In vitro expansion of hematopoietic stem cells by recombinant TAT-HOXB4 protein

Abstract

Hematopoietic stem cells (HSCs) can self-renew extensively after transplantation. The conditions supporting their in vitro expansion are still being defined. Retroviral overexpression of the human homeobox B4 (HOXB4) gene in mouse bone marrow cells enables over 40-fold expansion of HSCs in vitro. To circumvent the requirement for retroviral infection, we used recombinant human TAT-HOXB4 protein carrying the protein transduction domain of the HIV transactivating protein (TAT) as a potential growth factor for stem cells. HSCs exposed to TAT-HOXB4 for 4 d expanded by about four- to sixfold and were 8-20 times more numerous than HSCs in control cultures, indicating that HSC expansion induced by TAT-HOXB4 was comparable to that induced by the human HOXB4 retrovirus during a similar period of observation. Our results also show that TAT-HOXB4-expanded HSC populations retain their normal in vivo potential for differentiation and long-term repopulation. It is thus feasible to exploit recombinant HOXB4 protein for rapid and significant ex vivo expansion of normal HSCs.

Near-maximal expansions of hematopoietic stem cells in culture using NUP98-HOX fusions

“…Together with recent publication revealing the potency of another NUP98-HOX fusion to increase the numbers of human long-term culture-initiating cells [43,44], our findings raise optimism for future extensions of this technology to enable even greater levels of HSC expansion to be obtained in vitro after longer times and/or different growth factor conditions. The approach described here also appears ideally suited to the type of protein delivery system afforded using TAT-fusion protein technology [25], which might allow HSC expansion without gene manipulation and be of considerable interest for both basic and clinical applications to human HSCs. Figure 4A) was analyzed by Southern blotting for proviral integrants as described in the in the Methods.…”

“…A striking example of the latter strategy is the use of retrovirally engineered overexpression of the homeobox transcription factor HOXB4 to stimulate expansions of HSC numbers in vitro of up to 80-fold [16-20] with evidence of similarly enhanced HSC expansion in vivo [21][22][23][24]. Significant enhancement of HSC self-renewal has also been elicited by repeated delivery of HOXB4 protein to the cell as an exogenous supplied TAT-HOX fusion protein [25].The studies described here were designed to determine whether even greater HSC expansions might be achieved by forced overexpression of modified HOX-containing fusion proteins. Assuming a HSC cell cycle time of 12 to 14 hours [26,27], the continuous execution of symmetric self-renewal divisions, and no cell death, the maximum expansion of HSCs that would be predicted to occur over a period of 5 to 8 days is 1000-fold, i.e., greater than 10 times that found to be achieved by overexpressing HOXB4.…”

“…A striking example of the latter strategy is the use of retrovirally engineered overexpression of the homeobox transcription factor HOXB4 to stimulate expansions of HSC numbers in vitro of up to 80-fold [16][17][18][19][20] with evidence of similarly enhanced HSC expansion in vivo [21][22][23][24]. Significant enhancement of HSC self-renewal has also been elicited by repeated delivery of HOXB4 protein to the cell as an exogenous supplied TAT-HOX fusion protein [25].…”

Ex vivo expansion of hematopoietic stem cells

“…Conditional derivatives of certain growth factor receptors have also been used to support HSC expansion in culture [76,77]. The introduction of exogenous transcription factors such as homeotic protein HoxB4 can induce dramatic expansion of HSCs [54,56,[78][79][80].…”

HOXB4 et expansion des cellules souches hématopoïétiques humaines primitives

“…Nous avons montré que ces cellules amplifiées gardaient, au cours de leur différenciation chez l'animal, leur pluripotentialité intacte [4]. Cette capacité de HOXB4 d'amplifier les CSH sous la forme d'une protéine exogène ajoutée aux cultures a été confirmée dans un modèle où HOXB4 était fusionnée à la protéine transactivatrice TAT du VIH (cette protéine ayant les mêmes propriétés de transduction passive que l'homéodomaine des protéines HOX) et utilisée dans des cultures de cellules murines [5]. Au total, nous avons mis au point un système permettant l'amplification des CSH humaines primitives sans ajout de cytokines et sans modification génétique de ces cellules.…”