Reduced circulating MKP in PWH. Circulating hematogenic progenitor cells were analyzed in PBMCs from PWH (n = 17) and PWOH (n = 16) by flow cytometry. (A) Hematopoietic cell differentiation. CD34⁺CD38^– contains multipotent cells (bold gray text): HSC, MPP, and lymphoid-primed multipotent progenitors (LMPPs). The oligopotent progenitors CD34⁺CD38⁺ (black text): common myeloid progenitor (CMP), MEP, and granulocyte-monocyte progenitor (GMP) cells. MKPs originate from the bipotent progenitor MEP (black arrows). HSC-MKPs (stem-cell like) that originate from a subset of HSC-CD41⁺ (gray arrows). (B) Manual gating strategy. Free platelets (SSC^lowCD42b⁺) were excluded from the analysis followed by gating FSC vs SSC, live and single cells. The lineage negative cells (LIN^–) were gated by the exclusion of lineage positive cells using a cocktail of mAbs (CD2, CD3, CD4, CD7, CD8, CD10, CD11b, CD14, CD19, CD20, CD56, and CD235a). An additional CD14 mAb was used to eliminated potential contaminant events on the LIN^– population (supplemental Figure 1A). The LIN^– (gated as LIN^–CD14^– cells) were analyzed for progenitor marker expression CD34 and CD38: LIN^–CD34⁺CD38^– cells (bold gray text) contains multipotent progenitors: HSC, CD45RA^–CD90⁺; MPP, CD45RA^–CD90^–; and LMPP, CD45RA⁺. The LIN^–CD34⁺CD38⁺ cells (black text) contain oligopotent progenitors: CMP, CD45RA^–CD123⁺; MEP, CD45RA^–CD123^–; and GMP, CD45RA⁺ cells. MKPs derived from MEP were gated as MEPCD41⁺. The MKPs derived from HSC (“stem-cell like” or HSC-MKP) were defined as HSC-CD41⁺. Because of the low number of events, the analysis of CD41⁺ was performed in the MEP-CMP and HSC-MPP together. (C) Unsupervised clustering analysis using t-SNE and PhenoGraph of the total LIN^– cells. The heat map represents mean fluorescence intensity of markers (CD34, CD45, CD71, CD38, CD45RA, CD123, CD90, CX3CR1, CD41, CD42b, and CD62P). Bubble plots represent the median frequency of each cluster in PWOH (blue) and PWH (orange). (D) In vitro MK differentiation. LIN^–CD34⁺ and LIN^–CD34^low were sorted from PBMCs from PWH (n = 11) and PWOH (n = 12) and cultured in MK conditioned media. After 12 days of culture, differentiation was evaluated with a cocktail of mAbs: CD34, CD38, and CD42b. Sytox staining was used for exclusion of dead cells and differentiation was evaluated by positive expression of CD41 and CD42b. (E) MKs were gated on the high FSC-A and (F) platelets in the low FSC-A and negative nuclei staining (sytox^–) population. The graphs represented by box and whisker show the median value with first and third quartiles in the box, with whiskers extending to the minimum and maximum values. (G) Colony-forming unit assay: 500 sorted LIN^–CD34⁺ cells were cultured in MegaCult-C Collagen and Medium with Lipid in the presence of recombinant human thrombopoietin, recombinant human interleikin-3, and recombinant human interleikin-6. Colonies were evaluated at days 13 to 14 of culture by CD41 expression and Hoechst 33324 was used as nuclei staining. Representative individual images of MK colony including CD41 (green), Hoechst 33324 (blue), and differential interference contrast of the BF and merged images. MK colonies were identified by CD41 expression and represented in the graph as CD41⁺ colonies per 100 seeding cells (LIN^–CD34⁺ cells). Comparison between the groups was performed using a nonparametric unpaired Mann-Whitney test. P < .05 was considered significant. BF, brightfield image; DAPI, 4',6-diamidino-2-phenylindolem; FSC, forward scatter; FSC-A, forward scatter-area; mAb, monoclonal antibody; MKP, megakaryocyte progenitor; PBMC, peripheral blood mononuclear cells; SSC, side scatter; t-SNE, t-distributed stochastic neighbor embedding.