Connexins in the development and physiology of stem cells

ABSTRACT

Connexins (Cxs) form gap junction (GJ) channels linking vertebrate cells. During embryogenesis, Cxs are expressed as early as the 4–8 cell stage. As cells differentiate into pluripotent stem cells (PSCs) and during gastrulation, the Cx expression pattern is adapted. Knockdown of Cx43 and Cx45 does not interfere with embryogenic development until the blastula stage, questioning the role of Cxs in PSC physiology and development. Studies in cultivated and induced PSCs (iPSCs) showed that Cx43 is essential for the maintenance of self-renewal and the expression of pluripotency markers. It was found that the role of Cxs in PSCs is more related to regulation of transcription or cell-cell adherence than to formation of GJ channels. Furthermore, a crucial role of Cxs for the self-renewal and differentiation was shown in cultivated adult mesenchymal stem cells. This review aims to highlight aspects that link Cxs to the function and physiology of stem cell development.

Graphical abstract

Connexins (Cxs) are proteins in vertebrate cells that are encoded by a gene family with 21 and 20 members in humans and rodents, respectively.¹ The proteins are named by the abbreviation Cx (for connexin) followed by a numerical suffix that gives the approximate molecular weight of the protein. Accordingly, Cx26 is approximately 26 kDa. Similar to other membrane proteins, Cxs are synthesized in the membrane of the endoplasmic reticulum (ER), where they are inserted with a specific topology. The cytoplasmic N- and C-termini are connected to four transmembrane domains (TM1-TM4) by two extracellular loops (EL1 and EL2) and a cytoplasmic loop (CL). Along the secretory pathway through the ER and Golgi apparatus, Cx molecules oligomerize to form hexamers also called connexons, which are exported to the plasma membrane. Connexons of adjacent cells may dock to each other in the basolateral regions of adjacent cells and form cell-to-cell gap junction (GJ) channels that join the cytoplasmic spaces of interacting cells in tissue. GJ channels interact with the cytoskeleton through subsidiary proteins and are thereby assembled in so-called GJ plaques,² as observed by staining with anti-Cx antibodies or by the expression of GFP-labeled Cxs in cells.^3,4 When formed, GJ channels are large enough to allow a direct exchange of ions and metabolites such as glucose, nucleotides, second messenger, oligonucleotides such as microRNA, or peptides up to approximately 1.5 kDa.^5–8 GJ channels enable coupled cells to form synchronized functional units within a tissue.^9,10 Undocked Connexons in single cells can function as Cx hemichannels. In this constellation, Cx hemichannels permit exchange between the extracellular and intracellular space. Because of the large pores of connexons, hemichannels are permeable to metabolites up to approximately 1.5 kDa, which diffuse along their respective electrochemical gradient. When opened, the Cx hemichannels therefore offer a route for intracellular molecules such as nucleotides to diffuse in the tissue interstitium. The released molecules are mostly degraded in the extracellular space or actively retaken up into the cells. However, for a short time period and in the close vicinity of their release spot, intracellular agents such as NAD⁺, adenosine, or nucleotides may achieve concentrations high enough to stimulate, in a paracrine manner, the surrounding cells. Under normal conditions, the density of undocked Cx hemichannels in cells is strongly reduced, and their open probability is very low, since Cx hemichannels are already closed by external bivalent ions, such as Ca²⁺, in concentration ranges far below the concentrations observed in the interstitial space.^11,12 In addition to Cxs functioning as GJ channels or hemichannels, Cxs also display non-channel functions.¹³ A remarkable non-channel activity of Cx is the regulation of cell adhesion, cell motility and cell migration, which are essential for cell differentiation during tissue and organ formation.^14–21 Finally, Cxs are also found in organelles such as mitochondria. To date, the function of Cxs in organelles is not fully understood. However, it was shown that Cxs in mitochondria contributed to mitochondrial K⁺ and Ca²⁺ homeostasis, release and production of reactive oxygen species (ROS) and regulation of the respiratory complex. Therefore, Cxs mediate resistance to ischemic injury in tissues.²²

During mammalian development, Cxs are expressed beginning as early as the 4-cell stage, when the cells are totipotent, and is maintained in embryonic pluripotent stem cells (EPSCs).²³ As subsequent cell differentiation and migration takes place to form and sustain tissue homeostasis in developing embryo and later in an adult organism, the regulation of Cx expression changes at different developmental stages, from stem cells to fully differentiated somatic cells.²⁴ The role of Cxs in maintaining stemness and in the development of stem cells to mature somatic cells is not fully understood. In the following, we will briefly describe different types of stem cells and their differentiation into adult cells. We will also provide experimental evidence that shows the role of Cx in the physiology and induction of stem cell differentiation. Because of ethical concerns, many experiments have been conducted in mice, especially in the case of EPSCs. The conclusions concerning humans are extrapolated from these animal experiments or have been partly confirmed by experiments using human induced stem cells (iPSCs).

Stem cells

In mammals, stem cells are unspecialized cells capable of self-renewal and differentiation in different types of cells. Stem cells are canonically classified as either totipotent, pluripotent, or multipotent based on their degree of differentiation potency. As research has advanced, oligopotent and monopotent stem cells have been added as additional classifications.^25,26 In the present review, we will concentrate on the canonical classifications because they are univocally defined. A cell is called totipotent if it can recapitulate embryological development and produce a complete adult organism. With this definition, totipotent cell par excellence is a zygote that results from the fusion of a male and a female gamete. After fusion in mammals, the zygote divides by three successive cleavages, leading to the formation of 8 morphologically similar cells called blastomeres. In some species, including sheep, rats, cattle, pigs, horses, and monkeys, carefully isolated single cells at the 4–8 cell stage are able to start an embryo and generate a total organism,²⁷ indicating that the cells are totipotent. In mice, only the first two blastomeres are totipotent. In humans, it was shown that the four blastomeres at the 4-cell stage developed individually into blastocysts, each with an inner cell mass (ICM) and trophoectoderm (TE).²⁸ The aforementioned observations show that depending on the animal species, the totipotency of the zygote is maintained from the 2-cell stage to the 8-cell stage. For all species after the 8-cell stage, the cells begin to differentiate to form the morula followed by the blastula. At the blastula stage, which begins with the 32–64 cell stage, clear differentiation of the cells occurs. External cells, also called trophoblasts, are linked by tight junctions and form the TE, which isolates an inner cavity, the blastocoel, containing the ICM and the blastocoel liquid. The TE cells will not participate in further formation of embryonic tissues. Embryonic tissue will solely be formed by cells originating from the ICM. After multiple rounds of cell division, the ICM differentiates into two epithelia, the epiblast and the hypoblast, and implantation of the embryo in the maternal endometrium takes place. By different sequential migrations, the cells of these bilaminar germ discs (hypoblasts and epiblasts) generate primordial germ cells, which will be integrated into the gonads as primordial gametes for the next generation, and three germinal layers: endoderm, mesoderm and ectoderm. When isolated and cultured in vitro with appropriate care, the ICM remains immature. The cells can spontaneously differentiate and give rise to cells of the ectoderm, mesoderm, and endoderm, such as neurons, cardiomyocytes, and hepatocytes, respectively. Modern research, however, is more interested in establishing cultivation protocols that allow the induction of specific cell lineages. In animal (mouse) models, ICM cells can be cultured, multiplied and labeled for subsequent transfer into the blastocoel, where they are mixed with the ICM of the developing embryo. The transferred cells will participate in the formation of primordial germ cells as well as the three germinal layers of the developing embryo. However, ICM cells alone are not able to resume embryological development (cleavage to the 8-cell stage, formation of the morula, development of the blastula) to produce a complete organism. In animal models (mice), ICM cells, also called embryonic stem cells (ESCs), are pluripotent.²⁹ The capacity of ESCs to participate in the formation of all embryonic tissues of organisms is the most convincing criterion that qualifies ICM cells in embryos and in culture as pluripotent stem cells (PSCs). Because of ethical concerns, experiments to prove this property for human embryonic PSCs (EPSCs) cannot be performed. Likewise, it is problematic to envisage the continual production of human EPSCs. These obvious difficulties have pushed researchers to define other criteria that allow the characterization of PSCs. PSCs express a network of transcription factors. The most important are SOX2, OCT-4, and NANOG, which constitute the core of pluripotency factors and act in a mutual regulatory circuit to coordinate cell pluripotency.^30–32 These transcription factors are conserved between mammals and several vertebrates.^33–36 Auxiliary PSCs can differentiate into cells of the three germ layers. To obtain PSCs for research and medical applications, techniques to generate iPSCs have been developed. The techniques to induce a somatic cell to become a PSC are based on the introduction of specific transcription factors in the cells.³⁷ The applied protocols differ with respect to the required number of transcription factors and in the method used to introduce the transcription factors to somatic cells. Classically, DNAs coding for transcription factors have been transfected in somatic cells. In early studies, the DNA was integrated into the genome of the cells for stable expression of the transcription factors, but it was observed that even without integration, the transfected transcription factors were efficient enough for reprogramming of the cells.³⁸ These findings have led investigators to propose the usage of transcription factor mRNA or even transcription factors as already synthesized proteins.³⁹ Along with the historical development of reprogramming technology, it was found that the addition of a cocktail composed of small molecules such as valproic acid, sodium butyrate, or PD0325901 increased the probability of reprogramming the cells.³⁹ This has led to the development and optimization of a cocktail of small molecules that can be used for reprogramming somatic cells back to PSCs, now known as chemically induced pluripotent stem cells or CiPSCs, with acceptable efficiency and within a relatively short time.^40–42 All the methods are qualified by the efficiency to produce cell clones expressing the requested pluripotency markers and by the capability of the induced cells to self-renew and differentiate into cells of the three germ layers. iPSCs allow the scientists to analyze the mechanisms of cell differentiation, and we will consider their contribution to understanding the role of Cxs in cell differentiation.

After the formation of the three germinal layers, organs are formed by further embryological development, which means that cell proliferation, cell migration and differentiation occurs followed by organization in three dimensions according to the specificity of the respective organ. Therefore, undifferentiated cells proliferate and divide asymmetrically. This asymmetrical division results in an undifferentiated cell (self-renewable) and a cell that enters differentiation. The latter then develops into different organ-specific cells after further successive rounds of division and differentiation. In adult organs, undifferentiated cells are maintained in specific niches.⁴³ They are self-renewing and generate cells that, after proliferation and migration, undergo differentiation to sustain organ growth until the organism reaches adulthood. These undifferentiated cells also maintain organ homeostasis after the organism has completed its development. Undifferentiated cells in developing and adult organs can be isolated and maintained in culture for numerous passages. They maintain different characteristics: adherence to plastic in culture, expression of CD73, CD90, and CD105 and absence of CD34, CD45, HLA-DR, CD14, CD11b, CD79a, or CD19.^44–46 These cells can spontaneously differentiate and generate the different cell types of the tissue from which they were isolated. Because of these undifferentiated properties, the capacity to self-renew and generate different organotypic cell types, these cells are stem cells. However, they are not able to transdifferentiate from one germinal layer into another; thus, the differentiation potentiality is constrained to the specific germ layer of their origin.²⁵ These so-called adult stem cells are multipotent stem cells but are no longer pluripotent. Multipotent cells are, for example, stromal mesenchymal stem cells (MSCs), which are found in various organs, especially of mesodermal origin, such as the bone marrow and fat tissue. These MSCs are able to differentiate into osteoblasts, chondriocytes, myoblasts, adipocytes and hematopoietic supporting stromal cells.⁴⁷ MSCs are accessible and can easily be isolated and multiplied in culture without losing their stemness, confirmed by examination of their markers.^44–46 In culture of MSCs, mostly isolated from bone marrow or fat tissue, differentiation of neuroglial cells^48,49 as well as cells of the endodermal lineage, such as hepatocytes,^50,51 has been observed. Further, researchers have tried to induce transdifferentiation of MSCs, which means that these mesodermal cells can give rise to cells in the other germ layers. This can be achieved by transfecting with transcription factors and by using small molecules. Transdifferentiation makes it possible to analyze the cellular and molecular signaling that governs cellular differentiation. Transdifferentiated MSCs may be beneficial in regenerative medicine because of the possibility to use patients own cells to generate any cell needed for therapeutic propose.^52,53 In basic research, transdifferentiation allows the analysis of cell developmental mechanisms, e.g., the significance of Cxs in the different forms of pluripotency and in the differentiation of cells to somatic cells.⁵⁴ Methods and protocols to transdifferentiate specific lineages have been assessed in different laboratories.^55–58 Transdifferentiation methods have involved the introduction of specific transcription factors into MSCs^59,60 as well as the treatment of cells with small molecules.^61–64

Another example of adult stem cells is neuronal stem cells (NSCs). NSCs have astrocyte-like morphology and were identified in different regions of the developing and adult nervous systems. In adult animals, stem cells are mostly found in the subventricular zone (SVZ) along the lateral wall of the lateral ventricles, in the subgranular zone (SGZ) of the hippocampal dentate gyrus in the brain,⁶⁵ and in the ependymal channel of the spinal cord.^66,67 NSCs are self-renewing and can develop into neurons, astrocytes and oligodendrocytes.^68,69

As mentioned before, in addition to the canonical stem cell classifications, oligopotent and monopotent stem cell classifications were added to the literature. Oligopotent and monopotent cells are undifferentiated cells found in various tissues. They are precursor cells with differentiation capacities to become cells of a specific lineage in the specific tissue or a single cell type of the tissue, respectively. Myeloid progenitor cells that differentiate into basophils, neutrophils, eosinophils, monocytes, and thrombocytes as well as the hepatoblasts that develop into hepatocytes are classical examples of oligopotent and monopotent cells, respectively.⁷⁰

Connexin expression in stem cells

In pluripotent stem cells

Cx expression during the development of a fertilized oocyte into different tissues in an embryo has been studied extensively over many years. In mice, during the preimplantation development stage, Cx30, Cx31, Cx36, Cx43, Cx45, and Cx57 are expressed in the 2–4 cell stage up to the 8-cell stage.^23,71 The Cx proteins were also synthesized but mostly remained in the cytoplasm. Insertion in the plasma membrane and the formation of functional cell-to-cell GJ channels were induced when the cells started to adhere to each other and to form a compact morula.^72,73 In human EPSCs, the expression of 18 Cx isoforms, Cx25, Cx26, Cx30, Cx30.2, Cx30.3, Cx31, Cx31.1, Cx31.9, Cx32, Cx36, Cx37, Cx40, Cx43, Cx45, Cx46, Cx47, Cx59, and Cx62, has been demonstrated.^74–79 Quantitative comparison showed that the Cx43 and Cx45 isoforms were the most enriched Cxs in EPSCs in comparison to further developing cells.^1,80–82 Moreover, characteristic EPSC transcription factors, such as NANOG, directly upregulated the expression of Cx45.⁸³ At the protein level, Cx43 is synthesized and accumulates in cells during development from blastomeres to blastocysts.^71,73,80 Similar to what has been observed in mouse blastomeres, human Cx43 protein mostly remains in the cytoplasm of human blastomeres. When these cells adhere to each other and form a compact morula, Cx43 is then trafficked to the plasma membrane and form GJ channels.⁷³ By analyzing the expression of Cxs in the developing embryo, it has been assumed that good GJ communication is essential for morula compaction and embryo development through blastulation and gastrulation.^82,84

Induced pluripotent cells are typically generated using fibroblasts. The analysis of Cx expression has revealed that the reprogramming process of fibroblasts into pluripotent cells is accompanied by an upregulation of Cx25, Cx26, Cx30, Cx30.2, Cx30.3, Cx31, Cx31.1, Cx31.9, Cx32, Cx36, Cx37, Cx40, Cx43, Cx45, Cx46, Cx47, Cx59, and Cx62.^75,85 The findings suggest that ihPSCs (induced human PSCs) behave, to some extent, like EPSCs originating from ICMs with respect to Cx expression. ihPSCs can be produced and maintained in culture without ethical concerns, and these cells are a fantastic tool to analyze the role of Cx in the maintenance of stemness and further development in different directions.

Connexin expression during differentiation

EPSCs generate meso-, endo-, and ectoderm embryonic germ cell layers during the process of gastrulation. The onset of development of the germ cell layers in different tissues seems to correlate with the expression of different Cx isoforms. This has been studied in cardiac tissue, which represents the mesodermal system. During the differentiation of EPSCs into cardiomyocytes, Cx43 and Cx45 are already found in undifferentiated cells and embryoid bodies before the beating cells are differentiated.^86,87 Further differentiated beating cells express Cx40⁸⁶ as the cells develop and finish differentiation, other Cxs, such as Cx26, Cx37 or Cx57, are also expressed.⁸⁸ With regard to cells of different tissues of endodermal origin, such as hepatocytes or pancreatic cells, the study of EPSCs and iPSCs has revealed that Cx43 is required for the determination of EPSCs in the endodermal direction.⁸⁹ With further differentiation into somatic cells, the Cx expression pattern changes and adapts to the cell type. For example, in oval cells, which are progenitor cells that differentiate into different hepatic cells, such as hepatocytes or bile ductular cells, PCR and western blot analysis has revealed that during proliferation and the beginning of differentiation, the cells upregulate Cx43 and downregulate Cx32.^90,91 Hepatocyte differentiation correlates with an upregulation of Cx32 and a complete suppression of Cx43.⁹² Further analyses have shown that Cx32-mediated gap junction intercellular coupling (GJIC) is a prerequisite for the correct differentiation of EPSC-derived hepatocytes (hESC-Heps) during hepatic lineage restriction and maturation.⁹³ For ectodermal tissue, Cx43 is expressed in mice in the ectodermal cell layer⁸⁹ as well as in organs originating from the ectodermal cell layer, such as sensory organs or cells of the nervous system.⁹⁴

In multipotent stem cells

Adult multipotent stem cells originating from different tissues, such as the corneal limbus, periodontal ligament, skin, bone marrow, or fat tissue, have been analyzed.^95,96 With respect to Cx expression, Cx43, Cx32 and Cx31.9 are normally expressed.^97,98 Other additional Cx isoforms, such as Cx26, Cx37, and Cx45, are also expressed but to a lesser extent.^54,98,99 Among the Cx isoforms recognized by PCR in MSCs, only Cx43 is expressed, which has been revealed by western blotting or immunostaining experiments.^{54,97,100,101} The expression of other isoforms, such as Cx31.9, Cx32, and Cx45, has been sporadically reported.^97,101 Due to the availability of analytical tools such as antibodies, Cx43 has been intensively studied in the stem cell research field. The danger is to extrapolate the results on Cx43 to other isoforms or to consider the other Cx isoforms as unimportant for the physiology and differentiation of the different stem cells. Careful observation of the cells in development has revealed that other Cx isoforms play a role and should be considered as well. The challenge is to invest in the development of tools and to take time to understand the specific role of each Cx isoform on stem cell physiology and differentiation.

Neuronal stem cells are another example of adult stem cells that have been extensively studied.¹⁰² In the developing brain, Cx26, Cx36, Cx37, Cx43, and Cx45 are expressed. As adult stem cells are developed from the stem cell niche, the expression pattern of connexin isoforms changes. In particular, Cx36 and Cx43 are expressed in the cells of the SVZ, the region that represents the NSC niche.¹⁰³ As adult stem cells begin to develop, the expression of Cxs is downregulated and then upregulated and adapted to the destiny of the cells. For example, Cx36 is upregulated in cells that develop into neurons. Cells that develop into astrocytes mostly express Cx26, Cx30, and Cx43. Cells that develop into oligodendrocytes express Cx32 and Cx47.^104–106 A prominent role of Cx during development is the regulation of adhesion and migration of the developing cells from the SVZ to their definitive position in the organ. Developing neurons in the midbrain floor, where nigrostriatal dopaminergic neurons originate and differentiate, express Cx26, Cx32, Cx43, and Cx45.¹⁰⁷ Hippocampal neurons express Cx45 during memory consolidation.¹⁰⁸ Cx29 is expressed in oligodendrocytes or Schwann cells when these cells develop and start myelination.¹⁰⁹ Interestingly, adult astrocytes express Cx26, Cx30, Cx40, Cx43, Cx45, and Cx46.¹¹⁰

Functions of connexins in stem cells

In pluripotent stem cells

Preimplantation embryonic development starts with the first three divisions of the zygote producing eight totipotent cells. It continues with the formation of the compact morula and the differentiation of the TE and the ICMs, which are the EPSCs. The implantation of the embryo correlates with the generation of the three germ cell layers during gastrulation. It is assumed that good GJ communication is essential for the processes that occur during preimplantation development because Cx isoforms are expressed, e.g., during the compaction and the formation of the blastula.⁸² The usage of GJ inhibitors, such as 18a-glycyrrhentic acid or carbenoxolone (CBX), disrupts dye transfer and coupling but does not affect blastocyst formation or cell allocation to the TE or ICM.¹¹¹ However, recent experiments have shown that pharmacological inhibition of GJ communication delays blastocyst formation by extending the time from the second cleavage to the formation of blastocysts. Moreover, inhibiting GJIC induced aberrant division of the ICMs and led to a collapse of the blastocysts in more than 70% of the cases.¹¹² This result suggests a role of GJ communication during the early stage of embryological development. However, it was shown that a homozygous absence of Cx43, the most prevalent Cx in totipotent cells, does not suppress normal early embryonal development.¹¹³ Likewise, genetically engineered mice in which single Cxs that are usually expressed during early development are ablated and even Cx43/45 double knockout mice are able to complete blastulation and start the early stage of gastrulation. In developing mouse blastula without Cx43, the morphology is preserved, and the expression of pluripotency markers, such as OCT4 and NANOG, is maintained as is the expression of KRT8 and KRT18 early differentiation markers.¹¹⁴ These contradictory results regarding the significance of connexin and GJ coupling for preimplantation development may have different causes. Pharmacological inhibitors of GJ communication quickly affect gap junction intercellular communication (GJIC). However, the inhibition is nonspecific such that when applied for hours or days, other cellular reactions may be affected in addition to the GJIC. Thus, the effects of pharmacological inhibitors on the developing embryo may be caused by more than the blockade of GJIC. Therefore, studies using GJIC inhibitors and those using Cx gene ablation may not be always referring to the same effects. With Cx gene ablation, we observed that the cells express various Cx isoforms in addition to Cx43. Experiments in cells that are not stem cells have shown that Cx43 knockdown induces increased mRNA expression of other isoforms, such as Cx26 or Cx30.¹¹⁵ Moreover, it has been shown that Cx43 may be functionally replaced by Cx45 in Cx43^−/- embryos.¹¹⁶ Therefore, it is possible that other isoforms might compensate for ablated isoforms. Additionally, in developing animals, the stages of totipotency and pluripotency are dynamic. After the blastula stage, EPSCs enter the migration period of gastrulation and produce germ layer cells, while TE cells start to develop into extraembryonic parts of the placenta. In mice without Cxs expressed in early development, there are problems with tissue formation. In accordance with these observations, it is postulated that Cxs do not play a limiting role in the physiology of the establishment of ICM or in TE cells. Cxs are expressed as a preparation for the rapid diversification of cell types during gastrulation and further development of different tissues.^100,117 These observations suggest that analysis of PSCs as well as iPSCs in culture may allow us to decipher the significance of Cxs in the physiology of embryonic pluripotency as well as their development. Accordingly, it was shown that pharmacological inhibition of GJ coupling increased cell apoptosis and inhibited the formation of colonies by human EPSCs cultivated in Matrigel,⁷⁷ suggesting that Cx and GJIC are required for maintenance of EPSC stemness in humans. This may be related to the capacity of Cxs, such as Cx43, to couple PSCs with feeder cells.¹¹⁸ Moreover, it was reported that downregulation of Cx43 by siRNA results in downregulation of the human EPSC stemness markers OCT4, SOX2, and NANOG and loss of alkaline phosphatase.^118,119 However, Cx43 gene ablation does not suppress GJ coupling in EPSCs,¹¹⁸ although a reduction in GJ coupling has been observed.¹¹⁹ With respect to other aspects of pluripotency, it has been observed in culture that Cx43 ablation reduced proliferation capacity and impairs the formation of embryoid bodies. Suppression of Cx43 negatively affected the self-renewal of EPSCs.¹¹⁹ The data show that GJ coupling does not require Cx43 because other isoforms can compensate for GJ coupling. The data further suggest that Cx43 may transcriptionally regulate stemness markers, which is a role that other Cx isoforms may not be able to rescue.

The role of EPSCs is to generate the three embryonic germ cell layers during the process of gastrulation. As stated above, EPSCs express different Cxs, although Cxs and GJIC seem to play no role in EPSCs in developing embryo.¹¹⁴ However, gastrulation starts with downregulation of Cx26 and Cx43.¹²⁰ As gastrulation proceeds, Cx expression is upregulated, and the significance of Cxs becomes evident, showing that Cxs, as adhesion molecules and building blocks of GJIC, have an impact on the development of specific cell lineages. Knockdown of Cx43 in EPSCs does not affect expression of endodermal markers such as SOX7, FOXA2 and CXCR4. In contrast, the expression of mesodermal markers (CD56, CD34, and PDGFR-α) is downregulated.¹¹⁴ In culture, the knockdown of Cx43 and Cx45, which almost totally suppresses EPSC GJIC, completely disrupts the formation of primitive endoderm cells in embryoid bodies.¹²¹ For the ectodermal layer, in studies using the cell pair array technique, EPSCs expressing Sox1, an early marker of the neuroectodermal germ cell layer, are not able to promote neuroectodermal specification of naïve EPSCs that lack Cx43.¹²²

In summary, the abovementioned studies provide evidence that Cxs and GJIC are important for the specification of germ cell layers. Cx43 has been the most extensively studied Cx in the context of determination of the germ cell layers. It is possible that the other Cx isoforms that are expressed in the blastula may also play an interesting role, as was observed for Cx31.1, which was found to be necessary for the development of trophoblasts.¹²³

In developing organisms, pluripotency is closed after the formation of the three germ cell layers during gastrulation, which correlates with the implantation of the embryo in the maternal endometrium. Further development is relayed to stem cells in the germ layers that produce cells solely of germ cell lineage to form different tissues and organs. These stem cells in germ layers can be considered as adult stem cells. In culture, EPSCs and iPSCs do not resume gastrulation. Cells can be primed to differentiate into specific germ layer cells as mentioned above for the primitive endoderm in embryoid bodies.¹²¹ Thereafter, the cells develop into adult somatic cells corresponding to the germ cell layer. EPSCs and iPSCs are therefore a convenient model to analyze the role of Cxs and GJIC in the development of all cell types. For the nervous system, which represents the ectodermal germ layer differently, Cxs are expressed in NSCs as discussed above.

In iPSCs, it has been observed that the expression of Cx43 is upregulated in the process of reprogramming fibroblasts into PSCs.^85,124,125 Interestingly, Cx40 and Cx45 are also expressed in fibroblasts during reprogramming. These Cxs form functional GJ channels between cells even if Cx43 is silenced with specific iRNA. However, Cx43 knockdown reduces the efficiency of the reprogramming process. Likewise, the presence of pharmacological GJ coupling inhibitors does not alter the success of the reprogramming process, and ectopic expression of Cx43 increases the efficiency of the reprogramming process. In reprogrammed original fibroblasts, less than 1% of the cells form hematopoietic stem cell (HSC) colonies. The efficiency of HSC formation was more than doubled, when the cells ectopically expressed Cx43.⁸⁵ The results suggest a role of Cx43 in the induction and maintenance of stemness, but, as in EPSCs, GJ-dependent cell-to-cell communication does not seem to be the issue; properties other than channel formation are required. This suggests that Cx43 is necessary for an effective induction of stemness. A similar result has been observed for Cx45.¹²⁶ Similar to Cx43, the expression of Cx45 increases during the reprogramming process. This increase is observed at both the mRNA and protein levels. The knockdown of Cx45 reduces the degree of GJ coupling in cells, and similar to Cx43, the downregulation of Cx45 negatively affects cell proliferation and the efficiency of the reprogramming process. It is most striking that ectopic expression of Cx45 improves reprogramming efficiency by more than three times. Similar to Cx43, the effect is not necessarily related to GJ channels but to the regulation of proliferation.

Taken together, cultivation of EPSCs and iPSCs has changed our understanding of the role of Cxs in pluripotency. Cxs are involved in the regulation of the proliferation of cells and possibly in priming the cells for further differentiation. The fact that this role could not be studied in developing organisms is probably due to the passing character of the pluripotency state in developing organisms.

In adult stem cells

In adult (multipotent) stem cells, Cx43 is generally found and participates in the formation of extensive GJ channels found in the cells. It is considered to be a gene for self-renewal and is a characteristic marker of adult stem cells.^96,127,128 Accordingly, Cx43 has been used to isolate multipotent stem cells from human adult periodontal ligaments.⁹⁶ These cells express markers of pluripotency (OCT4, SOX2, and NANOG) and specific markers of neural crest cells such as SOX4, p75, as well as nestin. These cells are multipotent neuronal crest cells capable of differentiation into cells of ectodermal lineages. In HSCs, Cx43 and Cx40 are expressed.^127,129 Cx43 is essential for maintaining multipotency in skin-derived stem cells.¹³⁰ Skin stem cells isolated from Cx43 knockout mice exhibit reduced expression of OCT4 and NANOG compared to cells isolated from wild-type mice. In culture, HSCs from Cx43 knockout animals show reduced migration activity and an increased proliferation capacity in comparison to HCSs isolated from wild-type animals. When used in transplantation experiments after myeloablation depletion with 5-fluorouracil (5-FU), Cx43-deficient HSCs and progenitors (HSCs/P) have a low proliferation and survival capacity and are prone to senescence. The enhancement of senescence in Cx43-deficient cells seems to be related to an inability of the cells to evacuate ROS into the hematopoietic microenvironment, leading to an increase ROS content in HSCs.¹²⁹ Similarly, Cx43 protects MSCs from senescence and supports MSC survival.^131,132 It is possible that the same mechanism as described for HSCs is involved for MSCs. However, this mechanism is not the only mechanism by which Cxs regulate the physiology of mesenchymal cells. Bone marrow mesenchymal stem cells (BMSCs) isolated from a healthy person express lower Cx43 than BMSCs isolated from multiple myeloma (MM) patients. Co-cultivation of BMCs from a healthy person with MM cells increases the expression of Cx43 in BMSCs at both the mRNA and protein levels. Pharmacological inhibition of Cx channels impairs MM cell adherence to BMSCs, migration, and secretion of cell-derived factor-1α (SDF-1α). As a consequence, the migration of MM cells is impaired.¹³³ Additionally, in BMSCs, Cxs are involved in the maintenance of self-renewal in the bone marrow stem cell niche as well as in the osteogenic differentiation of MSCs. The mechanisms that participate in differentiation are related to cell-cell communication through GJ channels and to the Cx hemichannels that are used as release pathways of purine nucleotides, which participate in osteogenic, adipogenic and chondrogenic differentiation of MSCs.^134–136 Furthermore, the capacity of Cxs to regulate signaling and gene expression as well as the modulatory secretion of intercellular chemokines, such as CXCL12, are important.^137,138 Additionally, the adhesion activity of Cxs is essential for the physiology of MSCs and their differentiation in different bone cells. This also involves interactions with cells in the stem cell niche.¹³⁹

Regarding MSCs, different Cx isoforms, mostly Cx26, Cx37, Cx40, Cx43, and Cx45, are expressed. The function of these Cxs is not always understood; however, it is clear that GJ channels are formed and metabolically couple MSCs, as shown by dye transfer experiments.⁵⁴ Furthermore, transdifferentiation of MSCs in neuron-like cells using small molecules induces changes in the Cx expression pattern. Most prominently, the downregulation of Cx43 correlates with the upregulation of Cx26. However, pharmacological inhibition of GJIC does not stop transdifferentiation of the cells. The finding that inhibition of GJIC does not seem to affect the transdifferentiation of MSCs into neuron-like cells may be related to incomplete neuronal differentiation.⁵⁴ For the transdifferentiation of MSCs transplanted in cardiac tissue, GJs coupled with local cardiomyocytes are needed for development into cardiomyocytes.^140,141 Although functional GJ coupling seems to be necessary for the differentiation of cardiomyocytes, the nature of the exchanged signals or substances is not clear.

Taken together, the data of MSCs and HSCs reviewed above show that the expression of Cxs in adult stem cells and the other cells of the stem cell environment, the so-called stem cell niche, is fine-tuned to respond to the needs of the cells in the niche.¹⁴² Cxs are further involved in the interaction between developing cells and cells in their developmental environment. These interactions include the exchange of metabolites and the expression of adhesion molecules, such as cadherins, and ensure both stemness and control of the induction of differentiation.¹⁴³

For pancreatic cells, the expression of Cx43 is maintained until the cells developed into pancreatic progenitor cells and begin to express Cx32.⁸⁹ Both Cx isoforms are necessary for the proliferation of stem cells and for progenitor cells to form large β-cell colonies.¹⁴⁴ Finally, the significance of Cx for the differentiation of iPSCs into MSCs and MSC differentiation along adipogenic lineage mesenchymal cells has also been studied. Cx43 and GJIC are necessary for iPSC-derived MSC differentiation into chondrogenic lineages.¹⁴⁵ In other studies, using CRISPR-Cas9 technology, it has been reported that ablation of Cx43 strongly reduces GJIC but does not inhibit the differentiation of iPSCs in MSCs and the first stage of adipogenic differentiation. A role of Cx43 has been postulated for the entire adipogenic differentiation process. However, Cx is necessary to avoid early cell senescence¹³¹ and to close adipogenic development in a cell density-dependent mechanism.¹⁴⁶

Conclusion

As shown in the abstract Figure, in developing embryos, Cxs are expressed as early as the 4–8 cell stage when the cells are totipotent. They continue to be expressed in pluripotent cells and multipotent cells in tissue. In organisms, it seems that Cxs and GJIC play minor roles in embryonic cells. Knock out of Cxs does not hinder gastrulation. The imperative role of Cxs manifests when tissues start to differentiate during gastrulation. In culture, Cxs are necessary for the stemness and differentiation of PSCs, iPSCs and adult stem cells. Pharmacological inhibition of GJIC or knockdown of Cx convincingly reveals that Cxs participate in the physiology and differentiation of different types of stem cells. The role of Cxs in stem cells involves their capacity to form GJ channels and hemichannels as well as their function as regulators of cell adhesion, cell migration and gene expression. As channels, it is still not clear which signals and metabolites are exchanged with respect to the different stem cell types. Likewise, many studies have been performed on Cx43. More studies involving the other expressed isoforms, mainly Cx26, Cx32, or Cx45, are needed to completely clarify the significance of Cxs in the physiology and development of stem cells.

Acknowledgments

The authors thank Philip Palarz for helping by producing the figure.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This work was supported by the Federal Ministry of Education and Research (BMBF), Grant TRANS-LARA 02NUK051A and the Deutsche Forschungsgemeinschaft (DFG), Grant NG 4/10-1