The formation of tissues and organs from naïve stem and progenitor cells is controlled by combinatorial signaling of certain pathways in distinct temporal windows to progressively direct embryonic cells through a series of fate decisions into specific tissue lineages. Thus, stem cell functions in adults from quiescence through “activation” resulting in self-renewal, mobilization or differentiation require tightly control by these signaling networks and complex cross-talks between the different signaling pathways (Blank et al., 2008; Denner et al., 2010; Tanabe, 2015). Fibroblast growth factor (FGF) and leukemia inhibitory factor mediated signaling turned out to be of critical importance in regulating key transcriptional regulators of pluripotency, including sex determining region Y box 2 (SOX2), octamer-binding transcription factor (OCT) 3/4, krueppel-like factor 4 (KLF4), NANOG, and c-MYC (Coutu and Galipeau, 2011; Theunissen and Jaenisch, 2014; Tanabe, 2015; Mossahebi-Mohammadi et al., 2020). Signaling pathways that further regulate cell fate decisions particularly stem cell maintenance, self-renewal and differentiation, include: (i) Wingless related integration site (WNT) molecules supposed to act as niches factors maintaining a self-renewing state (Nusse, 2008; Van Camp et al., 2014), (ii) bone morphogenetic proteins (BMPs) that belong to the transformation growth factor beta (TGFβ) superfamily critically controlling differentiation (including epithelial and mesenchymal transitions) and cell death (Wagner, 2007; Sakaki-Yumoto et al., 2013), (iii) sonic hedgehog (SHH) signaling that is an important player for ASC maintenance (Petrova and Joyner, 2014), (iv) NOTCH signaling known to be responsible for maintaining balance between the different cell fates (Chiba, 2006; Wang et al., 2009), as well as (v) small molecules such as retinoic acid (RA) that orchestrate FGF signaling to drive differentiation, and thus decisively impact on balancing of stem cell quiescence and activation (Stavridis et al., 2010; Gudas and Wagner, 2011) (Figure 1). These signaling pathways in turn modulate the expression of homeotic selector (HOX) transcription factor genes, known as master regulators of cellular fate, in a spatio-temporal manner, finally balancing stem cell maintenance, proliferation, differentiation, and survival. However, it remains be unraveled how these signaling pathways and decisive molecular pathway candidates here mediate full colinear HOX activation and enable deterministic patterning of diverse cell-type specific HOX profiles, which could potentially be used to recapitulate HOX expression profiles e.g., for the generation of target cells or for an on-site manipulation of HOX signaling alterations in diseased states.

The positional identity that emerged from HOX gene expression patterns during embryonic development was found to be maintained in many adult tissues, particularly in ASCs, suggesting that the topographic specificity of these HOX codes as an intrinsic property is maintained during differentiation, and thus provides a mechanism for imposing cell identity and fate restriction (Yamamoto et al., 2003; Smith et al., 2019). Indeed, ASCs were found to exhibit characteristic HOX expression signatures that are heterogeneous but highly specific for their anatomical origin (Figure 3). The persistent expression of specific HOX genes in many adult tissues indicates that, in addition to the cellular identity, the function of these cells and respective organ tissues containing them is also determined by HOX genes meaning that HOX genes were co-opted for location-specific functions (Wellik, 2009; Krumlauf and Ahn, 2013; Quinonez and Innis, 2014). Due to the large number of findings in the ASCs, the following focus concerning stem cell-type specific HOX gene expression pattern and how this is related to stem cell function is rather on mesodermal stem cells: MSCs, HSCs and endothelial progenitor cells (EPCs).

Human MSCs can be isolated and expanded from almost every organ, preferred however from bone marrow, peripheral blood and various neonatal birth-associated tissues, blood vessels, and adipose tissue. Although these MSCs were phenotypically highly similar, MSC cultures are generally quite heterogenous. According to the minimal MSC criteria (as defined by the International Society of Cell Therapy) in vitro expanded MSC must adhere on plastic, express the MSC markers CD73, CD90 and CD105 while lacking expression of hematopoietic and endothelial lineage markers, and exhibit the capability of in vitro differentiation into adipocyte, osteoblast, and chondrocyte lineages (Dominici et al., 2006; Viswanathan et al., 2019).

Compared gene expression profiling of MSCs derived from bone marrow (BM), adipose tissue and cord blood identified 25 well-characterized genes including the two HOX genes HOXA5 and HOXB6 in all MSC preparations, irrespective of origin and culture conditions (Wagner et al., 2006). However, none of these genes alone was specific for MSCs and the definition of a unique marker, particularly a HOX gene or a HOX gene constellation, for MSCs remained elusive (Wagner et al., 2006; Ho et al., 2008). Investigations using murine MSCs (identified as colony forming unit-fibroblasts) from different organs firstly revealed that MSCs of different origins -although phenotypically highly similar- can be distinguished by their specific topographic HOX code depending on the tissue of origin (Ackema and Charite, 2008). Consistent with the assumption that the HOX code is an intrinsic property of ASCs, it was hypothesized that the MSC-type specific HOX code is part of a “blueprint” required for the tissue-specific (regenerative) action of MSCs (Ackema and Charite, 2008). Accordingly, it was suggested that MSC-specific HOX expression patterns as “biological fingerprint” could be used to distinguish human MSC populations of functionally distinct tissues (Liedtke et al., 2010). HOXA9, HOXB7, HOXC10 and HOXD8 (and to a lesser extend HOXA10 and HOXC6) were defined as potential molecular markers, which are highly expressed in fetal MSC derived from cord blood (CB-MSCs; compared to USSC) (Jansen et al., 2010; Liedtke et al., 2010). The HOXA cluster here turned out to be highly methylated, whereas the HOXB-D clusters are less methylated indicating that HOX genes from these clusters (HOXB6,7, HOXC4-10, HOXD3,4) are involved in CB-MSC maintenance and function (Liedtke et al., 2010). BM-MSCs showed a highly similar HOX code compared to CB-MSCs identifying HOXD9 and HOXD10 (being absent in BM-MSC) as decisive HOX genes enabling distinction (Liedtke et al., 2010). HOXC10 (being present in Decidua-derived MSCs) was further defined as a potential marker to distinguish amnion- and decidua-derived MSCs (Hwang et al., 2009). And HOXC10 (together with T-box 15 transcription factor) seemed to be a fundamental developmental transcription factor in adipose tissue-derived MSCs (AT-MSCs) (AD-MSCs) (Onate et al., 2013; Miklosz et al., 2022). Furthermore, increased expressions of HOXA10, HOXC6, HOXD8, and to a lesser extend of HOXA9, HOXB2, HOXB3, HOXC9, HOXC10 were reported in these cells (Kouidhi et al., 2015). BM-MSCs, as one of the commonly used sources for MSCs, usually show increased expressions of HOXA9, HOXA10, HOXB4, HOXB7, HOXC8, HOXC10, and HOXD8 (Coenen et al., 2015). By comparing different human BM-MSCs, namely MSCs isolated from bone marrow isolated from iliac crest, sternum, and vertebrae, it was shown that BM-MSCs at all express HOX genes in high numbers (35/39), with the different BM-MSC populations expressing specific sets of HOX genes in increased levels (Picchi et al., 2013). HOXC10 and HOXC11 were found to be upregulated in iliac crest-MSCs, HOXB8 and HOXB9 in vertebrae-MSCs, and HOXA3, HOXA5, HOXB6, HOXB7, HOXB9, HOXC4 as well as HOXC8 in sternum-MSCs (Picchi et al., 2013). Besides, so-called vascular wall-derived MSCs (VW-MSCs), which are located within the vascular stem cell niche, in the so-called “vasculogenic zone” of the blood vessel wall, were characterized by increased expression levels of HOXB7, HOXC6 and HOXC8; but also HOXA6, HOXA7, HOXB3, HOXB4 and HOXB5 as well as HOXC4 and HOXC5 expression can be detected (Klein et al., 2013). Even lung resident MSCs (LR-MSCs) were shown to express the VW-MSC specific HOX code of HOXB7, HOXC6 and HOXC8 (together with HOXB5), as these cells were found to be predominately located with the vascular adventitial stem cell niche (Klein, 2021; Steens et al., 2021). Accordingly, LR-MSCs turned out to be phenotypically and functionally indistinguishable from VW-MSC, a fact that further highlights the HOX code as master regulator of cellular identity.

Taken together, a distinct expression of HOXA9 and HOXA10, HOXB7, HOXC6, HOXC8, and HOXC10, as well as HOXD8 can frequently be observed in MSCs derived from different human tissues, and thus in certain combinations- potentially represent the MSC-type specific HOX code (Figure 3). It can also be stated that mainly central HOX genes, or HOX genes that are in the immediate vicinity of the central cluster region are expressed within tissue-specific MSCs. But besides their central role in defining segment identity, ensuring that the right structures are formed in the right body places, the activity HOX genes as well as their expression levels control segment-specific structures and cell types. Herein, other less frequently reported HOX genes, e.g. HOXA5, HOXA6 and HOXA7, HOXB6, HOXC4, HOXC5, and HOXC9, as well as HOXD3 might be important contributors to these actions.

The most common and reliable way to identify MSCs (in addition to surface marker analysis), is to verify their trilineage differentiation potential into adipocytes, osteoblasts, and chondrocytes in vivo and in vitro. HOX genes were of functional importance in regulating these differentiation processes [reviewed in (Seifert et al., 2015)]. As revealed from early mouse studies for example, the HOX candidate HOXC8 was shown to critically regulate the progression of cells along the chondrogenic differentiation pathway (Yueh et al., 1998). Particularly in MSCs, HOXC8 expressions were shown to be upregulated during chondrogenic differentiation and a fostered HOXC8 expression caused an enhanced expression of chondrogenic markers, promoted the chondrogenic differentiation and the formation of cartilage clumps (Yang et al., 2020a), while osteogenic differentiation was suppressed (Yang et al., 2020b). A forced expression of HOXC8 was even associated with adipogenesis inhibition (Mori et al., 2012). As another example, HOXA11 and HOXD11 functioned in regulating (the early steps) of chondrocyte differentiation (Gross et al., 2012). Loss of function studies further revealed that HOX11 impairments in MSCs of the bone marrow (and periosteum) at adult stages caused defects in differentiation, leading to an overall deficit in the cartilage production and thus defects in endochondral ossification (Rux et al., 2016). And in certain MSC types, namely in regional skeletal MSCs, which arose from the earliest stages of skeletal development, HOX11 expression turned out to be decisive for their self-renewal and for functioning as progenitors for osteoblasts, chondrocytes and adipocytes throughout lifetime (Pineault et al., 2019). Particularly HOXA cluster genes seem to play a central role here, because HOXA cluster negative cord blood MSCs failed to differentiate properly into the chondro-osteogenic lineages with the HOX candidates HOXA2 and HOXA10 being of special meaning (Liedtke et al., 2017). Herein, HOXA10 in BM-MSCs was already shown enabling RUNX2 activation, a central regulator of osteogenesis (Hassan et al., 2007). Mechanistically HOXA10 mediated chromatin hyperacetylation and H3K4 methylation of the bone-related RUNX2 P1 promoter by interacting through a HOX core motif, that caused activation of early osteogenic genes through the chromatin remodeling (Hassan et al., 2007). HOXC10 was further identified as a direct target of the lncRNA lncHOXC-AS3, which stabilized HOXC10 expression in BM-MSCs thereby regulating osteogenesis (Li et al., 2019).

Thus, it is clear that HOX genes exert MSC-specific functions particularly concerning the trilineage potential along mesodermal lineages. However, the observed differences of HOX genes in regulating similar processes maybe based in the difference of HOX codes orchestrating different but phenotypical highly similar MSC types, which in turn dependent of the tissue of origin. Interestingly, HOXB7 turned out to be an “universal MSC” HOX gene as it was reported to be expressed in all the different types of MSCs (Liedtke et al., 2010; Klein et al., 2013; Foppiani et al., 2019). An induced expression of HOXB7 was identified as a master player driving proliferation and differentiation of human BM- and AT-MSCs (Foppiani et al., 2019; Casari et al., 2021), and accounts for the high proliferation and sprouting potential of VW-MSCs (Klein et al., 2013; Klein, 2016). However, rather than accounting for stem cell type-specific functions, HOX genes specify various regional properties of a tissue along the rostral-caudal axis by regulating a wide range of cellular activities such as proliferation and differentiation, cellular adhesion, migration and invasion, as well as cell death (Parrish et al., 2009). The persisting HOX expression profiles of the different tissue-specific MSCs strongly suggest that those specific HOX profiles are of particular importance for the functioning of tissues throughout adult life with resident HOX-expressing MSCs as leading regulators of embedding stroma renewal and regeneration (Kulebyakina and Makarevich, 2020). Following a rough classification along the cranial-caudal direction, HOX1-4 paralogues were shown to act predominately in cranial tissues, followed by HOX5-6 in subsequent upper sternal tissues, and HOX7-8 in lower sternal and abdominal tissues, whereas the paralogues HOX9-13 represent HOX genes building up HOX codes in caudal body parts and extremities (Kulebyakina and Makarevich, 2020). An example for the association of HOX gene expression within the organization of tissue-specific structures and features comes from VW-MSCs (Klein et al., 2013). Under normal tissue homeostasis (“quiesence”), when residing in the adventitial vasculogenic stem cell niche, these cells express the HOX pattern HOXB7, HOXC6 and HOXC8 at high level that were shown to suppress the expression of transgelin (TAGLN), a TGFβ-inducible gene-inducible gene found to be essentiell for early smooth muscle cell differentiation. Upon commitment, VW-MSCs progress through tissue-specific differentiation events, where a silencing of the VW-specific HOX genes altered the TAGLN promotor methylation sites finally causing increased TAGLN expression, which induced then VW-MSC differentiation into tissue-typical smooth muscle cells (Klein et al., 2013).

Thus, modulation of cell-type specific HOX expression levels as well as respective patterns might offer potential druggable targets for innovative on-site tissue regeneration approaches or for the improved generation of stem cells in vitro, particularly of MSCs with superior repair capabilities. Within that scenario, enforcing trans-differentiation of pluripotent stem cells or even other somatic cell types towards MSCs by ectopic expression of HOX genes as MSC-specific transcription factors turned out to be a straight forward approach to generate MSCs in vitro in huge numbers desirable especially for regenerative purposes (Steens and Klein, 2018; Abdal Dayem et al., 2019). The in vitro generation of vascular wall-typical MSCs from (murine) iPSCs, based on a VW-wall MSC-specific HOX code was already reported (Klein et al., 2013; Steens et al., 2017; Steens et al., 2020a). A lentiviral vector expressing a small set of identified (human) VW-MSCs-specific HOX genes, namely HOXB7, HOXC6 and HOXC8 was here used to directly program murine iPSCs into MSCs, which then displayed classical MSC characteristics, both in vitro and in vivo. As HOX selector genes are highly conserved throughout evolution, it is assumed that forced expression of this HOX code also leads to MSC differentiation from human iPSCs, although the final proof for human iPSCs remains to be shown (Steens et al., 2017; Steens et al., 2020a). It could be shown accordingly, that the same triple combination of HOX genes as VW-MSC-specific gene code was sufficient to directly convert human skin fibroblasts towards MSCs, and thus directing cell fate conversion bypassing pluripotency (Steens et al., 2020b). The introduced HOX-code in primary human skin fibroblasts could further be linked to an increased colony formation and trilineage differentiation potential as (mesenchymal) stem cell characteristics (Steens et al., 2020b). However, it remains to be clarified whether the triple HOX combination approach of HOXB7, HOXC6 and HOXC8 actually retains the VW-MSC-specific master regulatory function to generate VW-MSCs, or whether the induced expression of individual (HOXB7, HOXC6 or HOX candidates already could serve as MSC-specific key transcription factor. Corresponding investigations could also shed light on which MSC characteristic(s) depend(s) on which introduced HOX gene.

HSCs can be classically found in the bone marrow and the peripheral blood being prerequisite for sustained hematopoietic reconstitution and the formation of blood cells. Whereas HSCs were known to be derived from mesodermal precursor cells called hemangioblasts during embryogenesis, HSC in adults (as identified phenotypically by tyrosine-protein kinase c-KIT/CD117 and stem cell antigen-1 (SCA1) expressions, while being lineage negative and by exhibiting functional hemangioblast activity) serve as a rich source for circulating EPCs in addition to the generation of blood cells (Cogle and Scott, 2004). During adult life, EPCs have been defined by their cell surface expression of the hematopoietic marker proteins CD133 and CD34 and the endothelial marker vascular endothelial growth factor receptor-2 (VEGFR2), and their capacity to incorporate into sites of neovascularization prior in situ differentiation into endothelial cells; thus, being decisive for new vessel formation (Asahara and Kawamoto, 2004; Ishige-Wada et al., 2016; Yoder, 2018). HSCs as well as their derived hematopoietic progenitors were known to be characterized by HOX genes in a pattern characteristic of the lineage and stage of differentiation of the cell (He et al., 2011), although total HOX expression levels of HSCs were suggested to be rather low (Bijl et al., 2006; Ackema and Charite, 2008).

The vigorous ability of HSC to produce huge numbers of lineage differentiated cells includes erythrocytes, (mega-) karyocytes, innate and acquired immune cells (Haas et al., 2018). And hematopoiesis is critically impacted by HOX gene expressions: anterior HOX genes (comprising the numbers 1–6 of the 3′ region) are maximally expressed in the more primitive HSCs, whereas posterior HOX genes (comprising the numbers 7–13 of the 5′ region) become prominent in the committed progenitors (Sauvageau et al., 1994; He et al., 2011). Quantification of HOX gene expression levels in the different hematopoietic cell populations isolated from human peripheral blood revealed that all these cells generally express HOXA and HOXC cluster genes at significantly higher levels compared to expression levels HOXB and HOXD cluster genes (10–100-fold lower) (Morgan and Whiting, 2008). High HOXA5 expression levels for example were suggested to account for hematopoietic lineage determination being able to promote differentiation along myelopoietic lineages (Fuller et al., 1999; Argiropoulos and Humphries, 2007). HOXB3, HOXB4, and HOXA9 were shown to play a crucial role for the presence of HSCs, as a combined deficiency in these HOX genes fostered severe effects on hematopoietic organs (Magnusson et al., 2007). Furthermore, the HOX candidates HOXA9, HOXB4, and HOXB6 were highly expressed in HSCs (Seifert et al., 2015; Bhatlekar et al., 2018). Particularly high HOXB4 expression levels seemed to be important for most potent HSCs, namely long-term reconstitution HSCs (Wang et al., 2005; Fan et al., 2012; Forrester and Jackson, 2012). A forced HOXB4 expression was shown to generate HSCs in vitro, and to account for the proliferative response of long-term repopulating HSCs as well as for HSC maintenance (Brun et al., 2004; Pilat et al., 2005). And, the other way around, a reduction of HOXB4 led to a reduction of HSC proliferation (and differentiation) and, to a reduction of neighboring HOX genes in an 3′to 5′order namely of HOXB2, HOXB3, HOXB5 (Brun et al., 2004). Similarly, HOXA4 and the HOX paralog group 4 at all were shown to be decisive for a functional HSC phenotype (Iacovino et al., 2009). Besides, HOXA5, HOXA9, and HOXA10 were identified as three of seven transcription factors (together with ERG, LCOR, RUNX1 and SPI1) to convert “pre-differentiated” tissue, namely haemogenic endothelium cells into HSCs (Sugimura et al., 2017; Fidanza and Forrester, 2021). Studies from the in vitro generation of HSCs using human pluripotent stem cells further revealed that HOXA cluster genes were significantly downregulated in (CD34-positive) HSCs that were incapable of long-term engraftment and repopulation, strongly indicating that HOXA genes critically regulate definitive hematopoiesis (Ng et al., 2016). Particularly, increased HOXA5 and HOXA7 expression levels were associated with the repopulation activity (Dou et al., 2016). In accordance with the 3′ to 5′ HOX direction, the HOX candidate HOXA9 then together with the ETS-family transcription factor ERG were further shown to re-specify lineage-restricted (CD34⁻ and CD45-positive) HSC precursors derived from human iPSCs into primitive (CD34-positive and CD38-negative) HSCs (Doulatov et al., 2013). Thus, stimulation of HOXA gene expression potentially improves HSC maintenance generating self-renewing HSCs from pluripotent stem cells (Collins and Thompson, 2018).

Decreases in HOX gene expressions were then observed upon HSC differentiation in a manner that seems to follow their anterior-posterior position with anterior HOX genes being downregulated earlier than posterior HOX genes (Guo et al., 2003; He et al., 2011). Generally, HOXB and single HOXC cluster genes were associated to hematopoietic cells with erythroid features, and HOXA cluster genes with myeloid features (Lawrence et al., 1996; He et al., 2011). Within that scenario, HOXA5 and HOXA9 were shown to be involved in the proliferation and differentiation of HSCs to common myeloid progenitors, with HOXA9 also regulating the differentiation of HSCs into common lymphoid progenitors (Bhatlekar et al., 2018). During the differentiation (of pre-B cells) into B cells, HOXB3 was found to be a relevant factor, while HOXC3 and HOXC4 crucially impacted on erythroid lineage differentiation (Bhatlekar et al., 2018). HOXA5 and HOXC8 further seemed to regulate erythroid differentiation of megakaryocyte-erythrocyte progenitors, with HOXA7 being expressed during megakaryocyte differentiation (Bhatlekar et al., 2018).

Conclusively, HOX gene expression does not only specify HSC identity, HOX genes significantly impact in HSC function as well as in stages of hematopoietic differentiation; and dysregulation of these HOX genes were associated with a number of leukocyte malignancies (as discussed elsewhere: (McGonigle et al., 2008; Alharbi et al., 2013)). However, a precise activation of indicated HOX genes following stimulation of different pathways orchestrating HSC characteristics were not reported up to now (Demirci et al., 2020). Thus, the sequential activation pattern of HOX genes following signaling induced HSC differentiation remains to be unraveled.

Although a number of transcriptomic profiles and EPCs characterization are available, the role of HOX genes in EPCs known to be involved in neovascularization processes remains nearly completely elusive (Medina et al., 2010; Wu et al., 2018; Abdelgawad et al., 2021). This may be due, at least in part, to the fact that many different cell subtypes are consistently grouped together under the term “EPC”, which in turn would argue in favor of setting minimum criteria for defining EPCs, e.g., detailed immunophenotyping and/or potency assays, similar to defining minimal criteria for characterizing MSCs (Medina et al., 2017). However, several HOX transcription factors were already associated with target gene expression known to promote the differentiation of mature endothelial cells, which might indicate that also EPCs exhibit a specific HOX code. HOXB5 might be an EPC HOX code candidate, as HOXB5 was involved in the in vitro differentiation of embryonic precursor cells towards the endothelial lineage (Wu et al., 2003). Endothelial cell differentiation of ESCs further showed that HOXA3 and HOXD3 are immediately expressed when differentiation is induced, whereas HOXA5 and HOXD10 are expressed in more mature and adult endothelial cells (Bahrami et al., 2011). Accordingly, high expressions of HOXD3 together with HOXD1, HOXD4, HOXD8 and HOXD9 were reported for less mature blood-derived outgrowth endothelial cells (Toshner et al., 2014). HOXD3 was further linked to endothelial activation (from a resting to an angiogenic state) (Boudreau et al., 1997), and HOXB3 seemed to be required for subsequent capillary morphogenesis (Myers et al., 2000). And HOXA13 was found to be essential for placental vascular patterning and labyrinth endothelial specification (Shaut et al., 2008). HOX genes involvement in endothelial differentiation was further revealed when the HOX expression profiles of BM-MSCs were investigated following endothelial differentiation (Chung et al., 2009). The expression patterns of the four HOX genes HOXA7, HOXB3, HOXA3, and HOXB13, significantly changed during endothelial cell differentiation with expression levels of HOXA7 and HOXB3 becoming increased, whereas those of HOXA3 and HOXB13 became decreased. According to the central role of HOXA9 for the cellular identity of HSCs, HOXA9 was shown to mediate maturation of endothelial cells and being a master switch to regulate the expression of typical endothelial-committed genes such as endothelial nitric oxide synthase, VEGFR2, and VE-cadherin (Rossig et al., 2005). HOXA9-deficiency was further reported to result in reduced EPCs numbers and thereby impaired the postnatal neovascularization capacity (Rossig et al., 2005). Likewise, reduced HOXA9 expression levels were estimated in CD34-positive cells of hypertensive patients, an effect that correlated with reduced EPC numbers (Pirro et al., 2007). EPCs isolated from umbilical cord blood showed that HOXD9 seemed to be required for EPC maintenance (Iordache et al., 2015). Thus, several HOX genes, especially HOX genes from HOXA and HOXD cluster, take part in EPC maintenance, endothelial cell differentiation and function, however an EPC-specific HOX code could not be defined up to now. It seems that within endothelial differentiation processes a complex interaction of various HOX genes are important. EPC or endothelial cell-specific HOX codes, and how respective HOX genes then contribute to EPC-endothelial cell functions as well as to the proper functioning of the adult vasculature need to be unraveled.

The diverse molecular signaling pathways and dependent transduction molecules known to orchestrate various stem cell characteristics regulate and control HOX gene expression. In addition to the involvement of HOX genes in the positional identity of stem cells along the stem cell hierarchy, HOX genes decisively function in all stem cell characteristics: self-renewal and maintenance, proliferation and survival, migration and invasion and lineage specification (differentiation). However, less is known how the main signaling pathways operating in stem cells regulate HOX expressions, and how single HOX transcription factors in turn impact on stem cell-related signaling pathways, and thus account for certain stem cell functions. Now we summarize the recent knowledge how the important stem cell-related signaling pathways such as WNT, BMP, FGF, SHH, NOTCH and RA, integrate HOX genes for maintaining cellular identity and regulation of differentiation.

Generally, during human embryogenesis, more 3′ located (anterior) HOX genes (HOX1-8) become activated and regulate paraxial mesoderm development with the formation of embryonic primordial segments called somites by modulating cell ingression into the primitive streak (Iimura and Pourquie, 2006). Subsequently activated more 5′ located (posterior) HOX genes (HOX10-13) limit mesoderm ingression by repressing WNT signaling, and mediating body axis termination. (Young et al., 2009; Denans et al., 2015). Concerning the signaling it is known that the paraxial regions of the mesoderm become specified following the action of BMPs, members of the FGF family and WNT molecules. Cross-regulation of these pathways, e.g., by the WNT effector molecule NOGGIN that inhibits BMP signaling, is not only important for the paraxial mesoderm specification but also for maintaining this identity (Aulehla and Pourquie, 2010; Budjan et al., 2022). As a model of human somitogenesis, paraxial mesoderm organoids were developed by differentiating human pluripotent stem cells towards paraxial mesoderm using a combination of WNT signaling activation together with BMP inhibition and following FGF2 treatment (Budjan et al., 2022). Thereby, the sequential activation pattern of the HOX genes was elegantly recapitulated. HOXA1 expression was early detected following WNT activation and BMP inhibition (with 24 h), followed by other cervical (HOX1-HOX5 paralogues) and thoracic (HOX6-HOX9 paralogues) HOX genes within the next 48–72 h, to HOXD9, a lumbosacral HOX gene in the somite-stage organoids of day 4–5, which was 24–48 h after the successive FGF2 treatment (Budjan et al., 2022). Concerning the mechanism it was revealed that WNT molecules activate HOX gene expression in a temporally collinear way as WNT-dependent enhancers, namely interaction regions with HOXA1 called “HOXA developmental early side (ADES)” that are located within the posterior region. WNT signals (particularly WNT3) caused thereby removal of H3K27 repressive marks on the 3′ region of the HOXA cluster that in turn promotes transcription of HOXA genes (Neijts et al., 2016). Subsequently to initial WNT-induced activation of 3′ HOX genes, coactivated caudal type homeobox genes (Cdx genes) were shown to act as crucial effectors for expression induction of central and 5′ HOX genes in a collinear manner (Neijts et al., 2017).

RA (a vitamin A metabolite)-dependent signaling is another central signaling pathway being essential for early embryonic development and the promotion of stem cell lineage specifications in pluripotent stem cells (Soprano et al., 2007; Gudas and Wagner, 2011). Studying the kinetics of transcriptional and epigenomic patterning responses of RA in ESCs revealed that RA plays even an essential role in HOX gene regulation by binding to specific retinoic acid receptors (NR1Bs) at RA responsive DNA elements (RAREs) located within the HOX clusters finally erasing H3K27me3 from repressed HOX genes during ESC differentiation (Kashyap et al., 2011). RA stimulation generally induces activation and recruitment of demethylases to HOX genes, which in turn become demethylated thereby promoting HOX expression and thus differentiation (Shahhoseini et al., 2013). Genes at the 3′ end of the complexes (e.g., the paralogs HOXA1, HOXB1, and HOXD1) display herein a higher responsiveness to RA while genes at the 5’ end (the paralogs HOXA13, HOXB13, HOXC13, and HOXD13) are more responsive to FGF signaling (Nolte et al., 2019). FGF signaling was shown to interact antagonistically with RA signaling during posterior development, finally resulting in morphogen gradients within the anterior-posterior-axis with high RA concentrations at the anterior and high FGF concentrations at the posterior end, which lead to an increased expression of the HOX genes HOXB9 and HOXA7 (Kumar et al., 2021).

Thus, stem cell fate decisions into specific tissue lineages require distinct temporal windows for the known stem cell-related signaling pathways and respective cross-regulations (Rankin et al., 2018). On the one hand, FGF-related signaling was shown to orchestrate the pluripotent stage (Mossahebi-Mohammadi et al., 2020). FGF family members, namely FGF2 and FGF4 were shown to function as intrinsic regulators of pluripotent stem cell self-renewal and survival by promoting MAPK and AKT signaling pathways. Particularly MAPK signaling seems to be required, whereas PI3K/AKT signals increase as pluripotency gets restricted (Lee et al., 2019; Mossahebi-Mohammadi et al., 2020). Moreover, FGF-related signaling being essential for pluripotency maintenance, especially FGF4 expression, depends on the presence of the pluripotency transcription factors OCT4 (and SOX2). On the other hand, FGF-related signaling, particularly through FGF2, has even the potential to induce lineage differentiation of pluripotent cells (Steens and Klein, 2018; Ma et al., 2019). An FGF2 activation was found to activate the downstream kinase Src, a non-receptor tyrosine kinase that in turn activates MEK1/2 (Mitogen-activated protein kinase 1) resulting in differentiation and/or increased proliferation particularly of mesodermal ASCs (Ma et al., 2019). Very recently it was shown that respective processes here were linked to HOX gene regulation (Tiana et al., 2022). The pluripotency factor OCT4 turned out to be important for maintaining HOX genes silent in the stage of pluripotency. Upon lineage commitment however, OCT4 switches from a repressor to an activator being required for the proper activation of HOX genes. Especially the genes of the HOXB cluster are coordinately regulated by OCT4 binding sites located at the 3′ end of the cluster (Tiana et al., 2022).

Concerning the time of induced HOX expressions, so how extrinsic factors influence the tempo of HOX expressions, it was recently shown that the progressive activation of HOX genes in pluripotent stem cells is controlled by a dynamic increase in FGF signaling (Mouilleau et al., 2021). Herein, an increase in FGF pathway activity was successfully associated with the sequential activation of HOX genes; and cells differentiated under accelerated HOX induction (Mouilleau et al., 2021). Combination of “cell type-specific” growth factors then are able to foster lineage specifications and thus further modulation of HOX expressions. Pluripotent stem cell differentiation towards mesodermal stem cells for example can be initiated by the induction of mesodermal differentiation, achieved by TGFβ/Activin/Nodal signaling inhibition (Mahmood et al., 2010). Subsequent treatment with cell type-specific growth factors particularly FGF2 and/or platelet-derived growth factor A/B, two known potent inducers of MSC differentiation under defined cell-culture conditions foster then MSCs differentiation (Steens and Klein, 2018; Abdal Dayem et al., 2019). For HSCs, VEGF together with hematopoietic cytokine cocktails as type-specific growth factors were generally used to drive hematopoiesis following mesoderm specification of pluripotent stem cells by the use of WNT agonists and BMP4 (Sturgeon et al., 2014; Alsayegh et al., 2019). However, reports are lacking, which follow the timely expression of mesodermal stem cell-related HOX genes induced by extrinsic signals. In contrast, it has become well established that deregulated signaling pathways and thus abnormal signal transduction as selective characteristics common to many cancers, are associated with deregulation of HOX genes. Increasing numbers of reports here revealed important molecular signaling interactions, mainly due to up-regulations of certain HOX candidates that finally result in cancer (stem cell) overpopulation, limited differentiation, and cellular disorganization of respective cancer tissues. For example, in HOXB7-overexpressing tumors, which were shown to be enriched in gene signatures classically characterizing ASCs as well as pluripotent stem cells, the pluripotency factor LIN28B was identified that sustained the expansion of a subpopulation of cells with stem cell characteristics (Monterisi et al., 2018). As another example, HOXC8 was shown to function as transcriptional activator of TGFβ signaling, finally mediating increased proliferation, anchorage-independent growth and migration of lung cancer cells (Liu et al., 2018). A more systematic evaluation of all human HOX gene target pathways being involved in biological processes associated with the cancer hallmarks revealed then, that five HOX genes (HOXC4, HOXB2, HOXB3, HOXC6, and HOXA13) and their respective targets contribute to sustained proliferative signaling (Brotto et al., 2020). As cancers are commonly characterized by an abnormal signal transduction associated with increased proliferation (and loss of apoptosis), the identification of the signal(s) that cause upregulation of certain HOX genes is still missing. It is generally assumed that a tumor-specific loss of control over the spatiotemporal expression pattern and levels compared to the expression pattern of related normal tissues occurs. Induced “tumor” HOX genes can also occur due to so-called gene dominance, when respective HOX gene expressions in normal tissues are missing. In addition, epigenetic changes in regulatory regions of HOX genes can lead to a loss of normal control (Abate-Shen, 2002; Brotto et al., 2020). The deregulated expression of HOX gene candidates however turned out to be cancer-type dependent and even show variations in respective clinical responses. Thus, unraveling the driving forces of HOX gene dysregulations may foster the development of new and additional therapeutic targets to improve cancer therapy. Within these scenarios, data sharing through the so-called blockchain technology is becoming increasingly necessary, which would improve not only the insights of researchers and clinicians; it could also serve as a suitable basis for an efficient and effective evidence-based decision-making process in therapeutic approaches, as reasoned from other diseases (Dubovitskaya et al., 2020; Fusco et al., 2020). For the current state of knowledge on the roles of HOX genes in human cancer the following review articles were recommended (Bhatlekar et al., 2018; Smith et al., 2019; Paco et al., 2020; Feng et al., 2021).