Here, we present a protocol to precisely form hemogenic endothelium from human pluripotent stem cells in a feeder- and xeno-free condition. This method will have broad applications in disease modeling and screening for therapeutic compounds.
Deriving functional hematopoietic stem and progenitor cells (HSPCs) from human pluripotent stem cells (PSCs) have been an elusive goal for autologous transplants to treat hematological and malignant disorders. Hemogenic endothelium (HE) is an amenable platform to produce functional HSPCs by transcription factors or to induce lymphocytes in a robust manner. However, current methods to derive HE are either costly or variable in yield. In this protocol, we established a cost-effective and precise method to derive HE within 4 days in a feeder- and xeno-free defined condition. The resultant HE underwent an endothelial-to-hematopoietic transition and produced CD34+CD45+ clonogenic hematopoietic progenitors. The potential capacity of our platform for generating functional HSPCs will have broad applications in disease modeling and screening for therapeutic compounds.
The development of new therapeutics for hematological and immunological disorders has been hampered by the lack of robust platforms for disease modeling and high throughput drug screening with autologous hematopoietic stem cells (HSCs) derived from patient pluripotent stem cells (PSCs). The breakthrough of induced (i)PSC technology holds promise to model diseases from patients' cells, but the establishment of functional HSCs from patient iPSCs has been elusive. In order to derive HSCs from human PSCs, the proper induction of embryonic patterning and transcriptional programs is key1,2,3,4,5,6,7,8. Recent progress in hematopoietic development has demonstrated the role of hemogenic endothelium (HE) in HSC development9. HE can be induced from PSCs and is amenable to downstream intervention such as gene transfer10,11,12,13. Using HE as a platform, the combination of 5 transcription factors (ERG, HOXA5, HOXA9, LCOR, RUNX1) successfully induced functional HSCs that engraft long-term and differentiate into multiple lineages14. However, variable and inefficient generation of HE from PSCs have hampered the systematic manipulation and analysis of cells along with the off-the-shelf production and large-scale induction of hematopoietic cells for clinical use.
Here, we describe a cost-effective and precise method to derive HE in 4 days with a feeder- and xeno-free defined condition. In this method, spheroid formation and spontaneous re-flattening on iMarix-511 ensures a consistent density of PSC colonies, which is crucial for the efficient induction of HE cells.
1. Reagents
2. PSC Colony Formation
3. Hemogenic Induction (Day 0)
4. Isolation of Hemogenic Endothelium (Day 4)
5. Induction of Endothelial-to-hematopoietic Transition (EHT)
6. Flow Cytometry Analysis of Hematopoietic Cells
7. Colony Forming Unit (CFU) Assay of Hematopoietic Cells
A schematic of forming PSC colonies is depicted in Figure 1A. PSC spheroids are formed on a micro-fabricated plastic vessel for one day. As a representative result, 20,000 409B2 cells could be handled per 96-well of micro-fabricated plastic vessel, although other cell lines may require a higher density (30,000-40,000 cells per 96-well of micro-fabricated plastic vessel). Those spheres are spontaneously flattened to a nearly two-dimensional culture when plated onto a culture dish in the presence of LM511-E8.
A schematic of hemogenic induction is illustrated in Figure 1B. When PSC colonies grow to a diameter of 750 µm, the medium is sequentially changed to induce mesodermal organoids. The PSC colonies will gradually become a sunny side up structure during differentiation to mesodermal organoids by sequential medium change until Day 4. On Day 4, hemogenic endothelia are specified from mesodermal organoids by magnetic sorting and subsequently plated onto fibronectin in EHT medium. 5-10 million CD34+CD45– cells are expected from spheroids containing 1 million PSCs (Figure 1C).
It is observed that the cells morphologically change from endothelial to hematopoietic cells (Supplementary Video 1). A representative phase-contrast image of hematopoietic progenitor cells upon stimulation with hematopoietic cocktail on day 7 is shown in Figure 2A. Hematopoietic cell colonies emerged in the culture.
A representative flow cytometry plot is shown in Figure 2B. The whole culture is stimulated with hematopoietic cocktail and on day 7 is analyzed for the expression of CD34 and CD45 by flow cytometry. Hematopoietic progenitor cells express CD34 and CD45.
A CFU assay showed the generation of granulocytes/macrophage colonies from the CD34+ CD45+ hematopoietic progenitor cells (Figure 2C). The estimated colony number is 38 CFU-G/M per 10,000 cells (Figure 2D)
Figure 1: Schematic of tiling PSC colonies and subsequent hematopoietic differentiation via hemogenic endothelia. (A) Schematic process of forming PSC colonies. PSCs are maintained on a LM511-E8-coated 6-well plate in PSC maintenance medium. On Day -4, cells are detached and dissociated to the single-cell level using dissociating solution, subsequently plated onto micro-fabricated plastic vessel in PSC maintenance medium with Y-27632 and cultured overnight to form spheroids. On Day -3, spheroids are plated in PSC maintenance medium with LM511-E8 and cultured for three days. By Day 0, spheroids are spontaneously flattened to a nearly two-dimensional culture. Scale bars = 200 µm. (B) On Day 0, the medium is replaced with Day0 differentiation medium and cultured in the hypoxic incubator. On Day 2 of the differentiation, the medium is replaced with Day2 differentiation medium. On Day 4 of the hemogenic endothelium enrichment, CD34+ cells are magnetically sorted and cultured on a fibronectin-coated 12-well plate in EHT medium for 6 days. (C) Representative flowcytometry plot of CD45 and CD34 expression of day 4 cells sorted by CD34. Please click here to view a larger version of this figure.
Figure 2: Analysis of hematopoietic property. (A) Representative phase-contrast image of hematopoietic progenitor cells after stimulation with hematopoietic cocktail on day 7. Scale bar = 200 µm. (B) Representative flow cytometry plot of CD45 and CD34 expression of whole culture upon stimulation with hematopoietic cocktail on day 7. (C) Representative phase-contrast image of a granulocyte/macrophage colony generated from Day 11 hematopoietic progenitor cells. Scale bar = 500 µm. (D) Number of CFUs per 10,000 cells. Please click here to view a larger version of this figure.
Supplementary video 1: EHT video. Please click here to download this file.
Our feeder- and xeno-free defined induction method offers a precise and cost-effective platform to induce HE cells in a scalable manner. The faithful formation of hemogenic endothelium in conventional protocols10,11,12,13,14 have been hampered by lack of precise control of PSC colony density and size, which affects efficiency of morphogen/cytokine-based directed differentiation. We had experienced inconsistency of hemogenic endothelium induction in each independent batch from conventional protocols. The new protocol enables tight regulation of PSC colony density and size, thus overcomes the inconsistency of hemogenic endothelium induction.
We exploited the uniformed formation of spheroids and subsequently conveyed a feeder- and xeno-free defined condition to form HE in a total of 4 days. We confirmed using three independent cell lines that the spheroid formation assures the consistent formation of HE cells. As a representative result, the 409B2 cell lines yielded 12.7%-23.6% CD34+ cell efficiency based on three independent experiments. The critical factor to tile PSC spheroids is LM511-E8. We do not recommend substituting this with other matrix, such as Matrigel or full-length laminin products in our experiences. We experienced that mesodermal organoids were easily detached from Matrigel and lost during differentiation process. And neither Matrigel or full-length laminin do not anchor cells without pre-coating.
The potential limitation of this protocol is that we depend on PSC maintenance medium to maintain PSCs. We have tested other media such as StemFit, but we compromised hemogenic induction. Presumably cells were resistant to exit from pluripotent state in our short time (4 days) induction protocol. If one maintains PSCs in other media, we recommend adapting cells in PSC maintenance medium and conduct a couple of passages prior to differentiation. This platform will allow the systematic manipulation and analysis of cells, and the off-the-shelf production and large-scale induction of hematopoietic cells for potential clinical use. A long-term goal of this system is to model immunodeficiency diseases in humanized mouse models to investigate the responsible cellular and molecular mechanisms and conduct drug screenings.
The authors have nothing to disclose.
We are grateful to Alina Li and Seiko Benno for their technical assistance. We would also like to thank Ms. Harumi Watanabe for providing administrative assistance and Dr. Peter Karagiannis for editing the paper. This work was supported by the Core Center for iPS Cell Research of Research Center Network for Realization of Regenerative Medicine from the Japan Agency for Medical Research and Development (AMED) [M.K.S.], the Program for Intractable Diseases Research utilizing. Disease-specific iPS cells of AMED (17935423) [M.K.S.] and Center for Innovation program of Japan Science and Technology Agency (JST) [R.O. and M.K.S.].
0.5 M EDTA | Thermo Fisher Scientific | 15575 | 0.5M EDTA |
40 μm cell strainer | Corning | 352340 | 40μM cell strainer |
6 well plate | Corning | 353046 | 6 well plate |
Antibiotic-antimycotic | Thermo Fisher Scientific | 15240-112 | Penicillin/ Streptomycin/Amphotericin B |
anti-CD34 antibody | Beckman Coulter | A07776 | anti-CD34 antibody |
anti-CD45 antibody | Biolegend | 304012 | anti-CD45 antibody |
BMP4 | R&D Systems | 314-BP-010 | BMP4 |
CD34 microbeads | Miltenyi | 130-046-703 | CD34 microbeads |
CHIR99021 | Wako | 038-23101 | CHIR99021 |
Essential 6 | Thermo Fisher Scientific | A15165-01 | Hemogenic basal medium |
Essential 8 | Thermo Fisher Scientific | A15169-01 | Mesodermal basal medium |
Ezsphere SP Microplate 96well | AGC | 4860-900SP | micro-fabricated plastic vessel |
FCS | Biosera | FB-1365/500 | FCS |
Fibronectin | Millipore | FC010-100MG | Fibronectin |
Flt-3L | R&D Systems | 308-FK-005 | Flt-3L |
Glutamax | Thermo Fisher Scientific | 35050-061 | L-Glutamine |
IL-6/IL-6Rα | R&D Systems | 8954-SR | IL-6/IL-6Rα |
iMatrix-511 | Matrixome | 892 001 | LM511-E8 |
ITS-X | Thermo Fisher Scientific | 51500-056 | Insulin-Transferrin-Selenium-Ethanolamine |
MACS buffer | Miltenyi | 130-091-221 | magnetic separation buffer |
Methocult 4435MethoCult™ H4435 Enriched | STEMCELL TECHNOLOGIES | 04435 | Methylcellulose-based media |
mTeSR1 | STEMCELL TECHNOLOGIES | 85850 | PSC maintenance medium |
PBS | nacalai tesque | 14249-24 | PBS |
SB431542 | Wako | 031-24291 | SB431542 |
SCF | R&D Systems | 255-SC-010 | SCF |
Stemline Ⅱ Hematopoietic Stem Cell Expansion Medium | Sigma Aldrich | S0192-500ML | Hematopoietic basal medium |
TPO | R&D Systems | 288-TPN | TPO |
TrypLE Express | Thermo Fisher Scientific | 12604-021 | dissociation solution |
VEGF | R&D Systems | 293-VE-010 | VEGF |
Y-27632 | Wako | 253-00513 | Y-27632 |