American Society of Hematology

Figure 6

$Figure 6. Knockdown of Mcl-1 in hPSCs shows a mechanistic conservation of Mcl-1 function as a regulator of self-renewal in human stem cells. (A) Experimental strategy used to examine whether Mcl-1 is required for survival and self-renewal in hPSC cultures. (B) Analysis of the effect of Mcl-1 knockdown on cell viability in the primitive self-renewing SSEA-3+ population of hPSC cultures. hPSC cultures were transduced with the empty vector or Mcl-1 shRNA-expressing vector and analyzed for the frequency of dead cells in the GFP+SSEA-3+ population at each passage. Error bars represent the mean ± SEM of 3 independent experiments. (C) Analysis of the effect of Mcl-1 knockdown on clonogenic self-renewal capacity in the primitive fraction of hPSC cultures. hPSC cultures were transduced with the empty vector or Mcl-1 shRNA-expressing vector and analyzed for the GFP expression in the viable primitive (SSEA-3+) fraction at each passage. The primitive clonogenic fraction is expressed as the frequency of GFP+SSEA-3+ cells per 10 000 cells. Error bars represent the mean ± SEM of 3 independent experiments. *P < .05, **P < .01. (D) Representative examples of hPSC cultures transduced with the empty vector, Mcl-1 shRNA-expressing vector, or untreated. Scale bar represents 50μM. (E) Effect of Mcl-1 knockdown on the frequency of hPSC colony-initiating cells. hPSC cultures were transduced with the empty vector or Mcl-1 shRNA-expressing vector, and the GFP+ SSEA-3+ fraction was analyzed for the frequency of hPSC colony initiating cells after the first passage. Error bars represent the mean ± SEM of 3 independent experiments. **P < .01.$

Knockdown of Mcl-1 in hPSCs shows a mechanistic conservation of Mcl-1 function as a regulator of self-renewal in human stem cells. (A) Experimental strategy used to examine whether Mcl-1 is required for survival and self-renewal in hPSC cultures. (B) Analysis of the effect of Mcl-1 knockdown on cell viability in the primitive self-renewing SSEA-3⁺ population of hPSC cultures. hPSC cultures were transduced with the empty vector or Mcl-1 shRNA-expressing vector and analyzed for the frequency of dead cells in the GFP⁺SSEA-3⁺ population at each passage. Error bars represent the mean ± SEM of 3 independent experiments. (C) Analysis of the effect of Mcl-1 knockdown on clonogenic self-renewal capacity in the primitive fraction of hPSC cultures. hPSC cultures were transduced with the empty vector or Mcl-1 shRNA-expressing vector and analyzed for the GFP expression in the viable primitive (SSEA-3⁺) fraction at each passage. The primitive clonogenic fraction is expressed as the frequency of GFP⁺SSEA-3⁺ cells per 10 000 cells. Error bars represent the mean ± SEM of 3 independent experiments. *P < .05, **P < .01. (D) Representative examples of hPSC cultures transduced with the empty vector, Mcl-1 shRNA-expressing vector, or untreated. Scale bar represents 50μM. (E) Effect of Mcl-1 knockdown on the frequency of hPSC colony-initiating cells. hPSC cultures were transduced with the empty vector or Mcl-1 shRNA-expressing vector, and the GFP⁺ SSEA-3⁺ fraction was analyzed for the frequency of hPSC colony initiating cells after the first passage. Error bars represent the mean ± SEM of 3 independent experiments. **P < .01.

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