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The stem cell marker CD133 meets the endosomal compartment – New insights into the cell division of hematopoietic stem cells
Blood Cells, Molecules, and Diseases 41 (2008) 194–195
Contents lists available at ScienceDirect
Blood Cells, Molecules, and Diseases j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y b c m d
Letter to the Editor The stem cell marker CD133 meets the endosomal compartment – New insights into the cell division of hematopoietic stem cells
To the Editor:
The self-renewal capacity of stem and progenitor cells relies on both extrinsic and intrinsic determinants involving for instance specific signaling pathways. Moreover, stem cells have the capacity to distribute asymmetrically certain of their protein components during cell division. In this context, we previously demonstrated that the widely used stem cell marker lipid raftassociated CD133 (prominin-1; AC133 antigen) [1,2] can be either symmetrically or asymmetrically distributed in mitotic human hematopoietic stem cells (hHSCs) cultured on primary human multipotent mesenchymal stromal cells (hMSCs) [3]. The same observation was made using hHSCs growing in stroma-free cultures, indicating that such phenomenon is independent of external clues [4]. More recently, Beckmann et al. have reported that four membrane proteins (CD53, CD62L, CD63 and CD71) segregate differentially in dividing hHSCs cultured in a stroma-free culture [5]. Since three of them (CD53, CD63 and CD71) are endosomal-associated proteins, these exciting results suggested a link between the endosomal compartment and symmetric/asymmetric cell division in rare primitive mammalian cells (see review Ref. [6] and references therein). Does CD133 associate with the endosomal compartment of mitotic hHSCs as described for the intracellular pool of CD63 and CD71 [5]? The answer is yes. We could observe, using a tripleimmunolabeling procedure on paraformaldehyde (PFA)-fixed, saponin-permeabilized hHSCs cultured on hMSCs [7], that CD133 and CD63 partly co-localized in the two nascent daughter cells during cytokinesis (Fig. 1A, filled arrows). More importantly, both CD markers are either equally or unequally distributed between the daughter cells (Fig. 1B, top and middle/bottom panels, respectively). Quantitative analysis revealed that CD133 was distributed asymmetrically in 22.3 ± 0.7% of cell divisions (n = 90; data obtained from three independent experiments using distinct donors). The absence of permeabilization prior to the CD133 labeling procedure likely explains why Beckmann et al. didn't detect CD133-positive intracellular structures in hHSCs [5]. Indeed, the authors only analysed the intracellular pool of CD63 and CD71 upon Triton X-100permeabilization (see Fig. 5B in Ref. [5]). However, it is interesting to note that the asymmetric distribution of these two proteins was observed in ≈ 20% of the mitotic cells, which is in agreement with our present data concerning CD133. We did not detect CD133 at the level of the hHSC midbody during cytokinesis (Figs. 1A, B, filled arrowheads), which is in contrast to the Beckmann' observation [5]. Such discrepancy might be explained, at least in part, by
1079-9796/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bcmd.2008.04.004
the fact that CD133 therein represents only a minute population of the total CD133 molecules and might therefore be under the detection level in our culture system. Alternatively, hHSCs derived from distinct sources, i.e. leukapheresis product (present study; see also Ref. [3,4]) versus umbilical cord blood [5], might behave differently. The symmetric versus asymmetric distribution of CD133 is not unique to hematopoietic cells since a similar phenomenon has been reported for the neuroepithelial progenitor cells [8]. However, the endosomal compartmentalization of CD133 described here is without precedent, and contrasts with its exclusive localization in apicalprotruding structures of dividing neuroepithelial progenitor cells [8]. This novel information is two fold of importance. First, it suggests that the endosomal compartment hosts potential stem cell determinants and thus might play an active role in the maintenance of certain stem cell properties. Second, it raises the possibility that CD133-containing lipid rafts, which might include other stem cell determinants, are segregated in mitotic stem cells (present study and Ref. [8]). The cholesterol-based lipid rafts are known to be implicated in several signaling cascades by allowing the formation of active signal transduction complexes, and their presence in the endosomal compartments was recently demonstrated [9]. The consequence of such segregation is that the daughter cell inheriting these “stem cellspecific microdomains” might maintain the stem cell features whereas its sister might undergo a differentiation process. Thus new insights into the self-renewal and differentiation processes of mammalian stem cells might be revealed by further studying the intracellular trafficking of CD133 in mitotic HSCs and by determining the lipid/protein composition of CD133-containing lipid rafts. Acknowledgments We thank Prof. Dr. med. Martin Bornhäuser for the primary hematopoietic stem and mesenchymal stromal cells, which were collected from healthy donors after informed consent and approval of the local ethics committee. Dr. Hella Hartmann and Jean-Christophe Olaya for their help with microscopic imaging. Dr. Christine A. Fargeas for critical review of the paper. D.C. is supported by the Deutsche Forschungsgemeinschaft (SFB/TR13-04 B1; SFB 655 A13). References [1] D. Corbeil, K. Röper, A. Weigmann, et al., AC133 hematopoietic stem cell antigen — human homologue of mouse kidney prominin or distinct member of a novel protein family, Blood 91 (1998) 2625–2626. [2] K. Röper, D. Corbeil, W.B. Huttner, Retention of prominin in microvilli reveals distinct cholesterol-based lipid micro-domains in the apical plasma membrane, Nat. Cell Biol. 2 (2000) 582–592. [3] C.A. Fargeas, A.-V. Fonseca, W.B. Huttner, et al., Prominin-1 (CD133): from progenitor cells to human diseases, Future Lipidol. 1 (2006) 213–225. [4] N. Bauer, A.-V. Fonseca, M. Florek et al., New insights into the cell biology of hematopoietic progenitors by studying prominin-1 (CD133), Cells Tissue Organs. In press: doi:10.1159/000112847.
Letter to the Editor
195
Fig. 1. Co-localization of CD133 and CD63 in symmetrically and asymmetrically dividing hHSCs. PFA-fixed, saponin-permeabilized hHSCs cultured on hMSCs were tripleimmunolabeled for CD133 (red), CD63 (green) and α-tubulin (white) followed by appropriate secondary antibodies (for technical details see Supplemental Materials available upon request) and observed by confocal laser scanning microscopy. The nuclei were visualized with 4,6-diamidino-2-phenylindole (DAPI; blue). (A) Three optical sections (1 μm each) from the top to the bottom of a mitotic hHSC revealed that CD133 and CD63 partially co-localized in intracellular structures (filled arrows). Note that some structures are only positive for either CD133 (outlined arrows) or CD63 (outlined arrowheads). (B) Both CD markers are either symmetrically (top panels) or asymmetrically (middle and bottom panels) distributed to the two nascent daughter cells. A composite of 6–8 optical sections is shown. Filled arrowheads indicate the midbody. DIC, differential interference contrast. Scale bars = 10 μm.
[5] J. Beckmann, S. Scheitza, P. Wernet, et al., Asymmetric cell division within the human hematopoietic stem and progenitor cell compartment: identification of asymmetrically segregating proteins, Blood 109 (2007) 5494–5501. [6] B. Giebel, J. Beckmann, Asymmetric cell divisions of human hematopoietic stem and progenitor cells meet endosomes, Cell Cycle 6 (2007) 2201–2204. [7] D. Freund, N. Bauer, S. Boxberger, et al., Polarization of human hematopoietic progenitors during contact with multipotent mesenchymal stromal cells — effects on proliferation and clonogenicity, Stem Cells Dev. 15 (2006) 815–829. [8] Y. Kosodo, K. Röper, W. Haubensak, et al., Asymmetric distribution of the apical plasma membrane during neurogenic divisions of mammalian neuroepithelial cells, EMBO J. 23 (2004) 2314–2324. [9] K. Sobo, J. Chevallier, R.G. Parton, et al., Diversity of raft-like domains in late endosomes, PLoS ONE 2 (2007) e391.
A.-V. Fonseca N. Bauer D. Corbeil⁎ Tissue Engineering Laboratories (BIOTEC) and DFG research center and cluster of excellence for Regenerative Therapies Dresden (CRTD), Medical Faculty, Technische Universität Dresden, Tatzberg 47-49, 01307, Dresden, Germany E-mail address: [email protected]. ⁎Corresponding author. Tissue Engineering Laboratories (BIOTEC), Technische Universität Dresden, Tatzberg 47-49, 01307 Dresden, Germany. Fax: +49 351 463 40244.
Contents lists available at ScienceDirect
Blood Cells, Molecules, and Diseases j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y b c m d
Letter to the Editor The stem cell marker CD133 meets the endosomal compartment – New insights into the cell division of hematopoietic stem cells
To the Editor:
The self-renewal capacity of stem and progenitor cells relies on both extrinsic and intrinsic determinants involving for instance specific signaling pathways. Moreover, stem cells have the capacity to distribute asymmetrically certain of their protein components during cell division. In this context, we previously demonstrated that the widely used stem cell marker lipid raftassociated CD133 (prominin-1; AC133 antigen) [1,2] can be either symmetrically or asymmetrically distributed in mitotic human hematopoietic stem cells (hHSCs) cultured on primary human multipotent mesenchymal stromal cells (hMSCs) [3]. The same observation was made using hHSCs growing in stroma-free cultures, indicating that such phenomenon is independent of external clues [4]. More recently, Beckmann et al. have reported that four membrane proteins (CD53, CD62L, CD63 and CD71) segregate differentially in dividing hHSCs cultured in a stroma-free culture [5]. Since three of them (CD53, CD63 and CD71) are endosomal-associated proteins, these exciting results suggested a link between the endosomal compartment and symmetric/asymmetric cell division in rare primitive mammalian cells (see review Ref. [6] and references therein). Does CD133 associate with the endosomal compartment of mitotic hHSCs as described for the intracellular pool of CD63 and CD71 [5]? The answer is yes. We could observe, using a tripleimmunolabeling procedure on paraformaldehyde (PFA)-fixed, saponin-permeabilized hHSCs cultured on hMSCs [7], that CD133 and CD63 partly co-localized in the two nascent daughter cells during cytokinesis (Fig. 1A, filled arrows). More importantly, both CD markers are either equally or unequally distributed between the daughter cells (Fig. 1B, top and middle/bottom panels, respectively). Quantitative analysis revealed that CD133 was distributed asymmetrically in 22.3 ± 0.7% of cell divisions (n = 90; data obtained from three independent experiments using distinct donors). The absence of permeabilization prior to the CD133 labeling procedure likely explains why Beckmann et al. didn't detect CD133-positive intracellular structures in hHSCs [5]. Indeed, the authors only analysed the intracellular pool of CD63 and CD71 upon Triton X-100permeabilization (see Fig. 5B in Ref. [5]). However, it is interesting to note that the asymmetric distribution of these two proteins was observed in ≈ 20% of the mitotic cells, which is in agreement with our present data concerning CD133. We did not detect CD133 at the level of the hHSC midbody during cytokinesis (Figs. 1A, B, filled arrowheads), which is in contrast to the Beckmann' observation [5]. Such discrepancy might be explained, at least in part, by
1079-9796/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bcmd.2008.04.004
the fact that CD133 therein represents only a minute population of the total CD133 molecules and might therefore be under the detection level in our culture system. Alternatively, hHSCs derived from distinct sources, i.e. leukapheresis product (present study; see also Ref. [3,4]) versus umbilical cord blood [5], might behave differently. The symmetric versus asymmetric distribution of CD133 is not unique to hematopoietic cells since a similar phenomenon has been reported for the neuroepithelial progenitor cells [8]. However, the endosomal compartmentalization of CD133 described here is without precedent, and contrasts with its exclusive localization in apicalprotruding structures of dividing neuroepithelial progenitor cells [8]. This novel information is two fold of importance. First, it suggests that the endosomal compartment hosts potential stem cell determinants and thus might play an active role in the maintenance of certain stem cell properties. Second, it raises the possibility that CD133-containing lipid rafts, which might include other stem cell determinants, are segregated in mitotic stem cells (present study and Ref. [8]). The cholesterol-based lipid rafts are known to be implicated in several signaling cascades by allowing the formation of active signal transduction complexes, and their presence in the endosomal compartments was recently demonstrated [9]. The consequence of such segregation is that the daughter cell inheriting these “stem cellspecific microdomains” might maintain the stem cell features whereas its sister might undergo a differentiation process. Thus new insights into the self-renewal and differentiation processes of mammalian stem cells might be revealed by further studying the intracellular trafficking of CD133 in mitotic HSCs and by determining the lipid/protein composition of CD133-containing lipid rafts. Acknowledgments We thank Prof. Dr. med. Martin Bornhäuser for the primary hematopoietic stem and mesenchymal stromal cells, which were collected from healthy donors after informed consent and approval of the local ethics committee. Dr. Hella Hartmann and Jean-Christophe Olaya for their help with microscopic imaging. Dr. Christine A. Fargeas for critical review of the paper. D.C. is supported by the Deutsche Forschungsgemeinschaft (SFB/TR13-04 B1; SFB 655 A13). References [1] D. Corbeil, K. Röper, A. Weigmann, et al., AC133 hematopoietic stem cell antigen — human homologue of mouse kidney prominin or distinct member of a novel protein family, Blood 91 (1998) 2625–2626. [2] K. Röper, D. Corbeil, W.B. Huttner, Retention of prominin in microvilli reveals distinct cholesterol-based lipid micro-domains in the apical plasma membrane, Nat. Cell Biol. 2 (2000) 582–592. [3] C.A. Fargeas, A.-V. Fonseca, W.B. Huttner, et al., Prominin-1 (CD133): from progenitor cells to human diseases, Future Lipidol. 1 (2006) 213–225. [4] N. Bauer, A.-V. Fonseca, M. Florek et al., New insights into the cell biology of hematopoietic progenitors by studying prominin-1 (CD133), Cells Tissue Organs. In press: doi:10.1159/000112847.
Letter to the Editor
195
Fig. 1. Co-localization of CD133 and CD63 in symmetrically and asymmetrically dividing hHSCs. PFA-fixed, saponin-permeabilized hHSCs cultured on hMSCs were tripleimmunolabeled for CD133 (red), CD63 (green) and α-tubulin (white) followed by appropriate secondary antibodies (for technical details see Supplemental Materials available upon request) and observed by confocal laser scanning microscopy. The nuclei were visualized with 4,6-diamidino-2-phenylindole (DAPI; blue). (A) Three optical sections (1 μm each) from the top to the bottom of a mitotic hHSC revealed that CD133 and CD63 partially co-localized in intracellular structures (filled arrows). Note that some structures are only positive for either CD133 (outlined arrows) or CD63 (outlined arrowheads). (B) Both CD markers are either symmetrically (top panels) or asymmetrically (middle and bottom panels) distributed to the two nascent daughter cells. A composite of 6–8 optical sections is shown. Filled arrowheads indicate the midbody. DIC, differential interference contrast. Scale bars = 10 μm.
[5] J. Beckmann, S. Scheitza, P. Wernet, et al., Asymmetric cell division within the human hematopoietic stem and progenitor cell compartment: identification of asymmetrically segregating proteins, Blood 109 (2007) 5494–5501. [6] B. Giebel, J. Beckmann, Asymmetric cell divisions of human hematopoietic stem and progenitor cells meet endosomes, Cell Cycle 6 (2007) 2201–2204. [7] D. Freund, N. Bauer, S. Boxberger, et al., Polarization of human hematopoietic progenitors during contact with multipotent mesenchymal stromal cells — effects on proliferation and clonogenicity, Stem Cells Dev. 15 (2006) 815–829. [8] Y. Kosodo, K. Röper, W. Haubensak, et al., Asymmetric distribution of the apical plasma membrane during neurogenic divisions of mammalian neuroepithelial cells, EMBO J. 23 (2004) 2314–2324. [9] K. Sobo, J. Chevallier, R.G. Parton, et al., Diversity of raft-like domains in late endosomes, PLoS ONE 2 (2007) e391.
A.-V. Fonseca N. Bauer D. Corbeil⁎ Tissue Engineering Laboratories (BIOTEC) and DFG research center and cluster of excellence for Regenerative Therapies Dresden (CRTD), Medical Faculty, Technische Universität Dresden, Tatzberg 47-49, 01307, Dresden, Germany E-mail address: [email protected]. ⁎Corresponding author. Tissue Engineering Laboratories (BIOTEC), Technische Universität Dresden, Tatzberg 47-49, 01307 Dresden, Germany. Fax: +49 351 463 40244.