pluripotency

human  |  mouse

maintenance of pluripotency in human and mouse embryonic and induced pluripotent stem cells

human pluripotent stem cells

In the search for conditions that maintain pluripotency in human pluripotency stem cells, a large number of media formulations have been described (the composition of most popular of which are shown in table 1), in general each of which targets 3 main signalling pathways.1

  • Activation of the PI3K/AKT/mTOR and MAPK/ERK pathways by FGF2 and/or neuregulin 1 (NRG1), which bind FGFR1/FGFR4 or ERBB3/ERBB4
  • Activation of the TGF-β signaling pathway by TGF-β1, NODAL, or Activin A, which bind TGFBR1/2 and/or ACVR2A/2B/1B/1C (NODAL is used less commonly in pluripotent medium formulations due to the expression of the NODAL antagonists LEFTY1/2 in hPSCs)
  • Activation of the PI3K/AKT pathway, promoting cell survival and growth, by insulin or insulin growth factor (IGF1), which bind INSR and IGF1R

table 1. the composition of four of the most popular human pluripotency medias

Components Recipe
mTeSR1 DMEM/F12, BSA, FGF2, TGFβ1, insulin, transferrin, cholesterol, lipids, pipecolic acid, GABA, b-mercaptoethanol Ludwig et al 20062
E8 DMEM/F12, FGF2, TGFβ1, insulin, transferrin, selenium, ascorbic acid Beers et al 20123
B8 DMEM/F12, FGF2-STAB® (FGF2-G3), TGFβ3, NRG1 (Heregulin-β1), insulin, ascorbic acid, transferrin, sodium selenite, sodium bicarbonate Lyra-Leite et al 20214
StemPro DMEM/F12, BSA, FGF2, TGFβ1, Activin A, IGF-1 LR3, NRG1 (Heregulin-β1), transferrin, Wang et al 20075

mouse pluripotent stem cells

LIF is often regarded as the most important growth factor involved in mouse pluripotent stem cell maintenance. LIF, part of the IL6 family, activates a number of pro-pluripotency signalling pathways including Jak-Stat3, PI3K-Akt, and YES-YAP. The JAK/STAT pathway regulates the core pluripotency regulatory circuit Oct4/Sox2/Nanog through activation of Klf4. PI3K-Akt blocks MAPK-Erk signalling, which is a driver of endoderm differentiation, and increases Tbx3 activity and Nanog expression. While the YES-YAP pathway induces Oct4 expression.6

However, LIF also activates the MAPK-Erk signalling pathway, which promotes differentiation. To prevent this differentiation, two methods can be employed; inhibition of ERK using inhibitors such as PD0325901, this is the method employed in the popular 2i+LIF conditions, or activation of BMP signalling.  BMP4 inhibits MAPK and ERK signalling and regulates pluripotency gene expression through SMADs.6

The third cytokine family shown to play a role in mouse pluripotency is the Wnt family. Activation of Wnt signalling stimulates self-renewal by inhibiting the repressor activity of endogenously expressed Tcf3. In the 2i+LIF conditions, inhibition of GSK3 activity (by CHIR99021) takes the place of Wnt signalling. Wnt3a has been shown to be capable of replacing GSK3 inhibition in mESC culture and has been shown to sensitise cells to LIF.7

Qkine proteins

references

  1. Kuo, Hui-Hsuan, et al. ‘Negligible-Cost and Weekend-Free Chemically Defined Human IPSC Culture’. Stem Cell Reports, vol. 14, no. 2, Feb. 2020, pp. 256–70.
  2. Ludwig, Tenneille E., et al. ‘Feeder-Independent Culture of Human Embryonic Stem Cells’. Nature Methods, vol. 3, no. 8, Aug. 2006, pp. 637–46.
  3. Beers, Jeanette, et al. ‘Passaging and Colony Expansion of Human Pluripotent Stem Cells by Enzyme-Free Dissociation in Chemically Defined Culture Conditions’. Nature Protocols, vol. 7, no. 11, 2012, pp. 2029–40.
  4. Lyra-Leite, Davi Marco, et al. ‘An Updated Protocol for the Cost-Effective and Weekend-Free Culture of Human Induced Pluripotent Stem Cells’. STAR Protocols, vol. 2, no. 1, Mar. 2021, p. 100213.
  5. Wang, Linlin, et al. ‘Self-Renewal of Human Embryonic Stem Cells Requires Insulin-like Growth Factor-1 Receptor and ERBB2 Receptor Signaling’. Blood, vol. 110, no. 12, Dec. 2007, pp. 4111–19.
  6. Chen, Chen-Yun, et al. ‘Mechanisms of Pluripotency Maintenance in Mouse Embryonic Stem Cells’. Cellular and Molecular Life Sciences, vol. 74, no. 10, May 2017, pp. 1805–17.
  7. Merrill, B. J. ‘Wnt Pathway Regulation of Embryonic Stem Cell Self-Renewal’. Cold Spring Harbor Perspectives in Biology, vol. 4, no. 9, Sept. 2012, pp. a007971–a007971.