CRISPR Gene Editing in Stem Cells

Consistent with this, detailed phenotypic characterization,

based on the phenotypic assays defined in the iPSC panel

above, confirmed a phenotype corresponding to a preleukemic

stage: correction of the emergence of CD45

cells, loss of

CD90 expression, re-emergence of a CD41a

/CD45 megakar-

yocyte progenitor population, partial rescue of clonogenicity,

and restored growth and viability (Figures 6B–6F).

Conversely, to model disease progression, we first started

with the preleukemic N-3.10 line, harboring a germline

GATA2

mutation.

GATA2

mutations found in patients with familial pre-

disposition syndromes are believed to be loss-of-function muta-

tions (Collin et al., 2015). Since the MDS clone of the same

patient from whom this line was derived (patient 3; Figure 1)

had acquired a second

GATA2

mutation in the other allele (Fig-

ure S1A) together with additional recurrent genetic abnormal-

ities, we sought to model the effects of inactivating the other

GATA2

allele in the disease phenotype. We designed two

distinct CRISPR/Cas9-based strategies and isolated two clones

Figure 3. iPSCs from Different Disease Stages Capture Stage-Specific Disease Phenotypes

(A) Methylcellulose assays on day 14 of hematopoietic differentiation. The number of colonies from 5,000 seeded cells is shown. (CFU-GEMM, colony-forming

unit-granulocyte, erythrocyte, monocyte, megakaryocyte; CFU-GM: CFU-granulocyte, monocyte; CFU-G: colony-forming unit-granulocyte; CFU-M: CFU-

monocyte; BFU-E, burst-forming unit-erythrocyte). Average of three to six independent experiments is shown for each line.

(B) Analysis of lineage markers (top) and morphologic assessment of cells generated in methylcellulose cultures. One iPSC line representative of each disease

stage is shown (from left to right: N-2.12, N-3.10, MDS-1.12, AML-4.16). High-risk MDS iPSCs do not give rise to colonies in methylcellulose and are therefore not

represented in this panel. Dysplastic changes are observed in preleukemic (arrows point to nuclear blebbing, whereas arrowheads point to pseudo Pelger-Huet

cells) and low-risk MDS cells (arrows point to hyper-segmented neutrophils, whereas arrowheads point to pseudo Pelger-Huet cells). Atypical monomorphic

myeloid cells (arrows) are the predominant cells observed in methylcellulose cultures from MDS/AML cells. Scale bars, 10

(C) Growth competition assay. The cells were mixed 1:1 with the N-2.12 line stably expressing GFP at the beginning of hematopoietic differentiation and followed

for 12 days by flow cytometry (schematic shown in Figure S5F). The relative population size was calculated as the percentage of GFP cells at each time point

relative to the population size at day 2. For lines differentiated more than once, the average value is shown.

(D) Cell viability measured by DAPI staining on day 14 of hematopoietic differentiation (extended data are shown in Figure S5E). Mean and SEM of different lines

are shown. For lines differentiated more than once (Figure S5E), the average value is shown.