STEMdiff™ Erythroid Kit

For differentiation of human ES or iPS cells to erythroblasts
STEMdiff™ Erythroid Kit

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

1 Kit
Catalog # 100-0074
1,290 EUR
Required Products
  1. 12-Well Flat-Bottom Plate, TC-Treated
    12-Well Flat-Bottom Plate, Tissue Culture-Treated

    Sterile, clear polystyrene, flat-bottom, tissue culture-treated multiwell plate with lid

  2. mTeSR™ Plus|100-0276
    mTeSR™ Plus

    cGMP, stabilized feeder-free maintenance medium for human ES and iPS cells

Overview

STEMdiff™ Erythroid Kit is designed for the serum-free and feeder-free differentiation of human embryonic stem (ES) and induced pluripotent stem (iPS) cells to erythroid progenitor cells (erythroblasts) expressing Glycophorin A and CD71. This simple 2D differentiation protocol is performed in two stages. During the first 10- to 12-day stage, STEMdiff™ Hematopoietic Supplement A and then Supplement E1 are added to basal medium to induce cells toward erythroid-biased hematopoietic progenitor cells. At the end of this stage, hematopoietic progenitor cells are easily harvested from the culture supernatant. During the second 14-day stage, cells are further differentiated to erythroid progenitor cells using Supplement E2 and StemSpan™ SFEM II (Day 10 - 24). The cells typically expand 241-fold +/- 190-fold (95% CI) during the 14-day erythroid differentiation stage and the Day 24 cell population typically contains 74% +/- 5% (95 CI) CD71+GlyA+ erythroblasts. Cells generated using STEMdiff™ Erythroid Kit can be further matured into normoblasts and reticulocytes when moved to appropriate culture conditions for maturation.

STEMdiff™ Erythroid Kit has been optimized for differentiation of cells maintained in mTeSR™1 (Catalog #85850), mTeSR™ Plus (Catalog #100-0276), and TeSR™-E8™ (Catalog #05990).
Advantages
⦁ Serum-free and feeder-free formulation
⦁ Robust generation of erythroblasts across multiple ES and iPS cell lines
⦁ Human iPSC/ESC-derived erythroblasts can be matured further
Components
  • STEMdiff™ Hematopoietic Basal Medium, 120 mL
  • STEMdiff™ Hematopoietic Supplement A, 225 μL
  • STEMdiff™ Erythroid Supplement E1 (10X), 10 mL
  • STEMdiff™ Erythroid Supplement E2 (10X), 20 mL
  • StemSpan™ SFEM II, 100 mL
Subtype
Specialized Media
Cell Type
Erythroid Cells, Hematopoietic Stem and Progenitor Cells
Species
Human
Application
Cell Culture, Differentiation, Expansion
Brand
STEMdiff
Area of Interest
Disease Modeling, Drug Discovery and Toxicity Testing, Stem Cell Biology, Transplantation Research
Formulation
Serum-Free

Scientific Resources

Product Documentation

Document Type Product Name Catalog # Lot # Language
Document Type
Product Information Sheet
Product Name
STEMdiff™ Erythroid Kit
Catalog #
100-0074
Lot #
All
Language
English
Document Type
Safety Data Sheet 1
Product Name
STEMdiff™ Erythroid Kit
Catalog #
100-0074
Lot #
All
Language
English
Document Type
Safety Data Sheet 2
Product Name
STEMdiff™ Erythroid Kit
Catalog #
100-0074
Lot #
All
Language
English
Document Type
Safety Data Sheet 3
Product Name
STEMdiff™ Erythroid Kit
Catalog #
100-0074
Lot #
All
Language
English
Document Type
Safety Data Sheet 4
Product Name
STEMdiff™ Erythroid Kit
Catalog #
100-0074
Lot #
All
Language
English
Document Type
Safety Data Sheet 5
Product Name
STEMdiff™ Erythroid Kit
Catalog #
100-0074
Lot #
All
Language
English

Educational Materials (9)

Brochure
Hematopoietic Stem and Progenitor Cells - Products for Your Research
Brochure
Products for Human Pluripotent Stem Cells
Technical Bulletin
Generation of Erythroblasts from Pluripotent Stem Cells Using STEMdiff™ Media and Supplements
Wallchart
Derivation and Applications of Human Pluripotent Stem Cells
Wallchart
Identification of Colonies Derived from Human Hematopoietic Progenitors
Wallchart
Human Hematopoietic Stem and Progenitor Cell Phenotyping
Webinar
Serum- and Feeder-Free Differentiation of Erythroid Progenitor Cells from hPSCs
31:39
Serum- and Feeder-Free Differentiation of Erythroid Progenitor Cells from hPSCs
Webinar
A Guide to Erythropoiesis - EPO Signaling and Erythroid Development
40:56
A Guide to Erythropoiesis - EPO Signaling and Erythroid Development
Scientific Poster
A Simple, Reproducible Method to Generate Red Blood Cells From Human Pluripotent Stem Cells

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

Figure showing the protocol for differentiating human pluripotent stem cells to erythroid cells using STEMdiff™ Erythroid Kit

Figure 1. Erythroid Differentiation Protocol

The protocol involves two main steps: (A) hematopoietic specification of human embryonic stem (hES) or induced pluripotent stem (hiPS) cells and (B) differentiation of hES or hiPS cell-derived HPCs into erythroid cells. One day prior to differentiation, hES or hiPS cells were plated as small aggregates (100 - 200 µm diameter) at a density of 15 - 20 aggregates/cm2 in mTeSR™1, mTeSR™ Plus, or TeSR™-E8™ medium on Corning® Matrigel®-coated plates. After attaching overnight, mesoderm differentiation was initiated by changing the medium to Medium A (STEMdiff™ Hematopoietic Basal Medium + STEMdiff™ Hematopoietic Supplement A). On day 3, the medium was changed to Medium E1 (STEMdiff™ Hematopoietic Basal Medium + STEMdiff™ Erythroid Supplement E1) to induce hematoendothelial and hematopoietic specification. On day 10, hematopoietic progenitor cells (HPCs) in suspension were harvested from the culture and counted. Following this, HPCs were replated in Medium E2 (StemSpan™ SFEM II + STEMdiff™ Erythroid Supplement E2) at a density of 4 x 10 4 cells/mL and cultured for 14 days to generate erythroblasts.

Figure showing generation of CD71+GlyA+ erythroid cells from pluripotent stem cells using STEMdiff™ Erythroid Kit

Figure 2. Robust and Efficient Generation of CD71+GlyA+ Erythroids Using STEMdiff™ Erythroid Kit

hES and hiPS cells were induced to differentiate into CD71+GlyA+ cells using the 24-day protocol shown in Figure 1. Following the initial 10-day culture period and at the end of the 24-day protocol, cells were harvested from the supernatant and analyzed by flow cytometry. Dead cells were excluded by light scatter profile and propidium iodide staining. (A) Representative flow cytometry plot for iPSC-derived (1C) cells analyzed on day 10. Cells were identified as CD34+CD43+ hematopoietic progenitors. (B) After 24 days of culture, cells were identified as having differentiated into CD71+ GlyA+ erythroblasts. The erythroid identity of the generated cells was additionally assessed by cell morphology and hemoglobin expression (see Figure 4).

Figure showing average cell numbers of erythroid cells generated from pluripotent stem cells using STEMdiff™ Erythroid Kit

Figure 3. Production of Erythroid Cells from hES and hiPS cells After 24 Days of Culture in the STEMdiff™ Erythroid Kit

(A) Shown are the average numbers of viable, GlyA+ erythroid cells generated per input cell after culturing hES and hiPS cells for 24 days using the STEMdiff™ Erythroid Kit. (B) The overall frequency of GlyA+ erythroid cells present in culture after 24 days is shown as a percentage of total nucleated cells present. Data are presented as mean and SEM (n = 5 - 21) for six individual hES and hiPS cell lines.

Figures showing erythroid cells generated using STEMdiff™ Erythroid Kit are hemoglobinized and display typical morphology

Figure 4. hES and hiPS cells-Derived Erythroid Cells Are Hemoglobinized and Display Typical Erythroid Morphology

(A) Erythroid cells generated with the STEMdiff™ Erythroid Kit express a mix of primitive (embryonic) and definitive (fetal, adult) hemoglobin. Shown are the results of qPCR analysis for globin gene expression after 24 days of culture. (B) A picture of the cell pellet shows that cells produced in culture are hemoglobinized. (C) Cells display typical basophilic erythroblast morphology after 24 days of culture using the STEMdiff™ Erythroid Kit (40X magnification, May-Grunwald Giemsa stain).

Figures showing erythroid cells generated using STEMdiff™ Erythroid Kit can be further matured into normoblasts

Figure 5. hES and hiPS Cell-Derived Erythroid Cells Can Mature into CD71low/GlyA+ Normoblasts

hES and hiPS cell-derived erythroblasts can mature further when cultured for an additional 7 days in maturation conditions (StemSpan™ SFEM II medium with human serum (3%) and EPO (3 U/mL)). (A) Further maturation of the erythroid cells results in smaller cells with condensed nuclei, which is typical for orthochromatic normoblasts (40X magnification, May-Grunwald Giemsa stain). (B) A representative flow cytometry plot shows that over 90% of the cells are GlyA+ and majority of cells have decreased CD71 expression, which is consistent with erythroid maturation. (C) Bar graphs summarize the average frequencies of GlyA+ erythroid cells on day 31 across all 6 hiPS cell lines. (D) Notably, fold expansion data shows that cell numbers are maintained during the maturation culture. Data are presented as mean and SEM (n=2-6).

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