This protocol describes the efficient induction of hemogenic endothelium and multipotential hematopoietic progenitors from human pluripotent stem cells via the forced expression of transcription factors.
During development, hematopoietic cells arise from a specialized subset of endothelial cells, hemogenic endothelium (HE). Modeling HE development in vitro is essential for mechanistic studies of the endothelial-hematopoietic transition and hematopoietic specification. Here, we describe a method for the efficient induction of HE from human pluripotent stem cells (hPSCs) by way of overexpression of different sets of transcription factors. The combination of ETV2 and GATA1 or GATA2 TFs is used to induce HE with pan-myeloid potential, while a combination of GATA2 and TAL1 transcription factors allows for the production of HE with erythroid and megakaryocytic potential. The addition of LMO2 to GATA2 and TAL1 combination substantially accelerates differentiation and increases erythroid and megakaryocytic cells production. This method provides an efficient and rapid means of HE induction from hPSCs and allows for the observation of the endothelial-hematopoietic transition in a culture dish. The protocol includes hPSCs transduction procedures and post-transduction analysis of HE and blood progenitors.
The unique ability of human pluripotent stem cells (hPSCs) to self-renew and to differentiate into cells of the three germ layers, including blood, make them a valuable tool for the mechanistic studies of hematopoietic development, modeling of blood diseases, drug screening, toxicity studies, and the development of cellular therapies. Because blood formation in the embryo proceeds from hemogenic endothelium (HE) through an endothelial hematopoietic transition1,2, the generation of HE in cultures would be essential to study the molecular mechanisms regulating the endothelial to hematopoietic transition and hematopoietic specification. Current methods for studies of HE are based on the induction of hematoendothelial differentiation in aggregates (EBs) with the addition of hematopoietic cytokines3-5, and coculture of hPSCs with hematopoiesis-supportive stromal cells6,7 or in two-dimensional cultures with extracellular matrices and cytokines8,9. These classical differentiation methods are based on the introduction of external signals acting at the cell surface and initiating cascades of molecular pathways that eventually lead to the activation of transcriptional program guiding hematoendothelial development. Thus, the efficiency of hPSCs differentiations in these systems relies on an effective induction of those signals, signal transduction to the nucleus, and the resulting activation of specific transcriptional regulators. In addition, the study of HE in conventional differentiation cultures requires the additional step of isolating HE cells using cell sorting. Here, we describe a simple protocol for the direct induction of HE and blood by overexpression of hematopoietic transcription factors. This method allows for the efficient induction of HE in a dish and direct observation of the endothelial to hematopoietic transition without the need for isolation of HE using a cumbersome cell sorting procedure.
Formation of HE and blood from human pluripotent stem cells can be efficiently induced by overexpressing just a few transcription factors (TFs). The optimal combination of TFs capable of inducing robust pan-myeloid hematopoiesis from hPSCs includes ETV2 and GATA1 or GATA2. In contrast, combination of GATA2 and TAL1 induces erythromegakaryocytopoiesis10. Programming hPSCs through overexpression of these factors differentiates hPSCs directly to the VE-cad+CD43–CD73– HE cells that gradually acquire the hematopoietic phenotype defined by the expression of the early hematopoietic marker CD437. This lentiviral-based method for the direct programming of human pluripotent stem cells method is applicable for the generation of HE and blood cells for mechanistic studies, studies of endothelial to hematopoietic transition, and transcriptional regulation of hematopoietic development and specification. Although the current protocol describes blood production using constitutive expression of the transgenes, similar results could be obtained using modified mRNA10.
1. Virus Preparations and Transcription Factor Combinations
2. hPSCs Culture Protocol
3. Induction of Hematoendothelial Pprecursors from hPSCs (day 0, Transduction of hPSCs)
4. Induction of Hematoendothelial Precursors from hPSCs (Day 1-7)
5. Analysis of Hemogenic Endothelium (HE) Stage of Differentiation.
6. Analysis of Induced Hematopoietic Precursors
The schematic diagram of HE and blood induction from hPSCs by overexpression of transcription factors is shown in Figure 1. ETV2 with GATA1 or GATA2 combination induces pan-myeloid hematopoiesis, while a GATA2, TAL1 +/- LMO2 combination induces predominantly erythro-megakaryocytic hematopoiesis. Both TF combinations directly induced HE cells that subsequently transformed into blood progenitors with a distinct spectrum of hematopoietic differentiation. Differentiation of hPSCs from the pluripotent state to blood producing HE takes on average 4 days. Round blood cells first emerge by day 5 of differentiation. Subsequently, numerous floating blood cells appear and expand in cultures. To maximize the number of floating blood cells, post-transduction cultures can be prolonged to 10-14 days. The desired lineage of cells can be expanded in low-adherent conditions with the appropriate combination of hematopoietic cytokines. Figure 2 demonstrates the induction of blood from hPSCs using ETV2 and GATA1 or GATA2 TFs. Following transduction with ETV2 and GATA2, cells acquire a typical endothelial morphology by day 4 of differentiation, and eventually transform into round blood cells (day 6-8 of differentiation (Figure 2A). Cells collected on days 3-4 of differentiation show a typical VE-cadherin+CD226+CD73– HE phenotype (Figure 2B, 3B). By day 7 of differentiation more than 50% of VE-cadherin+ cells acquire the hematopoietic CD43+ phenotype (Figure 2C). CFC-assays of GATA2/ETV2-induced cells reveal that colonies are comprised of high proliferative potential (HPP) CFCs, Erythroid (E) CFCs, macrophages (M) CFCs, and granulocytes (G) CFCs (Figure 2E). The hematoendothelial differentiation in hPSCs following overexpression of GATA2 and TAL1 alone or with LMO2 is shown in Figure 3. HE stage is best identified in cultures with GATA2 and TAL1. The addition of LMO2 accelerates differentiation and markedly contracts HE stage. The GATA2 and TAL1 combination alone or with LMO2 demonstrates differentiation and maturation of blood progenitors to mostly erythroid and megakaryocytic cells Figure 3D and 3E. Figure 4 shows optimization of transduction efficiency in H1 hESCs using GFP lentivirus (A) and cell death and differentiation efficiency in cultures transduced with GATA2/TAL1/LMO2 at high and low MOI.
Figure 1: Schematic diagram of protocol for induction of hemogenic endothelium and blood cells via the forced expression of TFs. Cartoon outlines major experimental steps and timelines. Following preparation of single cell suspension, hPSCs are transduced with TFs and seeded on matrix in complete commercial serum-free medium. Next day, medium is replaced with growth factor free commercial medium supplemented with bFGF, TPO and SCF (3F Medium). Following 3-4 days of culture, HE is formed. Subsequently, HE undergoes endothelial to hematopoietic transition with formation of multiple blood cells.
Figure 2: ETV2/GATA2-induced differentiation of hPSCs. (A) Phase contrast images of ETV2/GATA2-induced differentiation in H1 hESCs at days 2, 4, 6 and 8 after transduction; Scale bar, 100 µm. (B) Flow cytometric analysis of ETV2/GATA2-induced cells undergoing endothelial to hematopoietic transition at day 3 of differentiation: VE-cadherin+ HE cells express CD226 and lack expression of CD73. A small percentage of endothelial cells express CD73 (non-HE). (C) Flow cytometric analysis of ETV2/GATA1- and ETV2/GATA2-induced H1 hPSCs on day 7 post-transduction. (D) Flow cytometric analysis of ETV2/GATA2-induced hematopoietic cells expanded in medium supplemented with FBS and SCF, IL3, IL6, GM-CSF, G-CSF and EPO for 14 days. (E) Types of hematopoietic colonies formed from ETV2/GATA2 transduced cells in CFC-assay and corresponding Wright-stained cytospins representing Erythroid (E), Macrophages (M), Granulocytes (Gr) and High Proliferative Potential colonies (HPPs). Scale bars for CFC-assay, 250 µm, for cytospins, 20 µm. Please click here to view a larger version of this figure.
Figure 3: GATA2/TAL1 and GATA2/TAL1/LMO2-induced differentiation of hPSCs. (A) Phase contrast images of GATA2/TAL1/LMO2-induced differentiation in H1 hESCs at days 2, 4, 6 and 8; Scale bar, 100 µm. (B) Flow cytometric analysis of GATA2/TAL1-induced cells undergoing endothelial to hematopoietic transition on day 3 of differentiation: VE-cadherin+ cells acquire expression of HE marker CD226. A small percentage of endothelial cells express CD73 (non-HE). (C) Immunofluorescent staining of GATA2/TAL1-induced differentiation in H1 hESC, day 5; VE-cadherin+ cells (green) acquire expression of hematopoietic marker CD43 (red). (D) Flow cytometric analysis of GATA2/TAL1/LMO2-induced hematopoietic cultures in suspension on day 14 post-transduction following expansion in serum-free medium supplemented with SCF, TPO, EPO and bFGF; (E) Types of hematopoietic colonies formed in CFC-assay by GATA2/TAL1/LMO2 differentiated cellsand corresponding Wright-stained cytospins representing Erythroid (E), Macrophages (M), and Megakaryocytes (Mk). Scale bars for CFC-assay, 250 µm, for cytospins, 20 µm. Please click here to view a larger version of this figure.
Figure 4: Optimization of lentiviral transduction procedure. (A) Transduction of H1 hESCs with different MOI of GFP lentivirus. (B) Cell viability and differentiation efficiency in cultures transduced with GATA2/TAL1/LMO2 at low (1 for each virus) and high (2 for each virus) MOI.
The above-described method for hematopoietic differentiation of hPSCs by overexpression of TFs, represents a rapid and efficient approach for the generation of HE and myeloid and erytho-magakaryocytic progenitors from hESCs and iPSCs, thereby allowing the production of up to 30 millions blood cells from one million pluripotent stem cells10. This method exhibited consistent differentiation in multiple hESC and iPSCs lines10. During differentiation by ETV2 and GATA2, GATA1 factors as well as GATA2 and TAL1 factors, cells form blood producing endothelium, HE and undergo an endothelium to hematopoietic transition. Formation of HE is usually observed between days 3 and 4 of differentiation. Addition of LMO2, a transcriptional co-factor of TAL1, to the GATA2 and TAL1 combination, significantly increased the robustness of differentiation, while the HE stage became markedly contracted.
The success of directed differentiation of hPSCs by transcription factors mainly depends on (i) an effective delivery of nucleic acids into cells, (ii) cell viability following genetic manipulations, and (iii) an effective translation of exogenous transcripts inside the cell.
One of the key steps of the described method is the production of effective lentiviral units, which depends on a precise buffer pH for HEK 293T cell transfection, and the absence of endotoxin contaminants at all steps of viral production and transduction experiments. The integrity of all constructs, including packaging and envelope plasmids, must be verified if no signs of viral production in HEK 293T cells is observed13.
The differentiation protocol design using combinations of transcription factors should consider several variables. High efficiency of viral transduction is important for successful differentiation. Since transduction efficiency depends on the method used for virus production and purification, optimize hPSC transduction using a GFP-lentiviral vector is recommended (Figure 4A). Viral integration following transduction should be verified using genomic PCR with transgene-specific primers (Table 3). Differentiation optimization should also include adjustment of lentiviral concentration in the reaction mixture. A relatively low amount of virus, MOI of 0.5-1, is capable of inducing hPSC differentiation. While a higher MOI increases the efficiency of differentiation, it also increases cell death (Figure 4B). Additional optimization step may include MOI ratio adjustment for each virus in the reaction mixture. In GATA2/ETV2 transduced cultures, doubling the MOI for GATA2, relative to ETV2, increases the efficacy of differentiation. For GATA1/ETV2, GATA2/TAL1 and GATA2/TAL1/LMO2 equal MOI for each virus is optimal.
Following transduction with TFs, a vast majority of cells underwent angiohematopoietic differentiation while only a very few cells retained an undifferentiated morphology. Since pSIN-EF1α lentiviral vectors incorporate antibiotic resistance, treatment of cultures with puromycin can be used to eliminate residual undifferentiated cells.
The formation of cells with characteristic endothelial morphologies, and subsequent round blood cells, indicate a successful differentiation. On average, the GATA2/ETV2 combination produces 40-50% of CD43+VE-cadherin+/- blood cells by day 9-10 of differentiation with up to 30% cells remaining VE-cadherin+CD43–. GATA1/ETV2 combination induces CD43 expression more quickly and generates a greater number of CD43+ cells as compared to the GATA2/ETV2 combination. In these cultures, the majority of CD43+ cells coexpress VE-cadherin. In cultures transduced with GATA2/TAL1, the number of CD43+ cells usually reaches 40-50%. The addition of LMO2 to GATA2/TAL1 substantially accelerates and enhances the blood production, and markedly contracts the endothelial stage of development.
Colony-forming assays should be used to determine the frequency and type of induced hematopoietic progenitors. The nature of colony-forming cells can be confirmed by preparing Wright-stained cytospins from individual colonies. hPSCs transduced with ETV2 and GATA2 produce mostly large (greater than 0.5 mm in diameter) high proliferative potential (HPP) myeloid colonies composed of immature granulocytic and monocytic cells, with some mature myeloid cells and occasional erythroid and megakaryocytic cells. On average more than 80% of CFCs in GATA2/ETV2 transduced cultures are myeloid HPPs. In addition, these cultures generate erythroid and macrophage colonies (Figure 2E), which usually comprise less than 20% of total CFCs. hPSC transduction with ETV2 and GATA1 significantly increases the proportion of CFC-E to up to 50%. TAL1 and GATA2 transduced cultures produce mostly erythroid (more than 80% of total CFCs) and megakaryocytic colonies (more than 10% of total CFCs) with very few macrophage colonies (less than 5% of total CFCs). The addition of LMO2 to the TAL1 and GATA2 combination significantly increases the frequency of colony-forming cells with erythro-megakaryocytic potential10. The pattern of CFC activity following transduction with the described combinations of transcription factors is consistent among different hESC and hiPSC lines10.
In summary, lentiviral-mediated overexpression in hPSCs is a relatively quick and efficient tool for the induction of HE and blood using TFs. This system can also be used for assessing the differentiation capacity of other TFs and identify those that are required for endothelial and hematopoietic specification. However, the current protocol has a limited utility for studies of extracellular signaling involved in the induction of HE, as it uses TFs to bypass the surface receptor-mediated signaling. Although the current protocol describes blood production using the constitutive expression of a transgene, similar results could be obtained using modified mRNAs10.
The authors have nothing to disclose.
We thank Matt Raymond for editorial assistance. This work was supported by funds from the National Institute of Health (U01HL099773, R01HL116221, and P51 RR000167) and The Charlotte Geyer Foundation.
Table 1. Induction of hPSCs differentiation with trancription factors and analysis of hemogenic endothelium and blood cells.
pSIN4-EF1a-ETV2-IRES-Puro | Addgene | Plasmid #61061 | Lentiviral Vector |
pSIN4-EF1a-GATA2-IRES-Puro | Addgene | Plasmid #61063 | Lentiviral Vector |
pSIN4-EF1a-GATA1-IRES-Puro | Addgene | Plasmid #61062 | Lentiviral Vector |
pSIN4-EF1a-TAL1-IRES-Puro | Addgene | Plasmid #61062 | Lentiviral Vector |
pSIN4-EF1a-LMO2-IRES-Puro | Addgene | Plasmid #61064 | Lentiviral Vector |
Hexadimethrine bromide (Polybrene) | Sigma-Aldrich | 107689-10G | Cationic polymer used to increase the efficiency of infection |
Y-27632 (Dihydrochloride) ROCK inhibitor | STEMCELL Technologies | 72302 | RHO/ROCK pathway inhibitor Inhibits ROCK |
StemPro Accutase Cell Dissociation Reagent | Life Technologies | A11105-01 | Cell Dissociation Reagent |
Incomplete (growth factor- free) culture medium mTeSR1 Custom formulation | WiCell Research Institute (Madison, WI) | MCF | Serum-free mTeSR1 medium without bFGF and TGFb |
human SCF | Peprotech | 300-07 | Premium grade |
human TPO | Peprotech | 300-18 | Research grade |
human FGF-basic | Peprotech | 100-18B | Premium grade |
CD144 (VE-cad) FITC | BD Biosciences | 560411 | Endothelial marker (FACS) |
CD226 PE | BD Biosciences | 338305 | Hematopoietic (FACS) |
CD43 PE | BD Biosciences | 560199 | Hematopoietic (FACS) |
CD73 APC | R&D Systems | FAB5795A | Endothelial marker (FACS) |
CD45 APC | BD Biosciences | 555485 | Hematopoietic (FACS) |
7AAD | Life Technologies | A1310 | Live/Dead assay (FACS) |
Paraformaldehyde | Sigma-Aldrich | P6148-500G | Cell fixation |
Triton X-100 | Sigma-Aldrich | T9284-500ML | Permeabilization |
FBS | Fisher Scientific | SH3007003 | Fetal bovine serum |
Mouse anti-human CD43 | BD Biosciences | 551457 | Pure, primary antibody for Immunofluorescence (IF) staining |
Rabbit anti-human VE-cadherin | BenderMedSystem | BMS158 | Primary (IF) |
Anti-rabbit Alexa Fluor 488-conjugated | JacksonResearch | 715-486-152 | Secondary (IF) |
Anti-mouse Alexa Fluor 594-conjugated | JacksonResearch | 715-516-150 | Secondary (IF) |
DAPI nucleic acid stain | Life Technologies | D1306 | Live/Dead assay (IF) |
Clonogenic medium MethoCult H4435 Enriched | STEMCELL Technologies | 4435 | CFC-assay |
Wright Stain solution | Sigma -Aldrich | 32857 | Staining cytospins |
Table 2. hPSCs culture.
Human Pluripotent Stem Cells (hPSCs) | WiCell Research Institute (Madison, WI) | hESCs (WA01, WA09) human Embryonic Stem cells; iPSCs (DF-19-9-7T, DF-4-3-7T) transgene-free induced Pluripotent Stem Cells | hPSCs are able to self-renew and to differentiate into cells of three germ layers |
Complete serum-free medium for culture of hPSCs mTeSR1 media | WiCell Research Institute (Madison, WI) | M500 | Serum-free medium with growth factors for feeder free culture of ESC/iPSCs |
Matrigel | BD Biosciences/ Corning | 356234 | Matrix for maintenance of human ESC/iPSCs |
DMEM/F-12, powder | Life Technologies | 12500-062 | Basal Medium |
HyClone Dulbecco's PBS powder | Fisher Scientific | dSH30013.04 | PBS |
Dispase II, powder | Life Technologies | 17105-041 | Neutral protease, Cell dissociation |
Table 3. Primers for detection of virus genomic integration.
Primer's Name | Forward (Fwd) 5’ –>3’ | Reverse (Rev) 5’–>3’ | Discription |
pSIN EF1a Fwd | TTC CAT TTC AGG TGT CGT GA | — | EF1a promoter sequence |
GATA1 Rev | — | TCC CTG TAG TAG GCC AGT GC | Coding Region |
GATA2 Rev | — | GGT TGG CAT AGT AGG GGT TG | Coding Region |
TAL1 Rev | — | AGG CGG AGG ATC TCA TTC TT | Coding Region |
LMO2 Rev | — | GGC CCA GTT TGT AGT AGA GGC | Coding Region |
ETV2 Rev | — | GAA CTT CTG GGT GCA GTA AC | Coding Region |