STEMdiff™ Hematopoietic Kit

For differentiation of human ES or iPS cells into hematopoietic progenitor cells

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STEMdiff™ Hematopoietic Kit

For differentiation of human PSCs into the hematopoietic lineage

1 Kit
Catalog #05310
593 USD

Overview

STEMdiff™ Hematopoietic Kit includes a serum-free basal medium and supplements for the feeder-free differentiation of human embryonic stem (ES) and induced pluripotent stem (iPS) cells into hematopoietic progenitor cells expressing CD34, CD45, and CD43.
The simple, 12-day differentiation protocol is performed in two stages. During the first 3 days, STEMdiff™ Hematopoietic Supplement A is added to the basal medium to induce cells toward mesoderm. For the subsequent 9 days, mesodermal cells are further differentiated into hematopoietic progenitor cells using basal medium supplemented with STEMdiff™ Hematopoietic Supplement B. At the end of the 12-day protocol, hematopoietic cells can be easily harvested from the culture supernatant. This population typically contains 25 - 65% (average 43%) CD34+CD45+ progenitor cells, including progenitor cells that have the capacity to form hematopoietic colonies in the colony-forming unit (CFU) assay.
STEMdiff™ Hematopoietic Kit has been optimized for differentiation of cells maintained in mTeSR™1 (Catalog #85850) or TeSR™-E8™ (Catalog #05990).
Components:
• STEMdiff™ Hematopoietic Basal Medium, 120 mL
• STEMdiff™ Hematopoietic Supplement A (200X), 225 µL
• STEMdiff™ Hematopoietic Supplement B (200X), 375 µL
 
Subtype:
Specialized Media
Cell Type:
Hematopoietic Stem and Progenitor Cells; Pluripotent Stem Cells
Species:
Human
Application:
Cell Culture; Differentiation
Brand:
STEMdiff
Area of Interest:
Stem Cell Biology
Formulation:
Serum-Free

Scientific Resources

Educational Materials

(8)

Product Applications

This product is designed for use in the following research area(s) as part of the highlighted workflow stage(s). Explore these workflows to learn more about the other products we offer to support each research area.

Data and Publications

Data

Hematopoietic Cell Differentiation Protocol

Figure 1. Hematopoietic Differentiation Protocol

One day before the differentiation protocol, hPSC colonies are harvested and seeded as small aggregates (100 - 200 μm in diameter) at 10 - 20 aggregates/cm2 in mTeSR™1 or TeSR™-E8™. After one day, TeSR™ medium is replaced with Medium A (STEMdiff™ Hematopoietic Basal Medium containing Supplement A) to begin inducing the cells towards a mesoderm-like state (day 0). On day 2, a half medium change is performed with fresh Medium A. On day 3, the medium is changed to Medium B (STEMdiff™ Hematopoietic Basal Medium containing Supplement B) with half medium changes on days 5, 7 and 10, to promote further differentiation into hematopoietic cells. Typically, by day 12, large numbers of HPCs can be harvested from the culture supernatant.

Morphology of hPSC-Derived HPCs

Figure 2. Morphology of hPSC-Derived HPCs

Representative images of (A) hES (H1) cells and (B) hiPS (WLS-1C) cells on day 12 of differentiation to HPCs using the STEMdiff™ Hematopoietic Kit. Differentiated cells exhibit typical HPC morphology as round cells that float freely in suspension.

Efficient and Robust Generation of CD34+CD45+/CD43+ HPCs
Efficient and Robust Generation of CD34+CD45+/CD43+ HPCs
Efficient and Robust Generation of CD34+CD45+/CD43+ HPCs

Figure 3. Efficient and Robust Generation of CD34+CD45+/CD43+ HPCs

hES and hiPS cells were cultured for 12 days in single wells of 12-well plates using the STEMdiff™ Hematopoietic Kit. At the end of the culture period, cells in suspension were harvested and analyzed by flow cytometry for expression of hematopoietic cell surface markers: CD34, CD45 and CD43. (A,B) Example flow cytometry plots for hematopoietic cell surface-marker analysis of cultures of hES (H1 and H9) and hiPS (STiPS-M001) cells. (C,D) Percentages and total numbers of CD34+CD45+ cells in cultures of hES (H1 and H9) or hiPS (WLS-1C, STiPS-F016, STiPS-M001 and STiPS-B004) cells are shown. Data shown as mean ± SEM; n ≥ 3.

hPSC-Derived HPCs Produce Colonies of Multiple Lineages
hPSC-Derived HPCs Produce Colonies of Multiple Lineages
hPSC-Derived HPCs Produce Colonies of Multiple Lineages

Figure 4. hPSC-Derived HPCs Produce Colonies of Multiple Lineages

Cells in suspension were harvested from the cultures on day 12 of the hematopoietic differentiation protocol and assessed in colony-forming unit (CFU) assays using MethoCult™ H4435 Enriched (Catalog #04435) methylcellulose-based medium. (A) CFU frequencies in cultures of 6 different hPSC lines. (Data shown as mean ± SEM; n ≥ 3.) The CFU frequencies were variable between the different cell lines, with on average approximately 120 CFU per 10,000 hPSC-derived HPCs plated. (B) The progenitor cell types observed included granulocyte/macrophage (CFU-M, CFU-G and CFU-GM), erythroid (BFU-E and CFU-E) and occasional mixed (CFU-GEMM) colonies. Representative colony images are shown at 40X magnification.

Publications

(1)
Molecular neurodegeneration 2018 DEC

Development and validation of a simplified method to generate human microglia from pluripotent stem cells.

A. McQuade et al.

Abstract

BACKGROUND Microglia, the principle immune cells of the brain, play important roles in neuronal development, homeostatic function and neurodegenerative disease. Recent genetic studies have further highlighted the importance of microglia in neurodegeneration with the identification of disease risk polymorphisms in many microglial genes. To better understand the role of these genes in microglial biology and disease, we, and others, have developed methods to differentiate microglia from human induced pluripotent stem cells (iPSCs). While the development of these methods has begun to enable important new studies of microglial biology, labs with little prior stem cell experience have sometimes found it challenging to adopt these complex protocols. Therefore, we have now developed a greatly simplified approach to generate large numbers of highly pure human microglia. RESULTS iPSCs are first differentiated toward a mesodermal, hematopoietic lineage using commercially available media. Highly pure populations of non-adherent CD43+ hematopoietic progenitors are then simply transferred to media that includes three key cytokines (M-CSF, IL-34, and TGF$\beta$-1) that promote differentiation of homeostatic microglia. This updated approach avoids the prior requirement for hypoxic incubation, complex media formulation, FACS sorting, or co-culture, thereby significantly simplifying human microglial generation. To confirm that the resulting cells are equivalent to previously developed iPSC-microglia, we performed RNA-sequencing, functional testing, and transplantation studies. Our findings reveal that microglia generated via this simplified method are virtually identical to iPS-microglia produced via our previously published approach. To also determine whether a small molecule activator of TGF$\beta$ signaling (IDE1) can be used to replace recombinant TGF$\beta$1, further reducing costs, we examined growth kinetics and the transcriptome of cells differentiated with IDE1. These data demonstrate that a microglial cell can indeed be produced using this alternative approach, although transcriptional differences do occur that should be considered. CONCLUSION We anticipate that this new and greatly simplified protocol will enable many interested labs, including those with little prior stem cell or flow cytometry experience, to generate and study human iPS-microglia. By combining this method with other advances such as CRISPR-gene editing and xenotransplantation, the field will continue to improve our understanding of microglial biology and their important roles in human development, homeostasis, and disease.
STEMCELL TECHNOLOGIES INC.’S QUALITY MANAGEMENT SYSTEM IS CERTIFIED TO ISO 13485. PRODUCTS ARE FOR RESEARCH USE ONLY AND NOT INTENDED FOR HUMAN OR ANIMAL DIAGNOSTIC OR THERAPEUTIC USES UNLESS OTHERWISE STATED.