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Cell-based therapies using adult stem cells are promising options for the treatment of a number of diseases including autoimmune and cardiovascular disorders. Among these, vascular wall-derived mesenchymal stem cells (VW-MSCs) might be particularly well suited for the protection and curative treatment of vascular damage because of their tissue-specific action. Here we report a novel method for the direct conversion of human skin fibroblasts towards MSCs using a VW-MSC-specific gene code (HOXB7, HOXC6 and HOXC8) that directs cell fate conversion bypassing pluripotency. This direct programming approach using either a self-inactivating (SIN) lentiviral vector expressing the VW-MSC-specific HOX-code or a tetracycline-controlled Tet-On system for doxycycline-inducible gene expressions of HOXB7, HOXC6 and HOXC8 successfully mediated the generation of VW-typical MSCs with classical MSC characteristics in vitro and in vivo. The induced VW-MSCs (iVW-MSCs) fulfilled all criteria of MSCs as defined by the International Society for Cellular Therapy (ISCT). In terms of multipotency and clonogenicity, which are important specific properties to discriminate MSCs from fibroblasts, iVW-MSCs behaved like primary ex vivo isolated VW-MSCs and shared similar molecular and DNA methylation signatures. With respect to their therapeutic potential, these cells suppressed lymphocyte proliferation in vitro, and protected mice against vascular damage in a mouse model of radiation-induced pneumopathy in vivo, as well as ex vivo cultured human lung tissue. The feasibility to obtain patient-specific VW-MSCs from fibroblasts in large amounts by a direct conversion into induced VW-MSCs could potentially open avenues towards novel, MSC-based therapies.

Generation of hematopoietic stem cells (HSCs) from pluripotent stem cells, in vitro, holds great promise for regenerative therapies. Primarily, this has been achieved in mouse cells by overexpression of the homeotic selector protein HOXB4. The exact cellular stage at which HOXB4 promotes hematopoietic development, in vitro, is not yet known. However, its identification is a prerequisite to unambiguously identify the molecular circuits controlling hematopoiesis, since the activity of HOX proteins is highly cell and context dependent. To identify that stage, we retrovirally expressed HOXB4 in differentiating mouse embryonic stem cells (ESCs). Through the use of Runx1 (-/-) ESCs containing a doxycycline-inducible Runx1 coding sequence, we uncovered that HOXB4 promoted the formation of hemogenic endothelium cells without altering endothelial cell development. Whole-transcriptome analysis revealed that its expression mediated the upregulation of transcription of core transcription factors necessary for hematopoiesis, culminating in the formation of blood progenitors upon initiation of Runx1 expression., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

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.

The ability of embryonic stem (ES) cells to form cells and tissues from all 3 germ layers can be exploited to generate cells that can be used to treat diseases. In particular, successful generation of hematopoietic cells from ES cells could provide safer and less immunogenic cells than bone marrow cells, which require severe host preconditioning when transplanted across major histocompatibility complex barriers. Here, we exploited the self-renewal properties of ectopically expressed HOXB4, a homeobox transcription factor, to generate hematopoietic progenitor cells (HPCs) that successfully induce high-level mixed chimerism and long-term engraftment in recipient mice. The HPCs partially restored splenic architecture in Rag2(-/-)gamma(c)(-/-)-immunodeficient mice. In addition, HPC-derived newly generated T cells were able to mount a peptide-specific response to lymphocytic choriomeningitis virus and specifically secreted interleukin-2 and interferon-gamma upon CD3 stimulation. In addition, HPC-derived antigen presenting cells in chimeric mice efficiently presented viral antigen to wild-type T cells. These results demonstrate for the first time that leukocytes derived from ES cells ectopically expressing HOXB4 are immunologically functional, opening up new opportunities for the use of ES cell-derived HPCs in the treatment of hematologic and immunologic diseases.

Homeodomain-containing transcription factors are important regulators of stem cell behavior. HOXB4 mediates expansion of adult and embryo-derived hematopoietic stem cells (HSCs) when expressed ectopically. To define the underlying molecular mechanisms, we performed gene expression profiling in combination with subsequent functional analysis with enriched adult HSCs and embryonic derivatives expressing inducible HOXB4. Thereby, we identified a set of overlapping genes that likely represent "universal" targets of HOXB4. A substantial number of loci are involved in signaling pathways important for controlling self-renewal, maintenance, and differentiation of stem cells. Functional assays performed on selected pathways confirmed the biological coherence of the array results. HOXB4 activity protected adult HSCs from the detrimental effects mediated by the proinflammatory cytokine TNF-alpha. This protection likely contributes to the competitive repopulation advantage of HOXB4-expressing HSCs observed in vivo. The concept of TNF-alpha inhibition may also prove beneficial for patients undergoing bone marrow transplantation. Furthermore, we demonstrate that HOXB4 activity and FGF signaling are intertwined. HOXB4-mediated expansion of adult and ES cell-derived HSCs was enhanced by specific and complete inhibition of FGF receptors. In contrast, the expanding activity of HOXB4 on hematopoietic progenitors in day 4-6 embryoid bodies was blunted in the presence of basic FGF (FGF2), indicating a dominant negative effect of FGF signaling on the earliest hematopoietic cells. In summary, our results strongly suggest that HOXB4 modulates the response of HSCs to multiple extrinsic signals in a concerted manner, thereby shifting the balance toward stem cell self-renewal.

Ectopic expression of the homeodomain transcription factor HOXB4 expands hematopoietic stem and progenitor cells in vivo and in vitro, making HOXB4 a highly interesting candidate for therapeutic stem cell expansion. However, when expressed at high levels, HOXB4 concomitantly perturbs differentiation and thus likely predisposes the manipulated cells for leukemogenesis. We therefore asked whether the expression level of HOXB4 may be a critical parameter that influences the growth and transformation properties of transduced cells. Using a set of retroviral vectors which covered a 40-fold range of expression levels, we studied the consequences of HOXB4 expression at different levels in the well established Rat-1 fibroblast cell system. HOXB4 transformed Rat-1 fibroblasts beyond a certain threshold level of expression. Further escalation of HOXB4 expression, however, did not enhance transformation. Instead, HOXB4 mediated a dose dependent anti-proliferative effect on Rat-1 and NIH3T3 fibroblasts. This effect was aggravated under reduced serum concentrations and was, at least partially, due to an enhanced sensitivity of HOXB4 overexpressing cells to induction of apoptosis. Based on these results we propose that HOXB4 affects cell growth in a dose-dependent manner by sensitizing cells towards extrinsic signals.

Genetic manipulation of hematopoietic stem and progenitor cells is an important tool for experimental and clinical applied hematology. However, techniques that allow for gene targeting, subsequent in vitro selection, and expansion of genetically defined clones are available only for ES cells. Such molecularly defined and, hence, "safe" clones would be highly desirable for somatic gene therapy. Here, we demonstrate that in vitro differentiated ES cells completely recapitulate the growth and differentiation properties of adult bone marrow cells, in vitro and in vivo, when ectopically expressing HOXB4. Myeloid development was enforced and (T) lymphoid development suppressed over a wide range of expression levels, whereas only high expression levels of the transcription factor were detrimental for erythroid development. This indicates a close association between the amounts of ectopic HOXB4 present within a progenitor cell and and the decision to self renew or differentiate. Because HOXB4 mediates similar fates of ES-derived and bone marrow hematopoietic stem cells, the primitive embryonic cells can be considered a promising alternative for investigating hematopoietic reconstitution, in vivo, based on well defined clones. Provided that HOXB4 levels are kept within a certain therapeutic window, ES cells also carry the potential of efficient and safe somatic gene therapy.

The homeodomain transcription factor HOXB4 is one of the most attractive tools to expand hematopoietic stem cells in vitro and in vivo and to promote the formation of hematopoietic cells from in vitro differentiated embryonic stem cells. However, the expression levels compatible with the favorable effect of enhanced self-renewal without perturbing differentiation, in vivo, remain to be determined. In this paper, we discuss the necessity to define the "therapeutic width" of HOXB4 expression, based on observations from our lab and others that demonstrate that ectopic HOXB4 expression leads to a concentration-dependent perturbation of lineage differentiation of mouse and human hematopoietic cells. In summary, the combined results argue in favor of the existence of certain threshold levels for HOXB4 activity that control the differentiation and self-renewal behavior of hematopoietic stem and progenitor cells. Indeed, existing evidence suggests that dosage effects of ectopically expressed transcription factors may be more the rule than an exception.

Ectopic retroviral expression of homeobox B4 (HOXB4) causes an accelerated and enhanced regeneration of murine hematopoietic stem cells (HSCs) and is not known to compromise any program of lineage differentiation. However, HOXB4 expression levels for expansion of human stem cells have still to be established. To test the proposed hypothesis that HOXB4 could become a prime tool for in vivo expansion of genetically modified human HSCs, we retrovirally overexpressed HOXB4 in purified cord blood (CB) CD34+ cells together with green fluorescent protein (GFP) as a reporter protein, and evaluated the impact of ectopic HOXB4 expression on proliferation and differentiation in vitro and in vivo. When injected separately into nonobese diabetic-severe combined immunodeficient (NOD/SCID) mice or in competition with control vector-transduced cells, HOXB4-overexpressing cord blood CD34+ cells had a selective growth advantage in vivo, which resulted in a marked enhancement of the primitive CD34+ subpopulation (P =.01). However, high HOXB4 expression substantially impaired the myeloerythroid differentiation program, and this was reflected in a severe reduction of erythroid and myeloid progenitors in vitro (P <.03) and in vivo (P =.01). Furthermore, HOXB4 overexpression also significantly reduced B-cell output (P <.01). These results show for the first time unwanted side effects of ectopic HOXB4 expression and therefore underscore the need to carefully determine the therapeutic window of HOXB4 expression levels before initializing clinical trials.