Generation of Inducible CRISPRi and CRISPRa Human Stromal/Stem Cell Lines for Controlled Target Gene Transcription during Lineage Differentiation

Figure 2

Characterization of inducible CRISPRi and CRISPRa human skeletal stem cells (hMSC). (a) The morphology of control hMSC, inducible hMSC-CRISPRi, or hMSC-CRISPRa. (b) Short-term growth curve of control hMSC, hMSC-CRISPRi, or hMSC-CRISPRa. The cells were seeded in a 96-well plate and cultured in the absence or presence of doxycycline; cell viability assay was performed at different time points during 12 days in culture (D0–D12, D = day). (c–e) Control hMSC, hMSC-CRISPRi, and hMSC-CRISPRa were induced to osteogenic, adipogenic, and chondrogenic differentiation. The cells were cultured for 7 days in osteoblast induction medium (OIM) (c), 12 days in adipocyte induction medium (AIM) (d), or 18 days in chondrogenic induction medium (CIM) (e) as described in Methods. Alkaline phosphatase (ALP) activity and ALP staining (c), Oil Red O staining (d), or Alcian blue staining (e) were performed to visualize different hMSC lineage-differentiated phenotype. Data are expressed as the . Scale bar: 100 μm.

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