Stem cell Differentiation media Differentiation of Human iPSCs into microglia differentiation

Stem cells have the unique ability to self-renew or differentiate themselves into various cell types in response to appropriate signals. These cells are especially important for tissue repair, regeneration, replacement, or in the case of hematopoietic stem cells (HSCs) to differentiate into various myeloid populations. Appropriate signals refer to the growth factor supplements or cytokines that mediate differentiation of various stem cells into the required differentiated form. For instance, HSCs can be differentiated into dendritic cells (with IL-4 and GM-CSF), macrophages (with m-CSF) and MDSCs (with IL-6 and GM-CSF). Human pluripotent stem cells (hPSCs) and induced pluripotent stem cells (iPSCs) can be first cultured in neural differentiation media (GSK3𝛃-i, TGF𝛃-i, AMPK-i, hLIF) to form neural rosettes, which can be differentiated into neural or glial progenitors (finally differentiated into oligodendrocytes). Neural progenitors can be finally differentiated into glutaminergic (dibytyryl cAMP, ascorbic acid) and dopaminergic (SHH, FGF-8, BDNF, GDNF, TGF-𝛃3) neurons. Thus, it is important to first identify the self-renewing cell line: its source and its final differentiation state, followed by the supplements and cytokines required for the differentiation, and final use. Timelines are another thing that is considered. For instance, it takes 7-10 days to form neural rosettes from iPSCs and 3 days to differentiate neural progenitors to neurons. Finally, the stability for stem cell culture media varies. It is advised to make fresh media every time when differentiating HSCs to myeloid populations, whereas neural differentiation media may remain stable for two weeks when stored in dark between 2-8C.

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STEMdiff™ Hematopoietic Kit

STEMCELL technologies

Upstream tips
- Throughout the protocol, warm all media to room temperature (15 - 25°C) before use. Do not leave media at room temperature for extended periods of time.
Protocol tips
-Thaw STEMdiff™ Hematopoietic Basal Medium at room temperature (15 - 25°C) or overnight at 2 - 8°C. Mix thoroughly.
-Thaw Supplement A or B at room temperature (15 - 25°C) or at 2 - 8°C until just thawed. Mix thoroughly. If necessary, centrifuge for 30 seconds to remove liquid from cap.
-If it is difficult to anticipate the correct number of cells to plate (e.g. when testing a new hPSC line), use two or more numbers that differ by 2- to 3-fold (e.g. 5000 cells per dish and 10,000 cells per dish).
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