Monocytes are essential components of the innate immune system that provide defense against pathogens or tumors due to their ability to differentiate to macrophages and dendritic cells. While monocytes can be isolated from peripheral blood, human pluripotent stem cells (hPSCs) offer another, potentially unlimited, source of monocytes. These hPSC derived monocytes can be used in disease modeling, development of cell therapy applications, and research into basic biology.
STEMdiff™ Monocyte Kit facilitates the differentiation of hPSCs to monocytes under feeder-free and serum-free culture conditions.
Differentiate hPSCs to Monocytes
STEMdiff™ Monocyte Kit is used to differentiate hPSCs to monocytes
in a three-stage protocol as shown in Figure 1. In stage 1, Medium
A differentiates hPSCs to the mesoderm lineage after 3 days of
culture. In stage 2, the cells are cultured in Medium B for 4 days,
promoting their specification to hematopoietic progenitor cells.
During stage 3, Monocyte Differentiation Medium is used to promote
the differentiation of progenitor cells to monocytes. These can be
identified by the expression of CD14, as shown in Figure 2, and can
be repeatedly harvested. STEMdiff™ Monocyte Kit is optimized for
differentiation of multiple embryonic stem (ES) and induced pluripotent
stem (iPS) cell lines maintained in TeSR™ PSC maintenance medium.
After differentiation, hPSC-derived monocytes may be used in
additional downstream assays.
Why Use STEMdiff™ for Generating Monocytes?
Generate up to 7 million CD14+ monocytes per plate in just 14 - 23 days.
Eliminate variation introduced by serum and feeder-cells by using serum- and feeder-free conditions.
Produce monocytes in a simple monolayer culture for easier harvest of suspended cells.
Achieve robust generation of monocytes across multiple ES and iPS cell lines.
Protocol for Differentiation of hPSCs to Monocytes
This protocol is designed to promote the differentiation of hPSCs to monocytes over 17 - 23 days of culture in a 2-dimensional (2D) culture
system. The three stages — mesoderm formation (stage 1), hematopoietic specification (stage 2), and monocyte differentiation (stage 3) —
are shown in Figure 1.
Figure 1. Monocyte Differentiation Protocol
One day prior to differentiation, hPSC colonies are harvested and seeded as small aggregates (100 - 200 μm in diameter) at 10 - 20 aggregates/cm2 in mTeSR™1, TeSR™-E8™,
or mTeSR™ Plus. After one day, the medium is replaced with Medium A (STEMdiff™ Hematopoietic Basal Medium + Supplement A) to induce mesodermal specification
(stage 1). On day 3, the medium is changed to Medium B (STEMdiff™ Hematopoietic Basal Medium + Supplement B) to promote hematopoietic specification (stage 2).
On day 7, the medium is replaced with Monocyte Differentiation Medium (StemSpan™ SFEM II + STEMdiff™ Monocyte Differentiation Supplement) to promote the production
of CD14+ monocytes (stage 3). Monocyte Differentiation Medium is used for all medium changes for the remaining culture period. CD14+ cells can be detected in suspension
starting after day 14, and their frequency gradually increases until day 17 - 23. CD14+ cells can be harvested directly from the culture supernatant during medium changes.
Figure 2. Robust and Efficient Generation of CD14+ Monocytes Using STEMdiff™ Monocyte Kit
hPSCs were differentiated to monocytes using the 2D culture system described
in Figure 1. Between days 17 and 23, cells were harvested every 2 - 3 days and
analyzed by flow cytometry for CD14 expression. Representative flow cytometry
plots are shown for (A,B) iPS (WLS-1C)-derived cells and (C,D) ES (H9)-derived
cells. (E) The average frequency of viable CD14+ monocytes at the peak harvest
was 61 - 78%. The average yield of CD14+ monocytes produced per 6-well plate
at the peak harvest was between 1.6 x 106 and 7.1 x 106 cells. Data are shown
as mean ± SEM (n = 3 - 14).
Figure 3. STEMdiff™ Monocyte Kit Generates Monocytes That Are Capable of Differentiation to Macrophages
hPSC-derived monocytes were harvested after 21 days of culture. These were then differentiated to macrophages using ImmunoCult™-SF Macrophage Medium with
100 ng/mL M-CSF for 4 days. Macrophages were then incubated for an additional 2 days with either 10 ng/mL of LPS and 50 ng/mL of IFN-γ, or 10 ng/mL IL-4, to become
polarized to M1 or M2a macrophages, respectively. Representative flow cytometry plots of (A) M1 and (B) M2a macrophages produced from the WLS-1C iPS cell line are
shown. (C) To measure phagocytosis, hPSC-derived M2a macrophages and peripheral blood (PB) monocyte-derived M2a macrophages (primary M2a macrophages),
were incubated with pHrodo™ Red Zymosan A BioParticles® Conjugate and incubated at 37°C for 8 hours. Images were acquired using the IncuCyte® ZOOM every
30 minutes and analyzed for internalization of pHrodo™ Red Zymosan A BioParticles® (measured as red object/mm2). hPSC-derived and primary M2a macrophages show
similar phagocytic activity.
Figure 4. STEMdiff™ Monocyte Kit Generates Monocytes That Can Be Differentiated to Dendritic Cells
hPSCs were differentiated to monocytes, harvested after 21 days, and differentiated
to dendritic cells using ImmunoCult™ Dendritic Cell Culture Kit. Half of the dendritic
cells were harvested on day 7 and examined for CD14 and CD83 expression to
identify CD14−CD83−/lo immature dendritic cells. The remaining dendritic cells were
activated for 2 days and assessed for the presence of CD14−CD83+ mature dendritic
cells at day 7. Representative cultures initiated with ES (H9) cells are shown for
production of (A) immature dendritic cells and (B) mature dendritic cells.
Generation of Monocytes from Human Pluripotent Stem Cells Using STEMdiff™ Medium and Supplements
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