Figure 1.
New phenotypes identify cells at early stages of B and NM differentiation. (A) Experimental design used to generate phenotypically defined CD19+ (B lineage) and CD14/15+ (NM lineage) outputs in vitro from 1000 P-mix CB input cells in LEM cultures (LEM being composed of αMEM, FBS, and a StemSpan lymphoid expansion supplement; see “Methods” for additional details). (B) A representative flow cytometric profile of the surface marker expression of the cells present in the cultures described in panel A after 2 weeks (CD45RA [RA]; CLEC12A [C]). (C) Number of output cells per 1000 input P-mix cells analyzed weekly. The Lin– phenotypes were gated within the CD45+14–15–10–19– subset. Each bar shows the mean ± standard error of the mean (SEM) of values pooled from 5 different experiments. “n.d.” represents signals below the limit of detection (10 cells). (D) Experimental design used to examine the lineage potentials of the 3 input phenotypes shown. (E) Fluorescence-activated cell sorting (FACS) gating used to identify 3 phenotypes within the CD34+ cells present in day 7 cultures of CD45+14–15–10– cells generated from P-mix CB cells. (F) Percentages of output cell types (identified by their phenotypes) obtained in clonal cultures initiated with the 3 phenotypes shown in panel E. Each bar shows the mean ± SEM of values pooled from 4 different experiments with 150 to 350 total single cells tested per input subset. The statistical significance of differences in clone distribution between input phenotypes was assessed using t tests with Holm multitest correction (∗P < .05). (G) Reverse transcription quantitative polymerase chain reaction (PCR) analysis of mRNAs of various historically defined B-lineage (upper)– and NM-lineage (lower)–associated genes measured in sorted phenotypes generated from 2-week cultures initiated with P-mix CB cells. Each bar shows the mean ± SEM of values pooled from 3 to 4 different experiments. “n.d.” denotes signals below the limit of detection (PCR cycle, >40). Statistical differences in mRNA levels between the progenitor and all differentiated phenotypes were assessed using t tests with Holm correction (∗P < .05). GAPDH, glyceraldehyde-3-phosphate dehydrogenase; mRNA, messenger RNA.

New phenotypes identify cells at early stages of B and NM differentiation. (A) Experimental design used to generate phenotypically defined CD19+ (B lineage) and CD14/15+ (NM lineage) outputs in vitro from 1000 P-mix CB input cells in LEM cultures (LEM being composed of αMEM, FBS, and a StemSpan lymphoid expansion supplement; see “Methods” for additional details). (B) A representative flow cytometric profile of the surface marker expression of the cells present in the cultures described in panel A after 2 weeks (CD45RA [RA]; CLEC12A [C]). (C) Number of output cells per 1000 input P-mix cells analyzed weekly. The Lin– phenotypes were gated within the CD45+14–15–10–19– subset. Each bar shows the mean ± standard error of the mean (SEM) of values pooled from 5 different experiments. “n.d.” represents signals below the limit of detection (10 cells). (D) Experimental design used to examine the lineage potentials of the 3 input phenotypes shown. (E) Fluorescence-activated cell sorting (FACS) gating used to identify 3 phenotypes within the CD34+ cells present in day 7 cultures of CD45+14–15–10– cells generated from P-mix CB cells. (F) Percentages of output cell types (identified by their phenotypes) obtained in clonal cultures initiated with the 3 phenotypes shown in panel E. Each bar shows the mean ± SEM of values pooled from 4 different experiments with 150 to 350 total single cells tested per input subset. The statistical significance of differences in clone distribution between input phenotypes was assessed using t tests with Holm multitest correction (∗P < .05). (G) Reverse transcription quantitative polymerase chain reaction (PCR) analysis of mRNAs of various historically defined B-lineage (upper)– and NM-lineage (lower)–associated genes measured in sorted phenotypes generated from 2-week cultures initiated with P-mix CB cells. Each bar shows the mean ± SEM of values pooled from 3 to 4 different experiments. “n.d.” denotes signals below the limit of detection (PCR cycle, >40). Statistical differences in mRNA levels between the progenitor and all differentiated phenotypes were assessed using t tests with Holm correction (∗P < .05). GAPDH, glyceraldehyde-3-phosphate dehydrogenase; mRNA, messenger RNA.

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