Flow cytometry analysis of cord blood and postnatal BM Lin⁻CD34⁺ progenitors. Mononuclear cells from postnatal BM and CD34⁺-enriched cord blood were stained with a combination of lineage (Lin) markers and anti-CD34, anti-CD10, and anti-CD24 antibodies. (A) Representative analysis of CD10 and CD24 expression of gated Lin⁻CD34⁺ cells from cord blood and BM of young (<10 yr of age) and older (10–60 yr of age) donors. (B) CCR9 and CCR7 expression of gated Lin⁻CD34⁺CD10⁻, Lin⁻CD34⁺CD10⁺CD24⁻, and Lin⁻CD34⁺CD10⁺CD24⁺ cells (shaded histogram). The dotted line indicates control isotypes. Numbers in A and B represent percentages of cells.

Differentiation potential of CD10⁺CD24⁻ and CD10⁻CD24⁺ progenitors. (A) Flow cytometry analysis of 3-wk cultures on MS5 stromal cells in B or NK cell differentiation assays of CD10⁺CD24⁻ and CD10⁺CD24⁺ progenitors sorted from <10-yr-old BM. The data shown represent six independent experiments performed on cord blood (n = 2) and <10-yr-old (n = 2) and >10-yr-old (n = 2) BM samples. (B) CD4, CD8, CD3, and γδTCR expression analysis of 5-wk cultures on OP9-hDelta1 stromal cells of CD10⁺CD24⁻ progenitors sorted from cord blood or pediatric BM. The data shown represent five independent experiments performed on cord blood (n = 2) and <10-yr-old (n = 2) and >10-yr-old (n = 1) BM samples. After 5 wk, we recovered an average of 6,000 and 200 T cells (CD4⁺CD8⁺ and γδ T cells) from 1,000 CD10⁺CD24⁻ progenitors sorted from cord blood and BM samples, respectively. (C) Lin⁻CD34⁺CD10⁺ progenitors exhibit a DC potential. (left) A representative 10-d culture in DC differentiation conditions from cord blood–sorted CD10⁻ and CD10⁺CD24⁻ progenitors is shown. Bar, 10 μm. In the same culture conditions, CD10⁺CD24⁺ cells did not survive. (right) Flow cytometry analysis of 10-d cultures of CD10⁻ and CD10⁺CD24⁻ progenitors. HLA-DR expression is analyzed on CD1a⁺-gated cells. Numbers in A–C represent percentages of cells. (D) Limiting dilution assay analysis of CD10⁺CD24⁻ progenitors sorted from cord blood (n = 2) and <10-yr-old (n = 1) and >10-yr-old (n = 1) BM. After 4–5 wk of culture in 96-well plates, means of 20,000, 3,250, and 560 viable cells were recovered from 100 CD10⁺CD24⁻ progenitors sorted from cord blood, pediatric, and adult BM samples, respectively.

Flow cytometry analysis and differentiation potential of Lin⁻CD34⁺ cells in adult blood and postnatal thymus. (A) CD34⁺ enriched cells isolated from the blood of an adult healthy donor were stained with a combination of lineage (Lin) markers and anti-CD34, anti-CD7, and anti-CD10 antibodies. Data are representative of 5 pediatric and 11 adult blood samples. (B) CD10⁺ and CD10⁻ progenitors recovered from adult blood were plated in B/NK (left) or T (right) cell differentiation conditions. The culture was analyzed using flow cytometry. The results of one out of four experiments are shown. (C) CD34⁺-enriched cells from postnatal thymus were stained with a combination of lineage (Lin) markers and anti-CD34, anti-CD7, anti-CD1a, and anti-CD10 antibodies. (left) Analysis of Lin and CD34 expression in CD34⁺-enriched thymocytes. (right) CD10/CD7 expression on gated Lin⁻CD34⁺ cells is shown. CD1a expression is analyzed on Lin⁻CD34⁺CD10⁺CD7⁻ and CD7⁺ cells (histogram, bottom). Data are representative of 25 analyzed thymuses. (D) The lymphoid potential of Lin⁻CD34⁺CD10⁺ thymic progenitors. (left) Up to 8,000 Lin⁻CD34⁺CD10⁺CD7⁻ thymic progenitors were plated in B/NK cell differentiation conditions and analyzed after 3 wk. (right) Flow cytometry analysis of a representative 5-wk culture on OP9-hDelta1 stromal cells from postnatal thymus-sorted Lin⁻CD34⁺CD10⁺CD7⁻ cells. After 5 wk, we recovered an average of 4,000 T cells (CD4⁺CD8⁺ and γδ T cells) from 500 CD10⁺CD7⁻ progenitors sorted from thymus samples. Numbers in A–D represent percentages of cells.

Gene expression profile of CD10⁻, CD10⁺CD24⁻, and CD10⁺CD24⁺ progenitors. (A) BM or (B) cord blood Lin⁻CD34⁺CD10⁻, Lin⁻CD34⁺CD10⁺CD24⁻, and Lin⁻CD34⁺CD10⁺CD24⁺ populations and (C) thymic Lin⁻CD34⁺CD10⁺CD24⁻CD7⁻ cells were sorted directly into PCR tubes at 10 cells per well. RT followed by a quadruplex or triplex amplification PCR assay was performed. Each well was duplicated to perform a second qualitative PCR, and seven or eight representative wells are presented for each population. DNA fragment sizes are indicated. −, negative control.