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 ....

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 ....

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