-fold over 21 days when CD45 enrichment was performed; therefore, this approach was adopted for long-term studies. As an aside, CD19 selection may instead have had a detrimental effect in other similar studies, and thus maybe why B-cells are not routinely expanded from PSC sources by other labs. The expression of sIgM was assessed every 7 days in the extended MS-5 co-cultures after selection for CD45⁺ cells. On days 29 and 36 of culture, CD19⁺ cells were negative for sIgM (Fig. 4C); however, by day 43, an average of 3.74% (±1.47 SD) of CD19⁺ cells expressed sIgM. Specificity of sIgM staining was confirmed with FMO controls as shown in Supplementary Fig. S3. The proportion of hiPSC-derived B-cells expressing sIgM was highly robust and reproducible and analogous to B-cells that were generated from UCB-CD133/CD34⁺ cells in the same culture conditions and over the same 42/43 day culture period (Fig. 4D). hiPSC-B-cells can be differentiated to express cell sIgM, a phenomena that has not previously been reported.

FIG. 4.

hiPSC-derived B-cells mature to express cell surface IgM (sIgM). In (A), we show a schematic of long-term B-cell differentiation cultures. In (B), we show that when B-cells are enriched by MACS for CD19 or CD45 expression on day 21 of hiPSC-CD34⁺/MS-5 co-culture, and then re-plated onto fresh MS-5 cell, those B-cells selected on CD19 do not persist in long-term cultures. Thus for extended B-cell cultures, we routinely selected CD45⁺ populations. Values indicate mean±SD, from three independent experiments. Asterisk indicates statistical significance as determined by Student's t-test: *P≤0.05. In (C), we demonstrate that a culture period of 43 days is required for the expression of sIgM from hiPSC-CD34⁺ cells where dot plots show an increasing number of cells acquiring IgM expression. Specificity of staining is confirmed by FMO controls presented in Supplementary Fig. S3. Although a relatively small population, this is highly robust and reproducible (3.7%±1.5%, n=5), while the efficiency of sIgM⁺ cell generation is comparable between hiPSC-derived B-cells and B-cells generated from umbilical cord blood (UCB)-CD133⁺ cells under the same in vitro conditions (n=2), as shown in (D). In (E), we show that hiPSC-derived B-cells have undergone rearrangement of V,D,J gene segments at the IgM locus, where genomic DNA was amplified by polymerase chain reaction using FR1 and JH consensus primers (arrow). A specific band between 300 and 400 bp is also observed for B-cells derived from UCB-CD133⁺ cells (UCB-B) under the same conditions and the 0030 clonal control DNA (arrow). The human dermal fibroblast (hDF) and undifferentiated hiPSC cells (hiPSC) served as negative cellular controls, together with a no template control.

To express sIgM, B-cells must undergo rearrangement of V, (D), J gene segments at the heavy (IgH) and light chain loci (IgK and IgL). To confirm VDJ recombination, genomic DNA was extracted from B-cells derived from hiPSC-CD19⁺ cells and UCB-CD10⁺ cells on day 43 of co-culture. When DNA was amplified using FR1 and JH consensus primers that bind to conserved sequences in the V and J genes, a band between 290 and 360 bp could be observed in B-cell samples, but not in negative controls (Fig. 4E) and similar in size to a clonal control (0030). This confirmed that hiPSC-derived B cells had indeed undergone full rearrangement of VDJ gene segments, at the IgH locus.

The generation of pre-B cells from the hiPSC-CD34⁺ population was achieved by co-culture with MS-5 cells in media supplemented with IL-7, SCF, Flt3L, and IL-3 for the first 21 days (with no supplemented IL-3 from day 7 onward). We observed that if all these three cytokines were continually added throughout the extended cultures, sIgM⁺ cells were not generated (Fig. 5A). Therefore, to investigate this further, hiPSC-CD45⁺ cells were re-plated in the presence or absence of IL-7, SCF, and Flt3L. sIgM⁺ cells could be detected in cultures maintained in the absence of cytokines and when SCF or Flt3L had been added, however sIgM⁺ cells were not generated when IL-7 was included, as observed for the inclusion of all three factors together. Mean data are presented in Figure 5B. Notably, the addition of IL-7 prevented the generation of CD19⁺sIgM⁺ cells but did not impact total B-cell numbers (Fig. 5D). This supports the notion that IL-7 may inhibit Rag-mediated recombination as indicated by others [30, 31] and which indicates that hiPSC-derived B-cells faithfully mirror developmental B-cell lymphopoiesis.

FIG. 5.

The generation of hiPSC-derived sIgM⁺ B cells is inhibited by IL-7. hiPSC-CD34⁺ cells generate B cells, which mature to express sIgM after 43 days of culture. In (A), we show that expression of sIgM is not observed in cultures where recombinant IL-7 or IL-7/SCF/Flt3L have been added, where mean±SD are given in the dot plots and histograms are used to show significance in (B). Bright-field images of long-term B-cell cultures in the presence/absence of recombinant cytokines as indicated are also provided in (C), while cells with a cobblestone-like morphology are observed in all conditions except that in the presence of IL-7 (D), which are indicated by the arrows (magnification ×20). Values in (B) and (D) indicate mean±SD, from three independent experiments. Asterisks indicate statistical significance as determined by Student's t-test: ^NS P>0.05, **P≤0.01, and ****P≤0.0001. Cell counts were determined by total events in each gate, where all cells were analyzed.

hiPSC-derived B cells are highly homologous to their UCB HSC/HPC-derived counterparts

Since B lymphocyte differentiation from hiPSC-derived HPCs and from HPCs/HSC from UCB occurred with similar kinetics and efficiencies as analyzed by cell surface marker expression, we further assessed the comparability of B cells generated from hiPSC-HPC and UCB-HSC/HPC sources at a molecular level using transcriptome array profiling.

Four different hiPSC lines, generated from a range of cell sources with a variety of methods, were used to generate CD19⁺ B cells, and referred to as hiPSC-B. B cells were also isolated directly from un-cultured neonatal (UCB) and adult (PB) sources, as well as by the in vitro differentiation of UCB-CD133/CD34⁺ (HSC/HPCs) to CD19⁺ cells on MS-5 stroma (utilizing the same approach as hiPSC sources), and referred to as HSC-B. CD19⁺CD10⁺ cells were isolated by FACS from each source, from which RNA was prepared. RNA was also extracted from undifferentiated hiPSC as a negative control. All samples were analyzed with four biological replicates, although due to low cell number, one of the four PB B cell samples failed in the quality control assay and was excluded from further analysis. Gene array data were normalized and analyzed by significance analysis of microarrays, which identified 767 statistically significant genes across all groups. Hierarchical clustering ordered biological replicates within cell types (Fig. 6A). As expected, undifferentiated hiPSC samples were notably different from the B-cell populations. While there were notable similarities among all B-cell sources, hiPSC-B and HSC-B samples clustered together by hierarchical analysis. To further examine relatedness between groups, Principle Component Analysis was run using the same 767 gene data set. hiPSC-B, PB, and HSC-B were positioned proximally on the PC1 axis (Fig. 6B), with undifferentiated hiPSC samples being least related. Interestingly, UCB B-cells were less related than PB B-cells to cultured B-cells from both HSC and hiPSC sources. When PC2 was plotted against PC3, the hiPSC-B and HSC-B populations even overlapped (Fig. 6C), further demonstrating the high degree of holomology. When normalized genes were filtered on the basis of significance, genes that were highly differentially expressed (log FC of >1.5 or <−1.5) were considered. In a comparison of hiPSC-B and HSC-B samples, these criteria identified just 45 genes that were either up- or down-regulated (Tables 1 and 2). Of note, the number of genes described by these parameters in a comparison of HSC-B and UCB sources was 544. Of particular interest are those genes up-regulated in hiPSC-B versus HSC-B cells (Table 1) that included Lin28b and PDGFRα, which are of developmental significance, and genes such as CCR7 and B lymphoid kinase (BLK) that are known B-cell modulators. In comparison, genes up-regulated in HSC-B versus hiPSC-B were mainly associated with an increase in metabolism as assessed using gene ontology analysis (Table 2).

FIG. 6.

hiPSC-derived B-cells show a high level of similarity with B-cells generated from UCB-hematopoietic stem cell (HSC)/hematopoietic progenitor cells (HPCs). (A) Data from the gene array study were normalized and analyzed by significance analysis of microarrays (SAM). Sample populations include hiPSCs (yellow), UCB (CD19⁺) B-cells (green), peripheral blood (PB) (CD19⁺) B-cells (blue), hiPSC (CD19⁺) B-cells (red), and HSC(CD133⁺) B-cells (purple). The 767 significantly significant genes across all groups are displayed as a heat map to highlight relatedness (please see Tables 1 and 2 for details of the most differentially expressed genes between hiPSC B-cells and HSC(CD133⁺) B-cells). Undifferentiated hiPSC's demonstrate a highly different signature from all four B-cell populations. In (B) normalized gene array data were analyzed by Principle Component Analysis (PCA). PC1 (x-axis), plotted against PC2 (y-axis), reveals the high level of similarity between hiPSC-B and HSC-B (with populations color coded as above) while (C) shows PC2 (x-axis) plotted against PC3 (y-axis), where iPS-B and HSC-B samples overlap. The small number of genes that are significantly different between these groups is described in Tables 1 and 2.

Table 1.

Table of Genes Up-Regulated in hiPSC-B Cells in Comparison to HSC-B Cells

log FC	P value	Adj. P value	B	Probe_ID	Symbol	Chr	Definition
3.456159	2.91E−06	0.000463	4.894123	ILMN_3251587	LOC100008589		Homo sapiens 28S ribosomal RNA, noncoding RNA
2.157481	3.72E−06	0.000557	4.656703	ILMN_1733559	LOC100008589		Homo sapiens 28S ribosomal RNA, noncoding RNA
1.964523	2.07E−06	0.000362	5.228263	ILMN_2086470	PDGFRα	4	Homo sapiens platelet-derived growth factor receptor, alpha polypeptide
1.739929	1.83E−05	0.001614	3.091279	ILMN_2115862	ESPNL	2	Homo sapiens espin-like
1.683171	3.68E−09	5.89E−06	11.20305	ILMN_1662358	MX1	21	Homo sapiens myxovirus (influenza virus) resistance 1, interferon-inducible protein p78 (mouse)
1.631937	8.08E−07	0.000198	6.139794	ILMN_1715131	CCR7	17	Homo sapiens chemokine (C-C motif) receptor 7
1.626654	1.47E−05	0.001448	3.308951	ILMN_1795429	VCL	10	Homo sapiens vinculin, transcript variant 1
1.543455	2.37E−06	0.000407	5.094144	ILMN_2386790	KLRC3	12	Homo sapiens killer cell lectin-like receptor subfamily C, member 3, transcript variant 1
1.491172	6.57E−07	0.000174	6.34014	ILMN_1743205	ABCA7	19	Homo sapiens ATP-binding cassette, sub-family A (ABC1), member 7
1.303125	4.00E−08	3.31E−05	9.005695	ILMN_1668277	BLK	8	Homo sapiens B lymphoid tyrosine kinase
1.285123	1.91E−05	0.001655	3.053555	ILMN_2129505	CYBASC3	11	Homo sapiens cytochrome b, ascorbate dependent 3
1.24946	1.45E−05	0.001446	3.324628	ILMN_1768505	IL13RA1	X	Homo sapiens interleukin 13 receptor, alpha 1
1.22099	9.00E−07	0.000213	6.035435	ILMN_2414762	TLR10	4	Homo sapiens toll-like receptor 10, transcript variant 1
1.20158	6.00E−08	4.19E−05	8.624517	ILMN_1784300	TUBA4A	2	Homo sapiens tubulin, alpha 4a
1.166454	7.96E−06	0.000974	3.911464	ILMN_1797822	SEL1L3	4	Homo sapiens sel-1 suppressor of lin-12-like 3 (C. elegans)
1.109582	3.89E−08	3.31E−05	9.032013	ILMN_1748697	LIN28B	6	Homo sapiens lin-28 homolog B (C. elegans)
1.10723	9.66E−06	0.001109	3.721205	ILMN_1770290	CNN2	19	Homo sapiens calponin 2, transcript variant 2
1.064689	7.72E−08	4.52E−05	8.386704	lLMN_1695404	LY6E	8	Homo sapiens lymphocyte antigen 6 complex, locus E
1.032722	7.49E−07	0.000189	6.213286	ILMN_2311537	HMGA1	6	Homo sapiens high mobility group AT-hook 1, transcript variant 1

Data from Illumina Human-HT-12v4 expression array were normalized and the expression of hiPSC-B and HSC-B genes was compared. Significantly up-regulated genes in hiPSC-B cells were filtered based on the following criteria: adj. P value P<0.05, log FC >1.5 or 2.8 fold. Based on these criteria, 19 known genes were identified.

Table 2.

Table of Genes Down-Regulated in hiPSC-B Cells in Comparison to HSC-B Cells

log FC	P value	Adj. P value	B	Probe_ID	Symbol	Chr	Definition
−3.21467	5.48E−07	0.000158	6.515172	ILMN_1794927	LOC90925		Homo sapiens hypothetical protein LOC90925
−2.162	8.55E−12	1.88E−07	16.32902	ILMN_2201596	CYTL1	4	Homo sapiens cytokine-like 1
−2.06868	3.62E−07	0.000126	6.912718	ILMN_1684255	MYL4	17	Homo sapiens myosin, light chain 4, alkali; atrial, embryonic, transcript variant 2
−1.88424	1.57E−05	0.001508	3.245392	ILMN_1655595	SERPINE2	2	Homo sapiens serpin peptidase inhibitor, clade E (nexin, plasminogen activator inhibitor type 1), member 2
−1.71713	1.83E−05	0.001614	3.091087	ILMN_3308961	MIR1974		Homo sapiens microRNA 1974, microRNA
−1.57511	3.02E−08	3.02E−05	9.268396	ILMN_1748625	TCEAL4	X	Homo sapiens transcription elongation factor A (SII)-like 4, transcript variant 4
−1.54485	5.30E−06	0.000722	4.309865	ILMN_2370091	NGFRAP1	X	Homo sapiens nerve growth factor receptor (TNFRSF16) associated protein 1, transcript variant 1
−1.46865	1.06E−07	5.30E−05	8.085697	ILMN_2103761	TLE4	9	Homo sapiens transducin-like enhancer of split 4 [E(sp1) homolog, Drosophila]
−1.4292	4.63E−11	2.78E−07	14.98313	ILMN_1691476	MYLK	3	Homo sapiens myosin light chain kinase, transcript variant 8
−1.41958	1.19E−10	4.32E−07	14.20414	ILMN_1707491	KIAA0125	14	Homo sapiens KIAA0125
−1.38804	5.33E−07	0.000156	6.54165	ILMN_1734190	TCEAL3	X	Homo sapiens transcription elongation factor A (SII)-like 3, transcript variant 1
−1.37639	1.51E−06	0.000302	5.532312	ILMN_1752199	LHPP	10	Homo sapiens phospholysine phosphohistidine inorganic pyrophosphate phosphatase
−1.34356	1.46E−05	0.001448	3.318446	ILMN_2352097	GPR56	16	Homo sapiens G protein-coupled receptor 56, transcript variant 2
−1.29862	2.64E−11	2.11E−07	15.4373	ILMN_1795715	DPYD	1	Homo sapiens dihydropyrimidine dehydrogenase, transcript variant 1
−1.22773	1.26E−05	0.001331	3.458412	ILMN_3197097	TSTD1	1	Homo sapiens thiosulfate sulfurtransferase (rhodanese)-like domain containing 1, transcript variant 2
−1.21065	5.09E−08	3.94E−05	8.77929	ILMN_1768110	ZAK	2	Homo sapiens sterile alpha motif and leucine zipper containing kinase AZK, transcript variant 2
−1.19232	1.88E−07	8.06E−05	7.539998	ILMN_1700257	C4orf32	4	Homo sapiens chromosome 4 open 32
−1.16152	3.92E−08	3.31E−05	9.025336	ILMN_1757074	GNG10	9	Homo sapiens guanine nucleotide binding protein (G protein), gamma 10
−1.13081	1.08E−10	4.32E−07	14.27842	ILMN_1662795	CA2	8	Homo sapiens carbonic anhydrase II
−1.10287	5.22E−06	0.00072	4.324224	ILMN_2384122	GPR56	16	Homo sapiens G protein-coupled receptor 56, transcript variant 3
−1.09511	1.40E−09	2.58E−06	12.07045	ILMN_1737972	TSPYL5	8	Homo sapiens TSPY-like 5
−1.05809	6.17E−07	0.000168	6.400483	ILMN_1667966	Clorf24	1	Homo sapiens chromosome 1 open reading frame 24, transcript variant 2
−1.03976	3.26E−08	3.13E−05	9.196196	ILMN_2157240	MNS1	15	Homo sapiens meiosis-specific nuclear structural 1
−1.0328	1.84E−05	0.001614	3.090066	ILMN_1771385	GBP4	1	Homo sapiens guanylate binding protein 4
−1.02361	1.04E−05	0.001145	3.652633	ILMN_1715886	CNOT7	8	Homo sapiens CCR4-NOT transcription complex, subunit 7, transcript variant 1
−1.00786	1.43E−05	0.001443	3.335855	ILMN_1762436	UBB	17	Homo sapiens ubiquitin B

Data from Illumina Human-HT-12v4 expression array were normalized and the expression of hiPSC-B and HSC-B genes was compared. Significantly down-regulated genes in hiPSC-B cells were filtered based on; adj. P value P<0.05, log FC >1.5 or 2.8 fold. Based on these criteria 26 known genes were identified.

Discussion

Differentiation of PSCs in vitro frequently recapitulates characteristics and events known to occur during embryonic development [32]. Indeed, numerous blood cell types have been generated from PSCs in vitro, yet the production of HSCs has not been described. Hematopoietic ontogeny is a complex process, involving numerous anatomical locations and multiple “waves” (reviewed in Medvinsky et al. [33] and Cumano and Godin [34]). Therefore, understanding the potential of PSCs to generate defined hematopoietic progenitors with increased multi-lineage potential will aid progress toward the production of HSCs in vitro, for therapeutic application.

Definitive hematopoiesis that ultimately leads to development of the adult HSC has been shown to arise from the AGM and other major arteries [9 –11]. A number of recent reports have significantly progressed our understanding of how the first definitive hematopoietic progenitors are formed via a direct EHT [17 –20]. One challenge that has slowed progress has been the inability to distinguish HE from endothelial cells, but which has recently been demonstrated to diverge as distinct populations both in vivo [35] and in vitro [36]. While selection on day 3 cultures with KDR/CD235a has only recently been demonstrated to resolve primitive and definitive haematopoieis [3, 36], with definitive hematopoiesis defined by T-cell output, only cell surface expression of CD73 can distinguish HE from endothelium on day 8 of differentiating pluripotent cultures, and here the authors described only myeloid potential [22, 29]. Analysis of the B-cell potential of hiPSC-derived HE, as described here, is important as lymphoid potential is considered a distinguishing characteristic of definitive hematopoiesis [12]. Using the most stringent selection criteria for HE on day 8 (CD235a/CD43⁻CD144⁺CD73⁻), we have demonstrated a significant enrichment of B-cell potential (80-fold), over the hematopoietic fraction. However, since the frequency of cells able to give rise to B cells appeared low within the HE pool, this suggests that a yet smaller HE subpopulation has the potential to give rise to hematopoietic progenitors with a high-level multi-lineage potential that resembles the lymphoid-primed multipotential progenitors (LMPP), which is formed before the definitive HSC [14].

In this report, we have demonstrated that hiPSC-derived B cells have the capacity to undergo VDJ recombination and express sIgM. This potential enables the functional assessment of novel B-cell populations, and, as such, offers increased applications for the modeling of B-cell-associated diseases such as acute lymphocytic leukemia and, furthermore, as a potential source of cells for producing fully human antibodies. In addition, we report that the presence of recombinant IL-7 ameliorated the maturation of hiPSC-derived B-cells and their ability to express sIgM. The relevance of IL-7 in B-cell development has been both complex and contentious, potentially varying between species, the stage of hematopoietic ontogeny, and within the B-cell differentiation hierarchy. There is accumulating evidence that describes the inhibition of B-cell maturation by IL-7 [37]. This may prevent rearrangement at the immunoglobulin light chain locus [30, 31], which involves activation of Stat5 and degradation of Foxo1 [38]. Elevated IL-7 and STAT5 activity results in failure of cycling of pre-B-cells. These then undergo growth arrest and induction of Rag gene expression that promotes immunoglobulin light chain gene rearrangement is blocked, thus precluding further B-cell maturation. Interestingly, mouse IL-7, to which human cells are reactive, has been shown to be secreted by MS-5 stromal cells [39, 40]. Indeed, preliminary studies conducted here suggest that the addition of IL-7 neutralizing antibodies to hiPSC-B-cells cultures from the pre-B-cell stage may result in the ablation of the B-cell population. This is also analogous to murine signaling, where limiting concentrations of IL-7 are required for differentiation [41]. While the efficiency of maturing hiPSC-derived B-cells to express sIgM was low, this was highly comparable to the efficiency of B-cell maturation from UCB-HSC/HPCs as described here and by others. Our observation of a negative impact of excessive levels of IL-7 on B-cell maturation and IgM expression in hiPSC-derived B-cells therefore fits very well with the murine model.

Here, we sought to compare hiPSC sources of B-cells with their neonatal or adult counterparts by comparing B-cells generated in vitro from hiPSCs with those generated in vitro from UCB-HSCs, and with B-cells isolated directly from UCB and PB. Overall, B-cells derived from hiPSC-HPCs and UCB-HSC/HPCs, using the same culture conditions, showed an extremely high level of similarity. This is a particularly robust outcome, as B-cells from four separate hiPSC lines (generated in a variety of ways) were used, and compared with B-cells generated from four separate UCB CD133/CD34⁺ cell isolates. Of the 19 genes with differentially increased expression in hiPSC-derived B-cells (Table 1), those of interest include Lin28b with a potential role in conveying a “fetal-like” potential to PSC-derived populations and in fetal lymphoid development [42,43], PDGFRα as a well-described marker for the mesoderm lineage [44], BLK that may substitute for pre-BCR activity [45], and Ly6E, which is up-regulated in hiPSC derived B-cells and is an allelic variant of Ly6A, otherwise known as Sca-1 [46]. Twenty-six genes that are significantly down-regulated in hiPSC-derived B-cells (Table 2) appear to be metabolic and structural proteins as assessed by ontology analysis, and none are of particular significance in B-cell lymphopoiesis.

In summary, we have shown that hiPSCs can differentiate to HE with the potential to give rise to B lymphocytes. We specifically show that these hiPSC-B lymphocytes undergo VDJ recombination and mature to express sIgM, a process that is inhibited by the addition of recombinant IL-7. Moreover, hiPSC-B-cells show a strikingly high level of similarity with B-cells derived from UCB-HSC/HPCs at the global transcriptional level. This system can now be applied to advance our understanding of several critical areas such as those relating to the role of supportive stroma in B-cell lymphopoiesis, and elucidating molecular mechanisms around, for example, pro-B to pre-B maturation and class-switching. Such analysis can now be dissected much more readily in vitro, and understanding these aspects could have a huge impact on our understanding of B-cell lymphogenesis in development and disease.

Footnotes

Acknowledgments

The authors thank Dr. Polyanna Goh, University College London, for the PA hiPS cell line, as well as Kevin Clark and Paul Sopp at the WIMM, University of Oxford, and Dr. Kate Alford at NDM, University of Oxford, for flow cytometry services. They also thank the High-Throughput Genomics Group at the Wellcome Trust Center for Human Genetics (funded by Wellcome Trust grant reference no. 090532/Z/09/Z and MRC Hub Grant G0900747 91070) for performing microarray experiments and the generation of the Gene Expression data by Dr. Dilair Baban.

A.F. was funded by an MRC Studentship via the Oxford Stem Cell Institute. The authors would like to thank NHS Blood and Transplant, the Oxford Stem Cell Institute, the NHS Blood and Transplant Trust Fund, and the National Institute for Health Research for their support. This article summarizes independent research funded by the National Institute for Health Research (NIHR) under its Program Grants for Applied Research Program (grant reference nos. RP-PG-0310-1001, −1003, and −1004). The views expressed are those of the authors and not necessarily those of the NHS, the NIHR, or the Department of Health.

Accession Numbers

Gene expression data have been deposited in the Gene Expression Omnibus database [accession number (GSE53572).

Author Disclosure Statement

The authors declare no competing financial interests.

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Supplementary Material

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Human Induced Pluripotent Stem Cell-Derived B Lymphocytes Express sIgM and Can Be Generated via a Hemogenic Endothelium Intermediate

Abstract

Introduction

Materials and Methods

Generation, maintenance, and characterization of hiPSCs

MNC separation and isolation of CD133+ progenitors

Hemato-endothelial and B-lymphocyte differentiation

Hematopoietic colony-forming assay

Flow cytometry and fluorescent-activated cell sorting

Polymerase chain reaction analysis of VDJ recombination

Transcriptome profiling using gene array chips

Statistical test

Results

CD144+CD73−CD43/CD235a− putative HE is able to generate B lymphocytes

hiPSC-derived B-cells mature to undergo full V(D)J recombination and express cell sIgM

hiPSC-derived B cells are highly homologous to their UCB HSC/HPC-derived counterparts

Discussion

Footnotes

Acknowledgments

Accession Numbers

Author Disclosure Statement

References

Supplementary Material

MNC separation and isolation of CD133⁺ progenitors

CD144⁺CD73⁻CD43/CD235a⁻ putative HE is able to generate B lymphocytes