Applied StemCell, Inc. provides Control Human Induced Pluripotent Stem (iPS) cells at low passages (p16). These pluripotent cells were generated from normal human skin fibroblasts using episomal reprogramming methods. This method allows the transient expression of human transcription factors (OCT4, SOX2, KLF4, and c-MYC) that initiate the reprogramming process. The resulting human iPS cells (hiPSCs) were selected using morphological criteria without the use of either fluorescent markers or drug selection. These iPS cells have been tested for the expression of the pluripotency markers, including OCT4, SOX2, SSEA4, TRA-1-60, TRA-1-81, and alkaline phosphatase (AP) activity and normal female karyotype. The ASE-9209 control human iPSC line can be used for CRISPR/Cas9 genome editing and differentiation to somatic lineages in vitro. Detailed protocols for thawing, culturing under both feeder and feeder-free conditions, and cryopreservation of these iPS cells are provided.
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Karyotype analysis to rule out genetic aberrations. Cytogenic analysis was performed on G-banded metaphase cells from human iPSC line, ASE-9209. This iPSC line demonstrates a normal female karyotype.
Pluripotency Marker Analysis
Expression of pluripotency markers. ASC 9209 iPS cell line expresses common iPSC biomarkers (OCT4, SOX2, SSEA4, TRA-1-60, and TRA-1-81). The corresponding DAPI staining is below each marker staining image. All images were taken at 10x magnification.
Differentiation to Motor Neurons
Immunocytochemical staining for motor neuron marker, ChAT (green) and neuronal marker, MAP2 (blue) motor neurons differentiated from control iPSC line, ASE-9209 at 7 days post-thaw. DAPI (blue) was used for nucleus staining.
CRISPR Genome Editing in ASE-9209
Next generation sequencing of pooled clones from control iPSC ASE-9209 transfected with gRNA targeting a gene-of-interest (highlighted in orange) and Cas9 shows indel formation, indicating gRNA-mediated cleavage in this region. WT (Ref Seq): wild type reference sequence.
Teratoma Formation: Tri-Lineage Differentiation
Histological analyses using H&E staining of kidney and testis teratomas from mice injected with the ASE-9209 iPSC line show spontaneous differentiation of ASE-9209 to three germ layers (selected images, more available upon request). Abbreviations: EN: endoderm; ME: mesoderm; EC: ectoderm.