Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

Summary

Here, we present human pluripotent stem cell (hPSC) culture protocols, used to differentiate hPSCs into CD34⁺ hematopoietic progenitors. This method uses stage-specific manipulation of canonical WNT signaling to specify cells exclusively to either the definitive or primitive hematopoietic program.

Abstract

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem cells (hPSCs). Until recently, efforts to differentiate hPSCs into HSCs have predominantly generated hematopoietic progenitors that lack HSC potential, and instead resemble yolk sac hematopoiesis. These resulting hematopoietic progenitors may have limited utility for in vitro disease modeling of various adult hematopoietic disorders, particularly those of the lymphoid lineages. However, we have recently described methods to generate erythro-myelo-lymphoid multilineage definitive hematopoietic progenitors from hPSCs using a stage-specific directed differentiation protocol, which we outline here. Through enzymatic dissociation of hPSCs on basement membrane matrix-coated plasticware, embryoid bodies (EBs) are formed. EBs are differentiated to mesoderm by recombinant BMP4, which is subsequently specified to the definitive hematopoietic program by the GSK3β inhibitor, CHIR99021. Alternatively, primitive hematopoiesis is specified by the PORCN inhibitor, IWP2. Hematopoiesis is further driven through the addition of recombinant VEGF and supportive hematopoietic cytokines. The resulting hematopoietic progenitors generated using this method have the potential to be used for disease and developmental modeling, in vitro.

Introduction

Human pluripotent stem cells (hPSCs) are defined as encompassing both human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), and have the unique capability of not only undergoing self-renewal under appropriate growth conditions, but also, the capacity to differentiate into all cell types derived from the three germ layers: endoderm, mesoderm, and ectoderm¹. Due to these unique abilities, hPSCs hold great promise for regenerative medicine, disease modeling, and cell-based therapies². While multiple cell types have been successfully differentiated from hPSCs, one significant challenge is the in vitro specification of exclusively adult-like hPSC-derived hematopoietic stem cells (HSCs) and definitive hematopoietic progenitors.

One likely barrier to the development of human HSCs from hPSCs is the presence of multiple hematopoietic programs within the human embryo³. The first program which emerges, termed "primitive hematopoiesis," originates within the extraembryonic yolk sac tissue and is best characterized by its transient production of erythroblast progenitors (EryP-CFC), macrophages, and megakaryocytes. Notably, this program does not give rise to HSCs, nor does it give rise to T and B lymphoid progenitors. However, the yolk sac does transiently give rise to restricted definitive hematopoietic progenitors, such as the erythro-myeloid progenitor (EMP^4,5,6,7,8) and the erythroid-deficient lymphoid-primed multipotent progenitor (LMPP⁹). However, neither EMPs nor LMPPs are fully multipotent, or capable of HSC-like engraftment in adult recipients. In contrast, later in development, the classically defined "definitive" hematopoietic program is specified in the aorta-gonad-mesonephros region of the embryo proper, giving rise to all adult hematopoietic lineages, including the HSC. The specification of these intra-embryonic definitive hematopoietic cells occurs in a Notch-dependent fashion, via an endothelial-to-hematopoietic transition from hemogenic endothelium (HE)^{3,10,11,12,13,14}. Aside from reconstitution capacity, the multilineage potential and Notch-dependence of these cells can be used to distinguish these definitive hematopoietic progenitors from the EMP and the LMPP (reviewed in references^3,13).

Understanding the mechanism(s) governing primitive and definitive hematopoietic specification from hPSCs is likely critical to the reproducible production of definitive hematopoietic progenitors across a variety of hPSC lines. Until recently, hPSC differentiation protocols that could separate multipotent primitive and definitive hematopoietic progenitors did not exist^{15,16,17,18,19,20,21,22,23,24,25}. Many approaches using fetal bovine serum (FBS) and/or stromal co-culture first outlined the hematopoietic potential of hPSC differentiation, with mixtures of primitive and definitive hematopoietic potential^{15,16,17,19,22,23,25}. Further, many serum-free hematopoietic protocols have described the signal requirements for the specification of mesoderm from hPSCs that harbors hematopoietic potential^18,20,21,24. However, as these methods still gave rise to heterogeneous mixtures of both programs, their use in clinical applications and understanding developmental mechanisms may be limited.

We have recently built on these studies, having outlined the stage-specific signal requirements for ACTIVIN/NODAL and WNT signaling in primitive and definitive hematopoietic specification from hPSC-derived mesoderm^18,26. The latter was particularly unique, as its use of stage-specific WNT signal manipulation allows for the specification of either exclusively primitive or exclusively definitive hematopoietic progenitors²⁶. During mesoderm specification, the inhibition of canonical WNT signaling with the PORCN inhibitor IWP2 results in the specification of CD43⁺ EryP-CFC and myeloid progenitors, with no detectable lymphoid potential. In sharp contrast, stimulation of canonical WNT signaling with the GSK3β inhibitor, CHIR99021, during the same stage of differentiation resulted in the complete absence of detectable CD43⁺ EryP-CFC, while simultaneously leading to the specification of CD34⁺CD43⁻ HE. This population possessed myeloid, HBG-expressing erythroid, and T-lymphoid potential. Subsequent analyses identified this HE as lacking the expression of CD73^27,28 and CD184²⁸, and its hematopoietic potential was NOTCH-dependent²⁸. Further, single-cell clonal analyses demonstrated that these definitive hematopoietic lineages could be derived from multipotent single cells²⁸. Taken together, these studies indicate that stage-specific WNT signaling manipulation can specify either pure primitive hematopoietic progenitors, or multipotent NOTCH-dependent definitive hematopoietic progenitors.

Here, we outline our differentiation strategy that yields exclusively primitive or definitive hematopoietic progenitors, via manipulation of canonical WNT signaling during mesodermal patterning, and their downstream hematopoietic lineage assays. This protocol is of great value to investigators who are interested in the production of either primitive or definitive hematopoietic progenitors from hPSCs for regenerative medicine applications.

Protocol

1. Reagents

1. Obtain cell lines; hESCs or hiPSCs¹, mouse embryonic fibroblasts (MEFs)²⁹, OP9-DL4 stroma^30,31.
2. Reagent preparation
   1. Prepare a 0.1% w/v solution of gelatin in PBS. Sterilize by autoclaving and store at 4 °C after aliquoting.
      1. Prepare the gelatin-coated plasticware. Coat 6-well dishes with 1.5 mL of 0.1% gelatin solution. Incubate for 15 min at room temperature. Immediately before use, aspirate remaining gelatin solution.
   2. Prepare the MEF media by making IMDM with 15% v/v FBS and 1x antibiotics. Supplement with 2 mM L-glutamine and 400 µM monothioglycerol (MTG) prior to use and store at 4 °C.
   3. Prepare the hPSC culture media as a solution of DMEM/F12 with 20% v/v knockout serum replacement (KOSR), 1x non-essential amino acids (NEAA), 1x antibiotics, 55 µM β-mercaptoethanol, 2 mM L-glutamine, and 10 ng/mL bFGF. Filter to sterilize with a 2 µm filter and store in the dark at 4 °C.
   4. Prepare the serum-free Wash media as a solution of DMEM/F12 with 5% v/v KOSR and 4 mM HCl. Filter to sterilize with a 2 µm filter, aliquot, and store in the dark at 4 °C.
   5. Make a 1 mg/mL DNase I solution by dissolving DNase I for 3 - 4 h into ice-cold, sterile deionized water on ice. Filter to sterilize with a 2 µm filter and store aliquots at -20 °C.
   6. Prepare the STOP solution with WASH media: add 10% FBS and 10 µg/mL DNase I. The STOP solution should be prepared immediately prior to use.
   7. Prepare the basement membrane matrix (e.g., matrigel, referred to as MAT hereon) mixture solution.
      NOTE: All steps preparing and using the MAT mixture should be performed on ice or at 4 °C. Thaw frozen MAT in ice at 4 °C overnight. Dilute MAT 1:1 by adding 10 mL ice-cold IMDM and chill in ice at 4 °C overnight. Dilute MAT mixture with an additional 20 mL ice-cold IMDM and chill in ice at 4 °C overnight. Aliquot into pre-chilled tubes and store at -20 °C until use. When ready for use, thaw MAT mixture overnight at 4 °C.
      1. Coat the MAT cell culture plates.
         NOTE: All steps of coating MAT plates should be performed on ice. Pre-chill 6-well and 24-well plates at -20 °C for at least 1 h. When ready to coat plates, place them on ice. Coat each well with 4x-diluted MAT, removing and re-using any excess solution. Leave on ice for 30 - 60 min, and aspirate excess MAT. Dry the MAT coated plates in a 37 °C 5% CO₂ incubator for a minimum of 3 h. Use within 72 h of coating.
   8. Prepare the solution of 10% Bovine Serum Albumin (BSA) by dissolving 10% w/v BSA in ice-cold, distilled, deionized water at 4 °C for 3 - 4 h. Filter to sterilize with a 2 µm filter into a light-blocking bottle and store at 4 °C.
   9. Prepare the ascorbic acid solution. Slowly dissolve a 5 mg/mL solution of L-ascorbic acid in ice-cold, distilled, deionized water on ice for 3 - 4 h. Swirl occasionally until dissolved. Filter to sterilize with a 2 µm filter and store aliquots at -20 °C.
   10. Prepare the serum-free differentiation (SFD) media by making a solution of 75:25 of IMDM:Ham's F-12, then add 0.5% BSA, 1x B27, and 0.5x N2 supplements. Store at 4 °C. Add 1x L-glutamine, 50 µg/mL ascorbic acid, 400 µM MTG, and 150 µg/mL holo-transferrin immediately prior to use.
   11. Prepare serum-free stem cell culture media (e.g., StemPro, referred to as SP34 hereon) per the manufacturer's instructions. Store at 4 °C. Add 1x L-glutamine, 50 µg/mL ascorbic acid, 400 µM MTG, and 150 µg/mL holo-transferrin immediately prior to use.
   12. Prepare the 0.2% Collagenase II solution by dissolving Collagenase II to a final concentration of 0.2% w/v in 1:4 FBS:PBS solution. Dissolve in 37 °C water bath for at least 15 min. Filter to sterilize solution with a 2 µm filter and store aliquots at -20 °C.
   13. Prepare the Fluorescence Activated Cell Sorting (FACS) buffer as a solution of 5% FBS in IMDM immediately prior to use.
   14. Prepare OP9 media as a solution of α-MEM, 20% FBS, 2 mM L-glutamine, and 1x antibiotics. Filter to sterilize with a 2 µm filter and store at 4 °C.
   15. Obtain methylcellulose-based media (e.g., MethoCult, referred to as MeC hereon) as pre-aliquoted 3 mL quantities or as a 100 mL bottle. If using the 100 mL MeC, upon arrival, divide into 2.5 mL aliquots in 14 mL conical tubes.
       NOTE: All aliquots should be stored at -20 °C. MeC medium aliquots should be thawed immediately prior to use.

2. Mesoderm Differentiation of hPSCs

1. Grow the hPSC cell lines on MEFs to 70 - 80% confluency, in a 37 °C incubator with 5% CO₂.
   NOTE: The hPSCs should have sharp, undifferentiated borders.
2. Prepare MAT-coated plasticware 24 h before passaging the hPSCs.
   NOTE: The total number of MAT-coated 6-well dishes varies across hPSC lines. Normally, three 6-well dishes are sufficient per 6-well dish of confluent hPSCs on MEFs.
   1. Perform a trial feeder-depletion with different ratios of confluent hPSCs:MAT wells (1:4, 1:3, 1:2, etc.) before the first differentiation to determine how many MAT-coated 6-well dishes should be used for subsequent differentiations.
      NOTE: 24 h after plating onto MAT, the hPSCs should be 80 - 90% confluent, with cell clumps of approximately 10 - 20 cells in size.
3. Aspirate the hPSC media and add 1 mL 37 °C 0.25% Trypsin-EDTA to each well for 1 min. Aspirate and add 1 mL STOP media to each well. Using a cell scraper, scrape the cells off the plate. Add 1 mL WASH buffer to each well.
   1. With a 2 mL serological pipet, triturate 3 - 5 times to break up large clumps of cells and transfer the cells to a 50 mL conical tube. Centrifuge the hPSCs at 330 x g for 5 min.
4. Resuspend the cells in a total volume of hPSC media equivalent to 2 mL per well of the MAT-coated plasticware to be used. Add 2 mL/well of hPSCs to the plasticware and incubate overnight in a 37 °C incubator with 5% CO₂.
5. 24 h later, aspirate the media and replace with 37 °C 0.05% Trypsin-EDTA for 1 min. Aspirate the Trypsin-EDTA and add 1 mL STOP media. Scrape the cells vigorously with a cell scraper. Add 1 mL WASH media to each well. With a 2 mL serological pipet, triturate the cells 1 - 2 times and transfer to a 50 mL conical tube. Centrifuge cells at 220 x g for 5 min.
   NOTE: The length of time of Trypsin-EDTA treatment should be determined by the investigator for each hPSC line. The Trypsin treatment should be applied for as long as possible without causing single-cell dissociation. This step should detach all the hPSCs from the MAT-coated plates, but not result in single-cell dissociation. Resultant EBs should be 6 - 10 cells, on average.
6. Aspirate the media. Using a 5 mL serological pipet, gently resuspend the cells in 20 mL WASH media. Centrifuge at 220 x g for 5 min.
7. Gently resuspend the EBs in Day 0 media (Table 1). Dispense 2 mL of differentiation media containing EBs into each well of a 6-well low-adherent cell culture dish using a 10 mL serological pipet. Place into a 37 °C 5% CO₂ 5% O₂ (multi-gas) incubator.
   NOTE: Day 0 media: SFD media supplemented with 10 ng/mL BMP4 (Table 1). For every two plates of hPSCs, use one 6-well low-adherent cell culture dish.
8. 24 h later, add 2 mL Day 1 media (Table 1). Incubate the cells overnight in a multi-gas incubator.
   NOTE: Day 1 media: SFD media supplemented with 10 ng/mL BMP4 and 10 ng/mL bFGF to each well (Table 1).
9. 18 h later, gently harvest the EBs with a 5 mL serological pipet and spin at 100 x g for 5 min. Wash and combine the entire culture with 10 mL IMDM. Spin at 100 x g for 5 min. Aspirate the media.
   NOTE: Debris will exist in the cultures. This centrifugation step at a low speed (100 x g) will remove the debris.
10. Gently resuspend the EBs in SFD media supplemented with Day 2 media (Table 1), and then dispense 2 mL per well into 6-well low-adherent cell culture plates. Incubate in a 37 °C multi-gas incubator for 30 h.
    NOTE: Day 2 media: 10 ng/mL BMP4, and 5 ng/mL bFGF, and the appropriate WNT agonist or antagonist (Table 1).
    1. Add 3 µM CHIR99021 to specify definitive hematopoietic progenitors. Alternatively, add 3 µM IWP2 and 1 ng/mL Activin A to specify primitive hematopoietic progenitors.
11. Continue to Section 3 for hematopoietic progenitor specification.

3. Specification of CD34⁺ Hematopoietic Progenitors

1. After 30 h, isolate the EBs with a 2 mL serological pipet at Day 3 of differentiation. Transfer to a 50 mL conical tube and centrifuge at 330 x g for 5 min. Gently resuspend and wash with 10 mL of IMDM. Centrifuge the cells again at 330 x g for 5 min.
2. Resuspend the EBs in Day 3 media (Table 2) and dispense 2 mL into each well of low-adherent 6-well plates. Incubate cells in a 37 °C multi-gas incubator for 72 h.
   NOTE: Day 3 media: SP34, supplemented with 15 ng/mL VEGF and 5 ng/mL bFGF (Table 2). Add more media per well if the pH of the media harvested on Day 3 of differentiation has significantly changed (i.e., yellow-orange media). Alternatively, use fewer EBs per well on Day 0.
3. After 72 h of incubation, add 2 mL of Day 6 media (Table 2) to each well. Incubate in a 37 °C multi-gas incubator for an additional 48 h.
   NOTE: Day 6 media: SP34 media supplemented with 15 ng/mL VEGF, 5 ng/mL bFGF, 200 ng/mL SCF, 4 IU EPO, 20 ng/mL IL-6, 10 ng/mL IL-11, and 50 ng/mL IGF-1 (Table 2). If more media was added in step 3.1, then add the equivalent amount of media in step 3.2 so that the final cytokine concentrations remain the same.
4. Isolate the CHIR99021-treated EBs on Day 8 of differentiation. Proceed to Section 4.
5. IWP2-treated EBs are harvested on Day 8 of differentiation into a 50 mL conical tube. Centrifuge at 330 x g for 5 min. Gently resuspend in Day 8 media (Table 2). Incubate for 24 h in a 37 °C 5% CO₂ incubator in low adherent dishes. Proceed to Section 4.
   NOTE: Day 8 media: SP34 media supplemented with 100 ng/mL SCF, 2 IU EPO, 10 ng/mL IL-6, 5 ng/mL IL-11, 25 ng/mL IGF-1, 30 ng/mL TPO, 10 ng/mL Flt3-L, and 30 ng/mL IL-3 (Table 2).

4. Enzymatic Dissociation of EBs and Hematopoietic Progenitor Isolation

1. Isolate the EBs with a serological pipet into a 50 mL conical tube. Centrifuge at 330 x g for 5 min. Aspirate off the supernatant.
2. Add 2 mL of 0.25% Trypsin-EDTA to the EBs. Vortex for 5 s. Incubate in a 37 °C water bath for 8 min. Add 5 mL of STOP solution. Centrifuge at 330 x g for 5 min. Aspirate off the supernatant.
3. Resuspend the EBs in 5 mL of Collagenase II. Vortex for 5 s and incubate in a 37 °C water bath. After 60 min, vortex for 5 s and add 5 mL of STOP solution. Spin at 330 x g for 5 min. Aspirate off the supernatant.
4. In 1 mL of FACS buffer, resuspend the cells. Pass the cells through a 40 µm cell strainer. This removes any undissociated cell clumps.
5. Count the cells. Aliquot 1 x 10⁶ cells into a 14 mL conical tube. Spin the cells at 330 x g for 5 min. Resuspend the cells at a concentration of 5 x 10⁶ cells/mL in FACS buffer. Aliquot and pool 10% of the cells in each condition for flow cytometric color controls. Incubate the cells in antibodies for 30 min on ice, in the dark.
   NOTE: See suggested fluorophore combinations in Table of Materials.
   1. For IWP2-treated cells, use anti-CD34 and anti-CD43 antibodies.
   2. For CHIR99021-treated cells, use anti-CD34, anti-CD43, anti-CD73, and anti-CD184 antibodies.
6. Rinse the cells twice using FACS buffer. Spin at 330 x g for 5 min. Resuspend the cells at a final concentration of 5 x 10⁶ cells/mL.
7. Analyze or isolate cells by flow cytometry. For primitive hematopoietic progenitors, analyze the CD34 and CD43 expression (Figure 2A). For definitive hematopoietic progenitors, isolate the HE (CD34⁺CD43⁻CD73⁻CD184⁻) by FACS (Figure 2B). Collect the cells in tubes containing chilled FACS buffer.
8. To assess the definitive hematopoietic potential from isolated HE, continue to either Section 5 for the endothelial-to-hematopoietic transition, or Section 7 for T-lymphoid assay. For primitive hematopoietic CFC assays, continue to Section 6.

5. Endothelial-to-hematopoietic Transition

1. Spin the isolated CD34⁺CD43⁻CD73⁻CD184⁻ cells at 330 x g for 5 min. Resuspend cells in HE media at 200,000 cells/mL (Table 2). Distribute the cells in 50 µL aliquots in a 96-well low-adherent cell culture plate. Incubate the cells overnight in a 37 °C 5% CO₂ 5% O₂ multi-gas incubator.
   NOTE: HE media: SP34 media supplemented with 5 ng/mL VEGF, 5 ng/mL bFGF, 100 ng/mL SCF, 2 IU EPO, 10 ng/mL IL-6, 5 ng/mL IL-11, 25 ng/mL IGF-1, 30 ng/mL TPO, 10 ng/mL FLT3-L, 30 ng/mL IL-3, 10 ng/mL BMP4, 20 ng/mL SHH, 10 µg/mL Angiotensin II, and 100 µM Losartan potassium at 2 x 10⁵ cells/mL (Table 2).
2. Prepare the 24-well MAT-coated plates (see step 1.2.7.1), as needed.
3. The cells will aggregate into clumps of 6 - 10 cells overnight. For each 50 µL volume of reaggregated cells, gently transfer the cells into the center of a 24-well MAT-coated plate on the next morning. Gently place plate in 37 °C multi-gas incubator for 4 - 6 h, until the cells are attached.
4. Add 1 mL of fresh HE media to each well, after the cells are attached. Incubate the cells in a 37 °C 5% CO₂ 5% O₂ multi-gas incubator until the hematopoietic cells are visible (typically 5-7 days; Figure 2D). Visualize under 100X magnification using a light microscope.
5. Harvest the definitive hematopoietic progenitors by gently removing the HE media from cells. Pass this media through a 40 µm cell strainer and collect. To the adherent cells in the well, add 0.5 mL 0.25% Trypsin-EDTA to cells and incubate at 37 °C for 5 min. Add 0.5 mL STOP solution to each well. Pass through cells through the same 40 µm cell strainer to pool all adherent and non-adherent cells together. Spin at 330 x g for 5 min.
   1. To assess the CFC potential of this bulk culture, proceed to Section 6.
6. Wash the cells twice in FACS buffer. Spin at 330 x g for 5 min.
7. Resuspend the cells in FACS buffer at 5 x 10⁶ cells/mL. Stain the cells with anti-CD45 and anti-CD34 antibodies for 30 min on ice in the dark (suggested fluorophores are in the Table of Materials).
8. Wash the cells twice in FACS buffer. Spin at 330 x g for 5 min.
9. Isolate the CD34⁺CD45⁺ cells by FACS (Figure 2E). Sort the cells into chilled FACS buffer.
10. Assess the definitive CD34⁺CD45⁺ hematopoietic progenitors as needed (CFC assay in Section 6, lymphoid potential in Section 7, or another investigator-initiated assay).

6. CFC Assay

1. Thaw aliquots of MeC for each sample used for the CFC assay.
2. Add the cells to be assayed (steps 4.5, 4.7, 5.5, or 5.9) in MeC. Vortex thoroughly and let the MeC cultures stand for 5 - 10 min until air bubbles dissipate, as per the manufacturer's instructions. Using a 16 ½ gauge needle on a 3 mL syringe, carefully remove 2 mL of the MeC.
   NOTE: Typical plating densities range from 1,000 - 20,000 cells/mL, depending on the anticipated progenitor frequency.
3. Carefully, dispense 1 mL MeC cell mixture in each of two 35 mm tissue culture dishes. Evenly distribute the MeC in the 35 mm dishes by gently tapping and rotating the dish. Place each of the above dishes into a 15 cm tissue culture dish filled with water. Incubate in a 37 °C 5% CO₂ incubator.
4. Visualize the MeC cultures using a light microscope using 40X magnification. Quantify the various CFCs obtained. Analyze the Primitive CFC assays 8 - 10 days after plating (Figure 2C). Analyze the Definitive CFC assays 14 days after plating. Representative CFC assay colony morphologies can be seen in Figure 2F.

7. T Cell Assay to Establish Definitive Hematopoietic Potential

1. Grow the OP9-DL4 cells until confluent in a 100 mm tissue culture dish^30,31,32.
   1. Thaw the OP9-DL4 cells 72 - 96 h prior to plating the T cell assays. Resuspend the OP9-DL4 cells in 10 mL OP9 media and dispense into a 10 cm plate. Grow in a 37 °C 5% CO₂ incubator until 70 - 90% confluent.
   2. To harvest the OP9-DL4 cells from a 100 mm dish, remove the media and add 5 mL 0.25% Trypsin-EDTA for 5 min. Stop the trypsin activity with 5 mL OP9 media. Spin the cells at 330 x g for 5 min. Resuspend the cells in 1 mL OP9 media and count the cells.
      NOTE: One 10 cm plate of confluent OP9-DL4 cells will yield approximately 10 x 10⁶ OP9-DL4 cells.
   3. 48 h prior to the T cell assay, plate 2 x 10⁶ cells in 12 mL OP9 media in a 24-well dish (0.5 mL/well). Grow in a 37 °C 5% CO₂ incubator.
2. Add 2,000 - 10,000 cells to be assayed (as obtained in steps 4.5, 4.7, 5.5, or 5.9) to 1 well of OP9-DL4 cells in OP9 media supplemented with: 30 ng/mL SCF (first 6 days only), 5 ng/mL IL-7, and 5 ng/mL FLT3-L. Incubate in a 37 °C 5% CO₂ incubator. No media changes occur at this step.
3. Every 4 - 5 days, passage the T cell progenitors onto fresh OP9-DL4 stromal cells in a 6-well dish. Triturate the cells with a 1 mL pipet and pass through a 40 µm filter to remove clumps. Spin at 330 x g for 5 min. Aspirate off the media. Resuspend the cells in 2 mL of fresh OP9 media supplemented with 5 ng/mL IL-7 and 5 ng/mL Flt3-L, and place on fresh OP9-DL4 cells.
   NOTE: 48 h prior to passaging, plate 2 x 10⁶ fresh OP9-DL4 cells in 12 mL OP9 media, and distribute 2 mL/well into 6-well dishes.
4. After at least 21 days of co-culture, triturate the cells vigorously and pass through a 40 µm filter to remove cell debris. Spin at 330 x g for 5 min and aspirate the supernatant.
5. Wash the cells twice in cold FACS buffer. Spin at 330 x g for 5 min.
6. Resuspend the cells in FACS buffer at 5 x 10⁶ cells/mL. Stain cells with anti-CD45, anti-CD56, anti-CD4, and anti-CD8 antibodies for 30 min on ice in the dark (suggested fluorophore combinations are in Table of Materials). Apply a live/dead cell stain (DAPI, etc.) to remove debris from the analysis.
7. Wash the cells twice in FACS buffer. Spin at 330 x g for 5 min.
8. Analyze the cells using a flow cytometer to assess the presence of T-lymphocyte progenitors (Figure 3).

Results

A schematic depicting the induction of primitive and definitive hematopoietic progenitors from hPSCs is illustrated in Figure 1. Mesoderm patterning by canonical WNT signaling occurs during days 2 - 3 of differentiation, followed by hematopoietic progenitor specification.

Representative flow cytometric analysis and colony forming methylcellulose assays of hPSC-derived hematopoietic differentiation c...

Discussion

This protocol describes a rapid, serum-free, stroma-free method for the differentiation of either primitive or definitive hematopoietic progenitors. Mesodermal specification of either primitive or definitive hematopoietic progenitors can be reliably achieved using our protocol, which uniquely exploits small molecule inhibitors of canonical WNT signaling. Stage-specific WNT activation by the GSK3β inhibitor CHIR99021³³ gives rise to definitive hematopoietic mesoderm, whereas WNT inhibition by ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Iscove's Modified Dulbecco's Medium (IMD)	Corning	10-016
Fetal Bovine Serum (FBS), ES cell rated	Gemini Bioproducts	100-500
Fetal Bovine Serum (FBS)	Hyclone	SH30396.03
L-glutamine, 200 mM solution	Life Technologies	25030-081
Penicillin-streptomycin	Life Technologies	15070-063
0.25% Trypsin-EDTA	Life Technologies	25200056
0.05% Trypsin-EDTA	Life Technologies	25300054
Gelatin, porcine skin, Type A	Sigma-Aldrich	G1890
Alpha-MEM	Life Technologies	12000-022
DMEM-F12	Corning	10-092-CV
Knock-out serum replacement	Life Technologies	10828028	"KOSR"
Non-essential amino acids (NEAA)	Life Technologies	11140050
b-mercaptoethanol, 55 mM solution	Life Technologies	21985023
Hydrochloric acid	Sigma-Aldrich	H1758
Fraction V, Bovine Serum Albumin	Fisher Scientific	BP1605
Ham's F12	Corning	10-080
N2 supplement	Life Technologies	17502048
B27 supplement, no vitamin A	Life Technologies	12587010
Stempro-34 (SP34)	Life Technologies	10639011	"SP34"
Growth factor reduced Matrigel	Corning	354230	"MAT"
L-absorbic acid	Sigma-Aldrich	A4403
Human serum transferrin	Sigma-Aldrich	10652202001
Monothioglycerol (MTG)	Sigma-Aldrich	M6145
Collagenase B	Roche	11088831001
Collagenase II	Life Technologies	17101015
DNaseI	Calbiochem	260913
Phosphate Buffered Saline (PBS)	Life Technologies	14190144
bFGF	R&D Systems	233-FB
BMP4	R&D Systems	314-BP
Activin A	R&D Systems	338-AC
VEGF	R&D Systems	293-VE
SCF	R&D Systems	255-SC
IGF-1	R&D Systems	291-G1
IL-3	R&D Systems	203-IL
IL-6	R&D Systems	206-IL
IL-7	R&D Systems	207-IL
IL-11	R&D Systems	218-1L
TPO	R&D Systems	288-TP
EPO	Peprotech	100-64
Flt-3 ligand (FLT3-L)	R&D Systems	308-FK
CHIR99021	Tocris	4423
IWP2	Tocris	3533
Angiotensin II	Sigma-Aldrich	A9525
Losartan Potassium	Tocris	3798
CD4 PerCP Cy5.5 Clone RPA-T4	BD Biosciences	560650	Dilution 1:100; T cell assay
CD8 PE Clone RPA-T8	BD Biosciences	561950	Dilution 1:10; T cell assay
CD34 APC Clone 8G12	BD Biosciences	340441	Dilution 1:100; EHT assay
CD34 PE-Cy7 Clone 8G12	BD Biosciences	348801	Dilution 1:100; Hemogenic endothelium
CD43 FITC Clone 1G10	BD Biosciences	555475	Dilution 1:10; Hemogenic endothelium
CD45 APC-Cy7 Clone 2D1	BD Biosciences	557833	Dilution 1:50; T cell assay
CD45 eFluor450 Clone 2D1	BD Biosciences	642284	Dilution 1:50; EHT assay
CD56 APC Clone B159	BD Biosciences	555518	Dilution 1:20; T cell assay
CD73 PE Clone AD2	BD Biosciences	550257	Dilution 1:50; Hemogenic endothelium
CD184 APC Clone 12G5	BD Biosciences	555976	Dilution 1:50; Hemogenic endothelium
4',6-diamidino-2-phenylindole (DAPI)	BD Biosciences	564907	Dilution 1:10,000; T cell assay
OP9 DL4 cells			Holmes, R. and J.C. Zuniga-Pflucker. Cold Spring Harb Protoc, 2009. 2009(2): p. pdb prot5156
MethoCult H4034	Stemcell Technologies	4034	"MeC"
Milli-Q water purification system	EMD Millipore
5% CO2 incubator			Set at 37 C
Multigas incubator			Set at 37 C, 5% CO2, 5% O2
6 well tissue culture plate	Corning	353046
24 well tissue culture plate	Corning	353226
6 well low-adherence tissue culture plate	Corning	3471
24 well low-adherence tissue culture plate	Corning	3473
35 mm tissue culture dishes	Corning	353001
Blunt-end needle, 16 gauge	Corning	305198
3 cc syringes	Corning	309657
5 mL polypropylene test tube	Corning	352063
5 mL polystyrene test tube	Corning	352058
15 mL polypropylene conical	Corning	430791
50 mL polypropylene conical	Corning	430921
2 mL serological pipette	Corning	357507
5 mL serological pipette	Corning	4487
10 mL serological pipette	Corning	4488
25 mL serological pipette	Corning	4489
Cell scrapers	Corning	353085
2.0 mL cryovials	Corning	430488
5 mL test tube with 40 µM cell strainer	Corning	352235
40 µM cell strainer	Corning	352340
Cell culture centrifuge
Biosafety hood
FACS AriaII or equivalent
LSRii or equivalent
FlowJo software	TreeStar
Water bath			Set at 37 C
0.22 µM filtration system	Corning
Autoclave
4 C refrigerator
-20 C Freezer
-80 C Freezer