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1.
Figure 4.

Figure 4. From: Sox17 expression confers self-renewal potential and fetal stem cell characteristics upon adult hematopoietic progenitors.

Ectopic Sox17 expression in adult bone marrow cells expanded CD48+LSK hematopoietic progenitors and induced the expression of fetal HSC markers by these cells. (A) Representative flow cytometry plots of GFP+ bone marrow cells in primary recipient mice transplanted with MSCV-GFP control virus or MSCV-Sox17-infected bone marrow cells. (A–C) Sox17-expressing cells gave rise to a significantly higher frequency of LSK cells and CD48+LSK cells as compared with control cells in the bone marrow and spleen (shown in A,C) (*) P < 0.05; five to seven independent experiments. (B) The frequency of GFP+ CD150+CD48LSK HSCs was lower in recipients of Sox17-expressing cells as compared with control cells, although the difference was not statistically significant. (D–H) Compared with control CD48+LSK cells, Sox17-expressing CD48+LSK cells exhibited increased expression of the HSC markers ESAM (D) and Tie-2 (E), as well as the fetal HSC markers Mac-1 (F), VE-Cadherin (G), and AA4.1 (H).

Shenghui He, et al. Genes Dev. 2011 August 1;25(15):1613-1627.
2.
Figure 6.

Figure 6. From: Sox17 expression confers self-renewal potential and fetal stem cell characteristics upon adult hematopoietic progenitors.

Ectopic Sox17 expression in adult transiently reconstituting multipotent progenitors conferred long-term self-renewal potential. (A) Five-hundred adult bone marrow MPPs (CD150CD48LSK cells) were infected with MSCV-GFP control or MSCV-Sox17 virus and transplanted into lethally irradiated recipient mice along with 200,000 recipient bone marrow cells. While control MPPs gave transient multilineage reconstitution in most recipient mice (A,C), Sox17-expressing MPPs gave long-term multilineage reconstitution in five of 10 recipients and long-term erythroid and/or platelet reconstitution in another two recipients (C). Data represent mean ± SD from 13–15 recipients per treatment; (*) P < 0.05. (B) Five-hundred or 2000 CD48+LSK adult bone marrow cells were infected with MSCV-GFP control or MSCV-Sox17 virus and transplanted into lethally irradiated recipient mice along with 200,000 recipient bone marrow cells. These data reflect mean ± SD from 20–22 recipients per treatment; (*) P < 0.05. While control CD48+LSK cells gave transient multilineage reconstitution (in various combinations of lineages) in all recipient mice that showed donor cell reconstitution (B,C), Sox17-expressing CD48+LSK cells gave long-term reconstitution in various combinations of lineages in nine of 22 recipient mice and transient multilineage reconstitution (five of 22) or transient erythroid and/or platelet reconstitution in the remaining reconstituted recipients (four of 22; C). (C) No cell population infected with MSCV-GFP control virus gave long-term reconstitution in any combination of lineages. In contrast, nine of 16, five of 15, and one of 22 recipients of MSCV-Sox17-infected HSCs, MPPs, or CD48+LSK cells showed long-term multilineage reconstitution, respectively, and, in total, 15 of 16, seven of 15, and nine of 22 recipients of Sox17-expressing HSCs, MPPs, or CD48+LSK cells showed long-term reconstitution in the myeloid and/or erythroid lineages. Donor cell reconstitution by control and Sox17-expressing HSCs, GMPs, and Pre-MegEs can be found in Supplemental Figure 5.

Shenghui He, et al. Genes Dev. 2011 August 1;25(15):1613-1627.
3.
Figure 5.

Figure 5. From: Sox17 expression confers self-renewal potential and fetal stem cell characteristics upon adult hematopoietic progenitors.

Adult CD48+LSK cells give only low levels of transient multilineage reconstitution, but ectopic Sox17 expression in these cells confers the potential to give long-term multilineage reconstitution. (A) One-million unfractionated whole bone marrow cells from primary recipients of MSCV-GFP-infected control cells or MSCV-Sox17-infected cells were transplanted into lethally irradiated secondary recipients along with 200,000 radioprotective recipient bone marrow cells. Sox17-expressing cells gave rise to higher levels of long-term reconstitution in the myeloid (Mac-1 and Gr-1), erythroid (Ter119), and megakaryocyte (CD41) lineages, but significantly lower levels of reconstitution in the B-cell (B220) and T-cell (CD3) lineages. Data represent mean ± SD from four to 11 recipients per treatment; (*) P < 0.05; note that red lines indicate background, below which donor cell reconstitution cannot be detected. (B). Fifty GFP+CD48+LSK cells from primary recipients of MSCV-GFP-infected control cells or MSCV-Sox17-infected cells were transplanted into lethally irradiated secondary recipients along with 200,000 recipient bone marrow cells. While control CD48+LSK cells gave only low levels of transient multilineage reconstitution in the myeloid, erythroid, and lymphoid lineages, Sox17-expressing CD48+LSK cells gave rise to long-term multilineage reconstitution in the myeloid, erythroid, and megakaryocyte, but not the B- or T-cell lineages. The data represent mean ± SD from nine to 15 recipients per treatment; (*) P < 0.05. (C) Control CD48+LSK cells gave rise to transient multilineage reconstitution in almost all recipients (eight of nine), whereas Sox17-expressing CD48+LSK cells gave rise to long-term multilineage reconstitution in the erythroid and megakaryocyte lineages in most recipients (14 of 15 mice) and often in the myeloid lineage as well (nine of 15 mice).

Shenghui He, et al. Genes Dev. 2011 August 1;25(15):1613-1627.
4.
Figure 1.

Figure 1. From: Sox17 expression confers self-renewal potential and fetal stem cell characteristics upon adult hematopoietic progenitors.

Sox17 expression is restricted to immature hematopoietic stem/progenitor cells from fetal and neonatal mice. Representative histograms showing the distribution of Sox17 expression based on GFP fluorescence in Sox17GFP/+ mice at E13.5 (A), P0 (B), and 2 wk of age (C) in HSCs, MPPs, CD48+LSK cells, and GMPs. Histograms of the same cell populations from Sox17+/+ mice are shown as negative controls. (A) At E13.5, GFP expression was observed in HSCs, MPPs, and CD48+LSK cells, but not in GMPs or in most other fetal liver cells. (B) At P0, lower levels of GFP could still be observed in HSCs and MPPs, but not in CD48+LSK cells, GMPs, or most other newborn liver cells. (C) GFP expression was not detected in any hematopoietic cells from 2-wk-old Sox17GFP/+ mice. (D) Many GFP+ fetal liver cells from Sox17GFP/+ mice at E13.5 were LSK hematopoietic stem/progenitor cells. (E) Mean GFP fluorescence intensity for each sorted cell population. (*) Significantly different (P < 0.05) from the same cell population at E13.5; (§) significantly different (P < 0.05) from HSCs at the same developmental stage; (green numbers) significant difference from Sox17+/+ control cells at the same developmental stage. Data are from three to four independent experiments. All error bars indicate SD.

Shenghui He, et al. Genes Dev. 2011 August 1;25(15):1613-1627.
5.
Figure 2.

Figure 2. From: Sox17 expression confers self-renewal potential and fetal stem cell characteristics upon adult hematopoietic progenitors.

Ectopic Sox17 expression in adult bone marrow cells enhances their potential to give long-term multilineage reconstitution of irradiated mice. One-million donor (CD45.1) bone marrow cells infected with MSCV-GFP control virus, MSCV-Sox17, or MSCV-Sox17his virus were transplanted into 8-wk-old lethally irradiated CD45.2 recipients along with 200,000 CD45.2 bone marrow cells. (A–F) At 16-wk after transplantation, Sox17-expressing bone marrow cells gave rise to significantly higher levels of peripheral blood myeloid (Mac-1+ or Gr-1+, A,B), erythroid (Ter119+, C) and platelet (CD41+, D) reconstitution as compared with control virus-infected cells. MSCV-Sox17 but not MSCV-Sox17his virus-infected cells also exhibited significantly enhanced B-cell (B220+; E) reconstitution but no significant differences were observed in the levels of T-cell (CD3+; F) reconstitution. The data in A–F reflect three independent experiments with a total of nine to14 recipients per treatment. (*) P < 0.05; (**) P < 0.01. (G) Eighty-nine percent (eight of nine) of mice transplanted with MSCV-Sox17his-infected cells and 100% of mice transplanted with MSCV-Sox17-infected cells exhibited long-term multilineage reconstitution by donor GFP+ cells. In contrast, only 36% (five of 14) of recipients transplanted with control virus-infected cells showed long-term multilineage reconstitution. (H) Ectopic Sox17 expression significantly increased the ratio of myeloid (Mac-1+) to lymphoid (B220+ or CD3+) cells produced in recipient mice at 16 wk after transplantation. (*) P < 0.05. (I). Representative Wright-Giemsa stains of blood from E14 and adult wild-type mice, as well as primary recipients of MSCV-GFP or MSCV-Sox17 virus-infected adult bone marrow cells. Ectopic Sox17 expression did not lead to the appearance of nucleated red blood cells characteristic of primitive erythropoiesis.

Shenghui He, et al. Genes Dev. 2011 August 1;25(15):1613-1627.
6.
Figure 3.

Figure 3. From: Sox17 expression confers self-renewal potential and fetal stem cell characteristics upon adult hematopoietic progenitors.

Ectopic Sox17 expression in adult bone marrow cells biased hematopoiesis in adult recipient mice to resemble fetal hematopoiesis. Representative images of the spleens of primary recipient mice reconstituted with adult bone marrow cells infected with MSCV-GFP control virus, MSCV-Sox17, or MSCV-Sox17his virus (16 wk after transplantation). Sox17 expression significantly increased spleen size (A) and mass (B) as a consequence of extramedullary hematopoiesis (splenic architecture was normal and erythropoiesis was evident). (C) Sox17 expression significantly increased the frequency of Ter119+ erythroid cells and CD41+ megakaryocyte lineage cells in the spleen, and significantly reduced the frequencies of B220+ B cells and CD4+ or CD8+ T cells (four independent experiments with a total of four to six mice per treatment; [*] P < 0.05). (D,E) Representative flow cytometry plots showing increased erythroid cells (D; CD71+Ter119+) and decreased B cells (E; B220+) in the spleens of mice reconstituted by Sox17-expressing cells as compared with control cells. (F) Compared with control cells, Sox17-expressing cells gave rise to significantly fewer Mac-1+Gr-1+ myeloid cells, and myelopoiesis from Sox17-expressing cells phenotypically resembled fetal liver rather than adult bone marrow. (Numbers indicate the percentage of all GFP+ bone marrow cells that were Mac-1+Gr-1+.) (G) Compared with control cells, Sox17-expressing cells gave rise to significantly more c-kit+CD41+ megakaryocyte lineage cells, and thrombopoiesis by Sox17-expressing cells phenotypically resembled the fetal liver rather than adult bone marrow. (Numbers indicate the percentage of all GFP+ bone marrow cells that were c-kit+CD41+.) (H) All Sox17-expressing c-kit+CD41+ cells in primary recipients expressed CD45, and some expressed the megakaryocyte marker CD42d. (CD41 was excluded from the lineage cocktail in this experiment.)

Shenghui He, et al. Genes Dev. 2011 August 1;25(15):1613-1627.
7.
Figure 7.

Figure 7. From: Sox17 expression confers self-renewal potential and fetal stem cell characteristics upon adult hematopoietic progenitors.

Ectopic Sox17 expression in adult hematopoietic progenitors increased the expression of genes associated with fetal and adult HSCs and reduced the expression of genes associated with differentiation. (A–C) GSEA comparing Sox17-expressing (red) versus control bone marrow (blue) CD48+LSK cells. Sox17-expressing CD48+LSK cells showed significant enrichment of adult HSC (compared with bone marrow CD48+LSK cells) and fetal HSC (compared with adult bone marrow HSCs) gene sets (A,C), and significant depletion of a progenitor (CD48+LSK cell compared with HSCs) gene set (B). (NES) Normalized enrichment score. (D) Comparison of the gene expression profiles of Sox17-expressing and control bone marrow CD48+LSK cells revealed 376 significantly up-regulated and 264 significantly down-regulated genes in the Sox17-expressing cells (fold change >2 and P < 0.05). Ninety-one of the 376 up-regulated genes were preferentially expressed by HSCs (compared with bone marrow CD48+LSK cells, fold >2, P < 0.05), while 122 of the 264 down-regulated genes were preferentially expressed by CD48+LSK progenitors (compared with HSCs, fold >2, P < 0.05). (E) Using the more stringent cut-off of fold change >5 and P < 0.05, 82 genes were up-regulated and 33 genes were down-regulated in Sox17-expressing compared with control CD48+LSK cells. Twenty-one of the 82 up-regulated genes were preferentially expressed by adult HSCs as compared with CD48+LSK cells and only four were preferentially expressed by CD48+LSK cells. Seventeen of the 82 up-regulated genes were also preferentially expressed by fetal HSCs (compared with bone marrow HSCs), while only five of the 82 genes were preferentially expressed by adult HSCs (compared with fetal HSCs). Most of the down-regulated genes were preferentially expressed by progenitors (relative to HSCs) and by adult (relative to fetal) HSCs. (F) Quantitative RT–PCR of selected genes that were differentially expressed by Sox17-expressing versus control CD48+LSK cells by microarray analysis. (*) Transcripts not detected in control CD48+LSK cells after 40 cycles of amplification, fold change was estimated using 35 cycles as the detection limit; (**) transcripts not detected in Sox17-expressing CD48+LSK cells after 40 cycles of amplification, fold change was estimated using 35 cycles as the detection limit. Data represents mean ± SD from three independent experiments. (G). Kaplan-Meier survival curve of primary recipients that received 1 million control or Sox17 virus-infected whole bone marrow cells along with 200,000 bone marrow cells for radioprotection; (*) P < 0.01 versus control; n = 15–22 mice per group. (H) Kaplan-Meier survival curve of secondary recipients that received 3 million whole bone marrow cells from primary recipients in the treatments shown in G; (*) P < 0.01 versus control; n = 8–27 mice per group.

Shenghui He, et al. Genes Dev. 2011 August 1;25(15):1613-1627.

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