Abstract
Differentiation of pluripotent embryonic stem (ES) cells can recapitulate many aspects of hematopoiesis, in vitro, and can even generate cells capable of long-term multilineage repopulation after transplantation into recipient mice, when the homeodomain transcription factor HOXB4 is ectopically expressed. Thus, the ES-cell differentiation system is of great value for a detailed understanding of the process of blood formation. Furthermore, it is also promising for future application in hematopoietic cell and gene therapy. Since the arrival of techniques which allow the reprogramming of somatic cells back to an ES cell-like state, the generation of hematopoietic stem cells from patient-specific so-called induced pluripotent stem cells shows great promise for future therapeutic applications. In this chapter, we describe how to cultivate a certain feeder cell-independent mouse embryonic stem cell line, to manipulate these cells by retroviral gene transfer to ectopically express HOXB4, to differentiate these ES cells via embryoid body formation, and to selectively expand the arising, HOXB4-expressing hematopoietic stem and progenitor cells.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Punzel M, Moore KA, Lemischka IR, Verfaillie CM (1999) The type of stromal feeder used in limiting dilution assays influences frequency and maintenance assessment of human long-term culture initiating cells. Leukemia 13:92–97
Zhang CC, Kaba M, Ge G, Xie K, Tong W, Hug C, Lodish HF (2006) Angiopoietin-like proteins stimulate ex vivo expansion of hematopoietic stem cells. Nat Med 12:240–245
Zhang CC, Kaba M, Iizuka S, Huynh H, Lodish HF (2008) Angiopoietin-like 5 and IGFBP2 stimulate ex vivo expansion of human cord blood hematopoietic stem cells as assayed by NOD/SCID transplantation. Blood 111:3415–3423
Antonchuk J, Sauvageau G, Humphries RK (2002) HOXB4-induced expansion of adult hematopoietic stem cells ex vivo. Cell 109:39–45
Sauvageau G, Thorsteinsdottir U, Eaves CJ, Lawrence HJ, Largman C, Lansdorp PM, Humphries RK (1995) Overexpression of HOXB4 in hematopoietic cells causes the selective expansion of more primitive populations in vitro and in vivo. Genes Dev 9:1753–1765
Schiedlmeier B, Klump H, Will E, Arman-Kalcek G, Li Z, Wang Z, Rimek A, Friel J, Baum C, Ostertag W (2003) High-level ectopic HOXB4 expression confers a profound in vivo competitive growth advantage on human cord blood CD34+ cells, but impairs lymphomyeloid differentiation. Blood 101:1759–1768
Challen GA, Boles N, Lin KK, Goodell MA (2009) Mouse hematopoietic stem cell identification and analysis. Cytometry A 75:14–24
Giebel B, Punzel M (2008) Lineage development of hematopoietic stem and progenitor cells. Biol Chem 389:813–824
Kiel MJ, Yilmaz OH, Iwashita T, Terhorst C, Morrison SJ (2005) SLAM family receptors distinguish hematopoietic stem and progenitor cells and reveal endothelial niches for stem cells. Cell 121:1109–1121
McKenzie JL, Takenaka K, Gan OI, Doedens M, Dick JE (2007) Low rhodamine 123 retention identifies long-term human hematopoietic stem cells within the Lin-CD34+CD38− population. Blood 109:543–545
Kyba M, Perlingeiro RC, Daley GQ (2002) HoxB4 confers definitive lymphoid-myeloid engraftment potential on embryonic stem cell and yolk sac hematopoietic progenitors. Cell 109:29–37
Pilat S, Carotta S, Schiedlmeier B, Kamino K, Mairhofer A, Will E, Modlich U, Steinlein P, Ostertag W, Baum C, Beug H, Klump H (2005) HOXB4 enforces equivalent fates of ES-cell-derived and adult hematopoietic cells. Proc Natl Acad Sci U S A 102:12101–12106
Chan KM, Bonde S, Klump H, Zavazava N (2008) Hematopoiesis and immunity of HOXB4-transduced embryonic stem cell-derived hematopoietic progenitor cells. Blood 111:2953–2961
Pilat S, Carotta S, Schiedlmeier B, Kamino K, Mairhofer A, Schmidt M, von Kalle C, Steinlein P, Ostertag W, Baum C, Beug H, Klump H (2005) Hematopoietic repopulation, in vivo, with genetically defined clones derived from HOXB4 expressing ES-cells. ASH Annual Meeting Abstracts 106:196
Klump H, Schiedlmeier B, Baum C (2005) Control of self-renewal and differentiation of hematopoietic stem cells: HOXB4 on the threshold. Ann N YAcad Sci 1044:1–10
Bonde S, Chan KM, Zavazava N (2008) ES-cell derived hematopoietic cells induce transplantation tolerance. PLoS One 3:e3212
Schiedlmeier B, Santos AC, Ribeiro A, Moncaut N, Lesinski D, Auer H, Kornacker K, Ostertag W, Baum C, Mallo M, Klump H (2007) HOXB4’s road map to stem cell expansion. Proc Natl Acad Sci U S A 104: 16952–16957
Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, Yamanaka S (2007) Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131:861–872
Takahashi K, Yamanaka S (2006) Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126:663–676
Wernig M, Meissner A, Foreman R, Brambrink T, Ku M, Hochedlinger K, Bernstein BE, Jaenisch R (2007) In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state. Nature 448:318–324
Park IH, Arora N, Huo H, Maherali N, Ahfeldt T, Shimamura A, Lensch MW, Cowan C, Hochedlinger K, Daley GQ (2008) Disease-specific induced pluripotent stem cells. Cell 134:877–886
Kingston RE, Chen CA, Rose JK (2003) Calcium phosphate transfection. Curr Protoc Mol Biol Chapter 9:Unit 9 1
Robertson E, Bradley A, Kuehn M, Evans M (1986) Germ-line transmission of genes introduced into cultured pluripotential cells by retroviral vector. Nature 323:445–448
Bonde S, Zavazava N (2006) Immunogenicity and engraftment of mouse embryonic stem cells in allogeneic recipients. Stem Cells 24:2192–2201
Bonde S, Dowden AM, Chan KM, Tabayoyong WB, Zavazava N (2008) HOXB4 but not BMP4 confers self-renewal properties to ES-derived hematopoietic progenitor cells. Transplantation 86:1803–1809
Klump H, Schiedlmeier B, Vogt B, Ryan M, Ostertag W, Baum C (2001) Retroviral vector-mediated expression of HoxB4 in hematopoietic cells using a novel coexpression strategy. Gene Ther 8:811–817
Will E, Speidel D, Wang Z, Ghiaur G, Rimek A, Schiedlmeier B, Williams DA, Baum C, Ostertag W, Klump H (2006) HOXB4 inhibits cell growth in a dose-dependent manner and sensitizes cells towards extrinsic cues. Cell Cycle 5:14–22
Ferkowicz MJ, Starr M, Xie X, Li W, Johnson SA, Shelley WC, Morrison PR, Yoder MC (2003) CD41 expression defines the onset of primitive and definitive hematopoiesis in the murine embryo. Development 130: 4393–4403
Mikkola HK, Fujiwara Y, Schlaeger TM, Traver D, Orkin SH (2003) Expression of CD41 marks the initiation of definitive hematopoiesis in the mouse embryo. Blood 101:508–516
Tung JW, Parks DR, Moore WA, Herzenberg LA (2004) New approaches to fluorescence compensation and visualization of FACS data. Clin Immunol 110:277–283
Donnelly ML, Luke G, Mehrotra A, Li X, Hughes LE, Gani D, Ryan MD (2001) Analysis of the aphthovirus 2A/2B polyprotein ‘cleavage’ mechanism indicates not a proteolytic reaction, but a novel translational effect: a putative ribosomal ‘skip’. J Gen Virol 82:1013–1025
Acknowledgments
This work is dedicated to the memory of Wolfram Ostertag (1937–2010) and Hartmut Beug (1945–2011).
As well as Bernd Schiedlmeier, Christopher Baum (Department of Experimental Hematology, Hannover Medical School), and Bernd Giebel and Peter Horn (Institute for Transfusion Medicine, University Hospital Essen) for their steady support and continuing fruitful discussions.
This work was supported by grants of the Deutsche Forschungsgemeinschaft [KL 1311/4-2; KL1311/5-1, cluster of excellence “REBIRTH” (Exc 62)] and by the NIH (grant number RO1-HL073015-06, subaward No. 1000830668).
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2013 Springer Science+Business Media New York
About this protocol
Cite this protocol
Pilat, S., Carotta, S., Klump, H. (2013). Development of Hematopoietic Stem and Progenitor Cells from Mouse Embryonic Stem Cells, In Vitro, Supported by Ectopic Human HOXB4 Expression. In: Zavazava, N. (eds) Embryonic Stem Cell Immunobiology. Methods in Molecular Biology, vol 1029. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-478-4_10
Download citation
DOI: https://doi.org/10.1007/978-1-62703-478-4_10
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-477-7
Online ISBN: 978-1-62703-478-4
eBook Packages: Springer Protocols