Fig 2: Single-cell sequencing of MKPs allows the selection of further candidate markers.(A) UMAP of 10x Genomics scRNA-seq of D27 A1ATD1 MKs sorted for the top 50% MICB, top 26% PVR, and bottom 33% CD42 expression. (B and C) UMAP of single-cell transform–normalized MICB and PVR expression. (D to F) UMAP of un-normalized GATA1, TAL1, and FLI1 expression from lentiviral transgenes plotted on the gene expression UMAP. (G to J) UMAP of single-cell transform–normalized ANXA1, HMMR, ITGAV, and FLT1 expression.

Fig 3: Identification of a panel of surface markers that can further purify MICB+PVR+ MKPs.(A) Twenty index sorts of A1ATD1 MKs. Rank indicates the first occurrence of a marker in a depth-first search of the decision trees making up the bagging model; genes with consistently low rank are discriminative for a given replicate. (B) Determining cutoffs for MKP markers based on all index sorts of A1ATD1 MKs. Outliers were excluded, and the 20 datasets were normalized and combined. The resulting dataset was then divided into 60 bins based on PVR expression and the colonies and noncolonies counted in each bin. This allowed the calculation of (1 - proportion colony formation per bin) (y axis). Cells were then ordered with decreasing PVR expression, and the proportion of cells with higher than this value of PVR is plotted on the x axis. The dataset was then subsetted on this expression level of PVR (top 20%), and the analysis was repeated for MICB, CD51, CD168, and VEGFR1. Areas with maximum colony formation are shown with shaded boxes. (C) Final sort strategy for MKPs. Cells are first purified as MICBhiPVRhi, then as CD51hiCD168hi, and lastly as VEGFR1lo.

Fig 4: Identifying MKP surface markers by scRNA-seq.(A) UMAP of SMART-Seq scRNA-seq of a D44 A1ATD1 culture. Insert: Density clustering decision plot. (B) Normalized putative marker expression per cluster in (A). (C) Flow cytometry analysis of MICB, PVR, CD41, and CD42 expression during FFDK and QOLG3 MK differentiation. (D) qPCR analysis of MICB and PVR transcript levels during A1ATD1 MK differentiation. (E) Index sort schematic. (F) Index sort results: A1ATD1 MKs. Expression in cells that did not (N) and did form a colony (Y). CD41 (n = 7), CD42 (n = 7), MICB (n = 6), and PVR (n = 8). (G) Sorting scheme. (H to J) Colony counts (H), bright-field microscopy (I), and flow cytometry analysis (J) on D12 after D75 A1ATD1 MKs were sorted for MICB or PVR. (K) Sorting scheme. (L and M) Colony counts (L) and flow cytometry analysis (M) on D11 or D13 after D33 or D35 A1ATD1 MKs were sorted into CFU assays. (N) qPCR analysis of sorted D23 A1ATD1 MKs. Replicates are shown as points. For flow cytometry, viable cells were selected as DAPI negative and thresholds set on a corresponding isotype control for each sample. All colony counts were averaged per experiment (n = 2) and normalized to plated cell number and Live cell condition.

Fig 5: CD51, CD168, and VEGFR1 can further select MICB+PVR+ MKPs.(A and B) ITGAVhiHMMRhiFLT1lo cells’ location on the trajectory plot (A) and MKP UMAP (B). (C and D) Colony counts from CFU assay of A1ATD1 (C) or QOLG3 (D) MKs sorted as MICBhiPVRhi or MICBhiPVRhiCD51hiCD168hiVEGFR1lo. (E and F) Bright-field microscopy images (E) and flow cytometry analysis (F) of colonies. (G) Total viable cell count after one A1ATD1 and one QOLG3 CFU assay. (H) MYC expression levels on time-course UMAP. (I) qPCR analysis of MYC transcript levels. (J) qPCR analysis of telomeric repeats (D21 A1ATD1 and D34 QOLG3 MKs). Error bar, SD of three technical replicates, biological duplicates. (K and L) YTHDC2 and ATF2 expression on time-course UMAP. (M and N) qPCR analysis of YTHDC2 (M) and ATF2 (N) transcript levels. (O and P) Flow cytometry analysis of intracellular ATF2 (O) and YTHDC2 (P) levels in live and MICBhiPVRhiCD51hiCD168hiVEGFR1lo D38 QOLG1 MKs. (Q) Flow cytometry analysis of calcein+ platelet-like particles 72 hours after MKs were sorted into basal medium. **P < 0.01, *0.05 < P < 0.01, and .P = 0.05. Adjusted P value compared to Live cells by one-way analysis of variance (ANOVA) using Dunnett’s multiple comparisons test (C, D, and F) or two-tailed Mann-Whitney test (J). CFU counts were normalized to the total sorted cell number and the Live condition, shown as technical duplicates of biological triplicates. (I, M, N, and Q) D27 A1ATD1 and D34 QOLG3 MKs.