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Multipotent hematopoietic progenitors divide asymmetrically to create progenitors of the lymphomyeloid and erythromyeloid lineages.

Görgens A, Ludwig AK, Möllmann M, Krawczyk A, Dürig J, Hanenberg H, Horn PA, Giebel B - Stem Cell Reports (2014)

Bottom Line: Now, by using a novel anti-CD133 antibody (HC7), we confirmed the occurrence of asymmetric CD133 segregation on paraformaldehyde-fixed and living HSPCs.After showing that HC7 binding does not recognizably affect biological features of human HSPCs, we studied ACDs in different HSPC subtypes and determined the developmental potential of arising daughter cells at the single-cell level.Since MPPs hardly created daughter cells maintaining MPP characteristics, our data suggest that under conventional culture conditions, ACDs are lineage instructive rather than self-renewing.

View Article: PubMed Central - PubMed

Affiliation: Institute for Transfusion Medicine, University Hospital Essen, University of Duisburg-Essen, Virchowstraße 179, 45147 Essen, Germany; German Cancer Consortium (DKTK). Electronic address: andre.goergens@uk-essen.de.

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AC133- and HC7-Stained CD34+ Cells Reveal Comparable In Vitro and In Vivo Differentiation Potentials(A) CD34+ cells cultured for 50–60 hr were stained with either AC133 or HC7 and anti-CD34 antibodies and flow cytometrically analyzed before (presort) or after fluorescent cell sorting of CD133+CD34+ cells (postsort).(B) Experimental strategy for in vitro and in vivo analysis of sorted cell fractions using either AC133 or HC7 antibodies.(C and D) Total CFC frequencies of AC133- and HC7-stained CD133+CD34+ and CD133−CD34+ cells (C) as well as the total frequencies of the obtained colony subtypes (D).(E and F) CD34+ cells cultured for 50–60 hr were purified by fluorescent cell sorting according to the gating strategy shown in (A). Aliquots of 50,000 CD133+CD34+ cells were transplanted in four independent experiments to a total of 23 NOD/SCID mice. The total human cell engraftment was determined upon measuring the content of human CD45+ cells in the mice’s bone marrow 8 weeks after transplantation (E) and plotted in comparison to each other (F).(G) The content of hematopoietic subpopulations within the human CD45+ cell fractions was determined by using markers specific for the detection of B cells (CD19/20), natural killer cells (CD56), erythroid cells (CD71), myeloid cells (CD13), monocytes/macrophages (CD14), and granulocytes (CD15).(H) Obtained results were plotted in comparison to each other.n, number of independent experiments; N, number of mice; arrow bars are given as SD of the mean.
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fig4: AC133- and HC7-Stained CD34+ Cells Reveal Comparable In Vitro and In Vivo Differentiation Potentials(A) CD34+ cells cultured for 50–60 hr were stained with either AC133 or HC7 and anti-CD34 antibodies and flow cytometrically analyzed before (presort) or after fluorescent cell sorting of CD133+CD34+ cells (postsort).(B) Experimental strategy for in vitro and in vivo analysis of sorted cell fractions using either AC133 or HC7 antibodies.(C and D) Total CFC frequencies of AC133- and HC7-stained CD133+CD34+ and CD133−CD34+ cells (C) as well as the total frequencies of the obtained colony subtypes (D).(E and F) CD34+ cells cultured for 50–60 hr were purified by fluorescent cell sorting according to the gating strategy shown in (A). Aliquots of 50,000 CD133+CD34+ cells were transplanted in four independent experiments to a total of 23 NOD/SCID mice. The total human cell engraftment was determined upon measuring the content of human CD45+ cells in the mice’s bone marrow 8 weeks after transplantation (E) and plotted in comparison to each other (F).(G) The content of hematopoietic subpopulations within the human CD45+ cell fractions was determined by using markers specific for the detection of B cells (CD19/20), natural killer cells (CD56), erythroid cells (CD71), myeloid cells (CD13), monocytes/macrophages (CD14), and granulocytes (CD15).(H) Obtained results were plotted in comparison to each other.n, number of independent experiments; N, number of mice; arrow bars are given as SD of the mean.

Mentions: Next, we compared whether the HC7 or AC133 antibodies affect the development of HSPCs within the colony-forming cell (CFC) assay. We labeled 50–60 hr cultured CD34+ cells with the HC7 or AC133 anti-CD133 antibodies. Subsequently, fluorescent cell sorting was performed to separate CD133+CD34+ and CD133−CD34+ cells (Figure 4A) and transfer selected numbers of purified cells into the CFC assay (Figure 4B). Regardless of whether the CD34+ cells were labeled with the HC7 or AC133 antibody, the sorted cell fractions formed comparable colony numbers in all settings (Figure 4C). In good agreement with our previous studies (Görgens et al., 2013b), progenitors with colony-forming unit granulocyte-macrophage (CFU-GM) potentials were almost exclusively recovered within the CD133+CD34+ cell fractions, while progenitors revealing erythroid and colony-forming unit mixed (CFU-MIX) potentials were highly enriched within the CD133−CD34+ cell fractions (Figure 4D). Since no differences in the quality and quantity of colonies were observed in CFC assays of corresponding HC7- and AC133-labeled CD34+ cell fractions, we conclude that binding of these antibodies does not affect the colony-formation potential of UCB-derived HSPCs.


Multipotent hematopoietic progenitors divide asymmetrically to create progenitors of the lymphomyeloid and erythromyeloid lineages.

Görgens A, Ludwig AK, Möllmann M, Krawczyk A, Dürig J, Hanenberg H, Horn PA, Giebel B - Stem Cell Reports (2014)

AC133- and HC7-Stained CD34+ Cells Reveal Comparable In Vitro and In Vivo Differentiation Potentials(A) CD34+ cells cultured for 50–60 hr were stained with either AC133 or HC7 and anti-CD34 antibodies and flow cytometrically analyzed before (presort) or after fluorescent cell sorting of CD133+CD34+ cells (postsort).(B) Experimental strategy for in vitro and in vivo analysis of sorted cell fractions using either AC133 or HC7 antibodies.(C and D) Total CFC frequencies of AC133- and HC7-stained CD133+CD34+ and CD133−CD34+ cells (C) as well as the total frequencies of the obtained colony subtypes (D).(E and F) CD34+ cells cultured for 50–60 hr were purified by fluorescent cell sorting according to the gating strategy shown in (A). Aliquots of 50,000 CD133+CD34+ cells were transplanted in four independent experiments to a total of 23 NOD/SCID mice. The total human cell engraftment was determined upon measuring the content of human CD45+ cells in the mice’s bone marrow 8 weeks after transplantation (E) and plotted in comparison to each other (F).(G) The content of hematopoietic subpopulations within the human CD45+ cell fractions was determined by using markers specific for the detection of B cells (CD19/20), natural killer cells (CD56), erythroid cells (CD71), myeloid cells (CD13), monocytes/macrophages (CD14), and granulocytes (CD15).(H) Obtained results were plotted in comparison to each other.n, number of independent experiments; N, number of mice; arrow bars are given as SD of the mean.
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fig4: AC133- and HC7-Stained CD34+ Cells Reveal Comparable In Vitro and In Vivo Differentiation Potentials(A) CD34+ cells cultured for 50–60 hr were stained with either AC133 or HC7 and anti-CD34 antibodies and flow cytometrically analyzed before (presort) or after fluorescent cell sorting of CD133+CD34+ cells (postsort).(B) Experimental strategy for in vitro and in vivo analysis of sorted cell fractions using either AC133 or HC7 antibodies.(C and D) Total CFC frequencies of AC133- and HC7-stained CD133+CD34+ and CD133−CD34+ cells (C) as well as the total frequencies of the obtained colony subtypes (D).(E and F) CD34+ cells cultured for 50–60 hr were purified by fluorescent cell sorting according to the gating strategy shown in (A). Aliquots of 50,000 CD133+CD34+ cells were transplanted in four independent experiments to a total of 23 NOD/SCID mice. The total human cell engraftment was determined upon measuring the content of human CD45+ cells in the mice’s bone marrow 8 weeks after transplantation (E) and plotted in comparison to each other (F).(G) The content of hematopoietic subpopulations within the human CD45+ cell fractions was determined by using markers specific for the detection of B cells (CD19/20), natural killer cells (CD56), erythroid cells (CD71), myeloid cells (CD13), monocytes/macrophages (CD14), and granulocytes (CD15).(H) Obtained results were plotted in comparison to each other.n, number of independent experiments; N, number of mice; arrow bars are given as SD of the mean.
Mentions: Next, we compared whether the HC7 or AC133 antibodies affect the development of HSPCs within the colony-forming cell (CFC) assay. We labeled 50–60 hr cultured CD34+ cells with the HC7 or AC133 anti-CD133 antibodies. Subsequently, fluorescent cell sorting was performed to separate CD133+CD34+ and CD133−CD34+ cells (Figure 4A) and transfer selected numbers of purified cells into the CFC assay (Figure 4B). Regardless of whether the CD34+ cells were labeled with the HC7 or AC133 antibody, the sorted cell fractions formed comparable colony numbers in all settings (Figure 4C). In good agreement with our previous studies (Görgens et al., 2013b), progenitors with colony-forming unit granulocyte-macrophage (CFU-GM) potentials were almost exclusively recovered within the CD133+CD34+ cell fractions, while progenitors revealing erythroid and colony-forming unit mixed (CFU-MIX) potentials were highly enriched within the CD133−CD34+ cell fractions (Figure 4D). Since no differences in the quality and quantity of colonies were observed in CFC assays of corresponding HC7- and AC133-labeled CD34+ cell fractions, we conclude that binding of these antibodies does not affect the colony-formation potential of UCB-derived HSPCs.

Bottom Line: Now, by using a novel anti-CD133 antibody (HC7), we confirmed the occurrence of asymmetric CD133 segregation on paraformaldehyde-fixed and living HSPCs.After showing that HC7 binding does not recognizably affect biological features of human HSPCs, we studied ACDs in different HSPC subtypes and determined the developmental potential of arising daughter cells at the single-cell level.Since MPPs hardly created daughter cells maintaining MPP characteristics, our data suggest that under conventional culture conditions, ACDs are lineage instructive rather than self-renewing.

View Article: PubMed Central - PubMed

Affiliation: Institute for Transfusion Medicine, University Hospital Essen, University of Duisburg-Essen, Virchowstraße 179, 45147 Essen, Germany; German Cancer Consortium (DKTK). Electronic address: andre.goergens@uk-essen.de.

Show MeSH
Related in: MedlinePlus