Co-culture of MSCs and immune cells

The interactions between MSCs and immune cells are essential not only to maintain and restore tissue homeostasis but also to balance the inflammatory response during tissue regeneration. Within this scope, the crosstalk between MSCs and immune cells, namely neutrophils and macrophages, has been intensively investigated to understand how these interactions can be used to positively modulate and regulate the tissue regeneration process [10, 37, 46].

Neutrophils are usually the first inflammatory cells recruited at a site of injury, enhancing host defenses while removing contaminants [2, 16]. The existence of different subsets of neutrophils was first characterized according to their polarization states in the tumor microenvironment, namely N1 (pro-inflammatory subtype) and N2 (anti-inflammatory subtype) [47]. Whereas the N1 subtype has potent anti-tumor activity due to its capacity to release pro-inflammatory factors, namely IL-12, tumor necrosis factor-α (TNF-α), chemokine ligand 3 (CCL3, also known as macrophage inflammatory protein 1-alpha), and CXCL9, which facilitates recruitment and activation of CD8⁺ T cells, the N2 subtype has strong immunosuppressive and tumor-promoting activity characterized by secretion of oncostatin M, reactive oxygen species (ROS), reactive nitrogen species (RNS), matrix metalloproteinases (MMPs), and neutrophils elastase [48]. Despite the main properties of the N1/N2 subtypes being demonstrated in tumor microenvironments, their exact role, behavior, activation, recruitment, and temporal polarization during tissue repair are under investigation. Considering the importance of such cells, it has been demonstrated that MSCs are able to modulate the phenotype and activity of neutrophils in all phases of tissue repair and regeneration [44]. A co-culture of MSC-neutrophil showed that MSCs enhance the phagocytic ability of neutrophils, which contributes to the efficient removal of necrotic tissue and cellular debris [10]. Moreover, MSCs, via up-regulation of the extracellular superoxide dismutase (SOD3), prevent neutrophil death and enhance neutrophil-dependent elimination of microbial pathogens and cellular remnants. Furthermore, during the resolution phase of tissue repair, MSCs reduce the presence of N1 type neutrophils and stimulate their conversion to an N2 phenotype [10, 44]. In fact, the co-culture of MSC-neutrophil showed that MSCs secrete TNF-α stimulated gene/protein 6 (TSG-6), a key molecule correlated with the decrease of acute inflammation [46]. Notably, the reduction of ROS and the increased expression/secretion of IL-10 by neutrophils, favoured their polarization to an N2-like phenotype [46].

Compared to other immune cells, the crosstalk between MSCs and macrophages has become an attractive alternative for immunomodulation strategies [20, 40, 49, 50]. Similar to the N1/N2-like neutrophil paradigm, recent interest has grown around the concept of an M1/M2-like macrophage spectrum of polarization states during tissue regeneration. While M1 macrophages secrete monocyte chemoattractant protein-1 (MCP-1), MMP-12, NO, inflammatory cytokines (e.g., TNF-α, IL-1β, IL-12), and various other inflammatory chemokines, which stimulate the recruitment of circulating immune/stem cells to the site of injury [51], M2 macrophages, through the production of numerous growth factors, including PDGF, TGF-β1, insulin growth factor (IGF)-1, and VEGF-α, promote stem cells recruitment, neo-angiogenesis, deposition of collagen and ECM proteins [52]. Additionally, in case of severe injury, M2 phenotype also stimulates the activation and differentiation of tissue resident stem and progenitor cells [18, 51]. In this scope, different macrophage phenotypes play distinct roles at different stages of tissue repair and regeneration. Remarkably, MSCs support the phagocytic functions of M1 macrophages in the initial phases of tissue repair, while, in the last phases, promoting the expansion of M2 macrophages [2, 53].

For example, successful bone fracture healing is based on carefully coordinated crosstalk between macrophages and bone-forming cells, more specifically MSCs [52]. In particular, the key role that macrophages play in the recruitment, regulation, and differentiation of MSCs as well as the influence of MSCs in the macrophage function, activity, and polarization during bone tissue regeneration have been brought to focus. To clarify this, Zhang et al. [50] co-cultured MSCs with M0 (naive), M1, and M2 macrophages and measured alkaline phosphatase (ALP) activity at 7, 14, and 28 days. The results revealed that M1 and M2 macrophages were able to promote osteogenic differentiation at 7 and 28 days, respectively. Interestingly, the co-culture of MSCs with M2 macrophages, but not with M1 or M0 macrophages, resulted in a significant increase of MSC mineralization caused by secretion of soluble factors. Moreover, M2 macrophages also promoted proliferation and osteogenic differentiation of MSCs, while the subtypes M0 and M1 solely stimulated the osteogenic differentiation of MSCs in the early and middle stages of co-culturing. In terms of molecular mechanisms, the secretion of specific exosomes by the different subtypes of macrophages has been correlated with the osteogenic differentiation of MSCs [40]. However, in comparison to M2 macrophages, the exosomes secreted by M0 and M1 macrophages seem to be more effective in the osteoblastic differentiation of MSCs [49]. These results suggest that M0 and M1 phenotypes are essentials in early stages of bone injury playing a critical role in the osteogenic differentiation of MSCs, while M2 subtype can stimulate proliferation of MSCs, osteogenic differentiation, and mineralization in late stages of the bone tissue regeneration process.

Furthermore, Cho et al. [21] demonstrated that a transwell co-culture with MSCs and macrophages significantly prevented M1 macrophage polarization and induced M2 polarization. Additionally, another in vitro approach demonstrated that macrophages cocultured with MSCs consistently showed high-level expression of CD206, a marker of activated macrophages, while also expressing high levels of IL-10 and low levels of IL-12, compared to controls [54]. Functionally, these macrophages cocultured with MSCs showed a higher level of phagocytic activity and polarization to the M2 phenotype. Using three-dimensional (3D) models, a gelatin methacryloyl (GelMA) hydrogel encapsulating BM-MSCs and macrophages showed optimal effects on the proliferation, migration, osteogenic and chondrogenic differentiation, while also showing significant M2 macrophage polarization [55]. Inspired by these in vitro results, the in vivo implantation of hydrogels promoted osteochondral repair through an intrinsic crosstalk between MSCs and macrophages at the bone injury. Likewise, another approach showed that the transplantation of decellularized human amniotic membrane seeded with MSC-educated macrophages into animal models improved healing processes in incisional wounds by decreasing the inflammatory phase, while increasing angiogenesis, and reducing scar tissue development [56].

For instance, these findings support the powerful crosstalk between MSCs and myeloid cells, namely neutrophils and macrophages, during different phases of the regenerative process. The capacity of MSCs to switch the polarization of these cells conjugated with the effect of myeloid cells exert in MSCs can be used to locally inhibit conventional pro-inflammatory pathways while enhancing alternative anti-inflammatory responses at the injury site. Consequently, several in vitro/in vivo combinations are still necessary to explore different cellular parameters with the phase of the regenerative phase. This know-how could provide the development of several alternatives for immunomodulatory strategies using specific pool populations of MSCs, neutrophils, or macrophages, according to the individual necessity, target tissue, and regenerative phase.

Culture of MSCs in the presence of inflammatory factors

It is known that during the inflammatory phase, the amount of pro-inflammatory cytokines is significantly elevated. Taking into consideration that the biological activities of MSCs are not only influenced by the direct contact with immune cells but also by the presence of biomolecules, several in vitro studies have investigated the survival of MSCs, as well as their migration, proliferation, differentiation, and secretome profile in the presence of pro-inflammatory factors [36, 38, 57, 58].

In this sense, Wedzinska et al. [57] explored the biological functions of MSCs derived from Wharton’s jelly (WJ-MSCs) exposed to pro-inflammatory cytokines, namely interferon-γ (IFN-γ), TNF-α, and IL-1β at different concentrations and under different aerobic conditions (21% O₂ vs. 5% O₂), key factors of the inflammatory phase. While both aerobic conditions did not influence the dynamic proliferation of MSCs, the presence of pro-inflammatory signals stimulated the secretion of anti-inflammatory cytokines, namely IL-4 and IL-10 by MSCs.

Similarly, human BM-MSCs cultured with a cocktail of pro-inflammatory cytokines induced a significant upregulation of proteins involved with angiogenesis and resolution of inflammation [38]. Of note, priming MSCs with pro-inflammatory molecules, such as IFN-γ and TNF-α elicits an immunomodulatory phenotype by these cells characterized by an upregulation secretion of chemokines such as CXCR3, CCR5, CXCL9, CXCL10, and CXCL12, which attract immune cells via chemotaxis; anti-inflammatory cytokines (e.g., IL-10), and many other soluble immunosuppressive factors (e.g., IDO, COX-2, PGE-2, TGF-β) [2, 42].

Notably, these findings have been used to potentiate the immunomodulatory effect of MSCs before their in vivo administration [58]. However, a limitation of this strategy is that some pro-inflammatory signals, for example, IFN-γ, also upregulates the expression of genes associated with programmed cell death, reflecting a negative impact on MSC survival and proliferation after implantation [36]. Likewise, only cytokine-activated MSCs, but not non-activated MSCs, are effective in the reduction of an exacerbated inflammatory response through the release of anti-inflammatory factors [59].

Culture of MSCs under hypoxic conditions

MSC-based therapies typically rely on the ex vivo expansion of cells under ambient oxygen and high glucose levels that favour mitochondrial respiration for energy generation [5]. However, upon transplantation, donated MSCs are exposed to a hypoxic microenvironment which significantly impacts stem cell biology, particularly its metabolism. Given the microenvironment of hypoxia usually created at the site of tissue injury, the culture of MSCs under hypoxic conditions has been gaining substantial interest to understand how such conditions can influence the regenerative properties of MSCs.

Since the therapeutic functions of these cells can be impacted by physical conditions, several in vitro studies have shown higher viability and proliferative rate of MSCs cultured under hypoxia (1–10% O₂) in comparison to normoxia (± 21% O₂) [39, 45, 60]. Additionally, hypoxic conditions induce distinct changes in the biological activities of MSCs, influencing their metabolism, secretion of cytokines, growth factors, and EVs. For instance, the pre-incubation of MSCs for 2 days or more in 1% oxygen reduced serum deprivation-mediated cell death, while expressing significantly low levels of cytochrome c and heme oxygenase 1 compared to controls [60]. The consumption of glucose and secretion of lactate at a slower rate in comparison to controls, possibly promoted cell survival, as glucose remained available for longer periods of time. Most importantly, MSCs showed enhanced survival in vivo after intramuscular injection into immune-deficient mice. These findings suggest that the preincubation of MSCs under hypoxia induces key metabolic changes that yield higher retention after transplantation. The role of metabolism in regulating the function of MSCs has opened new avenues to improve the effectiveness of stem cell-based therapies for tissue regeneration. As a consequence, strategies to mitigate the metabolic shock experienced by MSCs after transplantation have become an attractive alternative to alleviate stem cell death while improving the antioxidant, cytoprotective defense, and immunomodulatory effects of MSCs [42].

Despite metabolic changes, the co-culture of MSCs and human umbilical vein endothelial cells (HUVECs) under normoxic (21% O₂) vs. hypoxic conditions (5% O₂) showed that hypoxia significantly increased the tube formation capacity of these cells compared to normoxia [45]. This result was correlated with the secretion of more functionally potent MSCs-derived EVs cultured under hypoxia, indicating that the angiogenic potential of MSC is facilitated by EVs release rather than by soluble factors under hypoxic compared to normoxic conditions.

Similar results were reported from MSCs cultured in serum-free media under hypoxia [6]. The release of anti-inflammatory EVs was also accompanied by an increased hypoxia-inducible factor 1-alpha (HIF-1-alpha). To corroborate these results, pre-treated MSCs cultured under hypoxic conditions demonstrated greater therapeutic effects after implantation in mice with liver cirrhosis [61]. The regenerative properties were correlated with local secretion of prostaglandin E synthase, macrophage polarization to M2 phenotype, and reduced hepatocyte cell death.

Although the preconditioning of MSCs under hypoxic conditions significantly impacts their angiogenic and immunomodulatory potential before implantation, the use of MSC-secretome has also emerged as another kind of MSCs-based therapy. Currently, MSCs-free therapy, more specifically MSC-secretome therapy, presents key advantages compared to direct MSC transplantation, including: (i) decrease the risk of immune reactions and carcinogenesis; (ii) facility to be manufactured in a large scale; (iii) safety has been confirmed in vivo and in some clinical trials [35].

The use of biomaterials to create pro-regenerative strategies

In contrast to the cell-only regenerative medicine paradigm, biomaterials have been extremely useful to recreate biomimetic 3D microenvironments resembling key properties of the regenerative process while also directing tissue-specific repair. Basically, a critical mode of action by which biomaterials can promote tissue repair is by influencing the differentiation of stem cells and modulation of the immune response. To this end, several strategies have combined numerous components of tissue engineering tools (e.g., cells, bioactive molecules, biomaterials, and technologies) [62]. In this sense, various biomaterials with tunable biophysical and biochemical characteristics have been designed to facilitate and improve the attachment, migration, proliferation, and differentiation of MSCs into tissue-specific cell types, while modulating the immune response in favour of a pro-regenerative state [63, 64].

Basic approaches have focused on the interaction between biomaterials and cells, namely stem cells and immune cells, using several types of monoculture or co-culture systems [20, 65–67]. The use of biomaterials as a 3D platform can both preserve the tissue architecture and provide a 3D biomimetic milieu for MSCs, which enhances their paracrine functions, including their immunomodulatory potential [68]. The dimensionality, physical, topographical, and biochemical cues, and nano/microstructure can be used to improve the regenerative properties and immunomodulatory capacity of MSCs [64, 68–70]. For example, the use of liquefied capsules containing MSCs and macrophages has been proposed as a promising immunomodulatory platform, which favours the osteogenic potential of MSCs and macrophage polarization toward a regenerative profile, through the up-regulation and release of anti-inflammatory molecules [20]. Despite the direct effect of biomaterials on MSCs’ regenerative potential, the use of biomaterials has been extremely useful to elucidate the main upstream effector immune cells and their functions throughout the whole regeneration process [71].

In this line, studies have suggested that biomaterials can play a major role in influencing the polarization of both macrophages and T-helper cells, which have significant crosstalk during tissue healing [72, 73]. Therefore, among immune cells, macrophages are the dominant effector cells, playing a prominent role in tissue repair through the polarization from M1 to M2 phenotype. This can be illustrated by a study showing that T-helper cells are necessary for macrophage polarization to an M2 phenotype seeded in decellularized ECM scaffolds [65]. Translating for in vivo models, titanium implants of varying wettability (rough, and rough-hydrophilic) placed in the femur of mice showed that surface modifications applied to titanium implants polarized the adaptive immune response towards a type 2 helper cells (Th2), characteristic of a pro-wound healing phenotype, with increased recruitment of MSCs and macrophages, both in vitro and in vivo [71].

Remarkably, these kinds of approaches exemplify the use of biomaterials as a key tool for the development of promising exploratory platforms with several applications, such as (i) investigation of the involvement and crosstalk of many cellular components during tissue repair; (ii) combination of different biomaterials, properties, and technologies to design strategies with high biological complexity and regenerative capacity; (iii) synchronization of cells with biochemical and physical cues in a regulated manner able to guide and improve new tissue formation, both in vitro and in vivo; (iv) design of effective immunomodulatory strategies; and (v) personalized therapies for tissue regeneration [62].

In this perspective, the design of bioengineering microenvironments which recapitulate crucial characteristics of the different regeneration phases holds great promise to provide new treatment concepts for replacement, regeneration, and tissue healing. However, in vitro, strategies that diversify the cellular components to understand the host responses under different stimuli need to be intensively explored.

In summary, despite all the approaches presented here have supported the use of MSCs as “multi-talented cells” for regenerative therapies with a broad clinical application (Figure 2), most of the overwhelmingly positive results seen in preclinical studies have not yet translated into clinical [74]. Maybe the use of these cells in the clinic should consider the conjugation of MSCs-parameters such as tissue source, number of cells, activated vs. nonactivated-MSCs, study design (preconditioning/non-preconditioning, cell-based therapy, cell-free therapy, absence/presence of biomaterials) with individual parameters, namely age, gender, patient health conditions, and the stage of the regenerative process [41]. As a consequence, the conjunction of multiple parameters and standard protocols may facilitate the development of new therapeutic tools based on the use of MSCs or MSCs-derived products that positively influence the immune response while improving tissue regeneration in a more personalized way.

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Figure 2. 

Representation of the main regenerative properties of the MSC. A) This panel shows the most important effects of MSCs during tissue regeneration; B) MSC can be isolated from BM aspirate, dental pulp, UC tissue, and adipose tissue

Evaluation of the selective migration of HSPCs after tissue injury

Normally, HSPCs reside in specialized niches in the BM microenvironment, in which these cells usually stay in a quiescent state [12, 95, 96]. However, in some cases, such as infection or inflammation, there is a selective mobilization and migration of rare populations of HSPCs to injured regions [81, 94]. In this line, there is the necessity to clarify the molecular mechanisms by which specific HSC subpopulations home to the tissue of interest, and what is the exact role of these cells at the injured tissue.

It is well recognised that platelets are the first line of defence after a tissue trauma or injury. These cells rapidly adhere and aggregate upon exposure to subendothelial components such as collagen and von Willebrand factor [97]. Evidence has demonstrated that platelets release specific cytokines for the recruitment of adult stem cells, namely MSCs and HSPCs, to site of vascular injury [81]. This again confirms the indispensable role of both stem cells for an effective resolution of the regenerative process [29]. Similarly, Powerski et al. [94] demonstrated that after a musculoskeletal surgery, the total circulating CD133⁺/CD34⁺ HSCs significantly increased in the postsurgical period (after 8 h). These cells selectively migrated to the injured site and stimulated tissue repair through differentiation to parenchymal cells and/ or by post-traumatic immunological response.

Another approach using systemic administration of HSCs in murine models showed that the migration of these cells to the hepatic ischemia-reperfusion (IR) injury done via alpha(4)beta(1)/vascular cell adhesion molecule-1 (VCAM-1) pathway [98]. Interestingly, HSCs adhered in sinusoidal capillaries, suggesting sinusoids are the initial point of entry to the injured liver for HSCs and that the alpha(4)beta(1)/VCAM-1 pathway is responsible to drive the hematopoietic migration mechanism. Likewise, White et al. [99] explored the molecular mechanisms governing HSCs recruitment to injured kidney. Both murine models and HSCs were pre-treated before HSC infusion with blocking antibodies, hyaluronidases, or cytokines. CD49d, CD44, VCAM-1, and hyaluronan governed HSC adhesion to the IR-injured kidney. Remarkably, both pre-treatment strategies with keratinocyte-derived chemokine (KC), murine functional homologue of human IL-8 (released after acute renal injury), and stromal cell-derived factor-1α (SDF-1α, released following renal injury) significantly increased HSC adhesion within the injured kidney, whilst SDF-1α also increased numbers continuing to circulate. However, SDF-1α and KC did not increase CD49d or CD44 expression but increased HSC adhesion to VCAM-1 and hyaluronan, respectively. SDF-1α increased CD49d surface clustering, as well as HSC deformability. Increasing HSC adhesive capacity for its endothelial counter-ligands may explain their enhanced renal retention in vivo. Moreover, increasing HSCs’ deformability with SDF-1α treatment could explain the prolonged systemic circulation, while HSCs can also continue to survey the damaged tissue instead of becoming entrapped within non-injured sites. Taken together these results suggest that both autologous and exogenous HSCs are capable to migrate to damaged tissues, even in low amounts. For instance, some works have shown that it is possible to manipulate the mechanisms of HSC mobilization/migration through the activation of specific HSC signaling pathways by using antibodies, growth factors, or cytokines. Currently, the recruitment of these rare cells to injured tissues may also be a new window of opportunities in the medicine regenerative field.

HSCs biology and inflammation

Similar to MSCs, significant interest has emerged to understand how an inflammatory microenvironment influences the fate of HSCs and HSCs-derived progenitors. Notably, it has been demonstrated that the biology and behavior of HSPCs can be directly affected by inflammatory signaling [100]. During inflammation, the hematopoietic equilibrium is disrupted and quiescent HSCs are directly or indirectly activated to generate more mature immune cells, especially cells of the myeloid lineage [95]. Therefore, HSPCs can be activated directly through toll-like receptors (TLRs) or indirectly through cytokine/growth factor receptors to mediate their hematopoietic responses [101].

Recent findings show that HSCs are the first responders to infection and that pro-inflammatory factors released during infection or inflammation, namely TNF-α, granulocyte colony-stimulating factor (G-CSF), macrophage colony-stimulating factor (M-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-1, IL-6, IL-8, and IFNs are key regulators of HSCs [82, 100]. As a consequence of such stimuli, downstream signaling cascades initiate the mobilization of HSPCs in order to provide effector immune cells for the infection/inflammation place [101].

Despite inflammatory factors having different effects on HSCs activation and differentiation, studies have shown that such signaling usually activates the differentiation of HSCs towards myelopoiesis rather than lymphopoiesis [95]. In this scope, a murine model which resembles the pathogenesis of human inflammatory bowel disease (IBD) showed that acute gut inflammation expands HSCs and MPPs in the BM while enhancing myelopoiesis via up-regulation of GM-CSF ligands in local tissue and its receptor on HSPCs [79]. At the same time, MPPs migrated to gut-associated lymph nodes to expand, and potentially differentiated into myeloid cells, suggesting a key role of these cells in gut tissue repair. On the contrary, the block of MPP migration exacerbated the colitis symptoms and gut tissue atrophy.

Similarly, studies performed in zebrafish revealed that TNF promotes HSCs survival and myeloid differentiation by activating a specific p65/NF-κB-dependent gene program that primarily prevents hematopoietic necroptosis [102]. However, dysregulation of TNF production has been related to directly inhibiting growth and inducing apoptosis of HSCs, as well as indirectly changing the BM microenvironment.

Additionally, IFN-γ, one of the critical cytokines controlling inflammation, seems to have oppositive effects on regulating HSCs proliferation and differentiation, possibly in a context-dependent manner [101]. While IFN-γ was able to activate HSC proliferation and differentiation during infections in vivo models [77, 103], contrary findings have demonstrated that IFN-γ also acts as a negative regulator of HSCs and their progenitors, directly reducing their proliferative capacity and complicating the restoration of HSC numbers upon viral infection [104]. In fact, not only IFN-γ but also IFN-α have the ability to play dual effects in the hematopoietic system [105]. While these molecules can be beneficial to induce proliferation and differentiation of HSCs, increasing myelopoiesis, and enhancing immune responses in response to acute infections [105], the same molecules are also capable of inducing deleterious long-term effects, namely HSC exhaustion and selection pressure for clonal stem cells during chronic infection [106].

Persistent cytokine stimulation via TNF, IFN, and IL-6 has been correlated with HSC dysfunction and may readily impact the initiation and progression of hematologic malignancies and BM failure [107]. Notably, chronic immune stimulation induces cell stress, DNA damage, and other dysfunctions in the hematopoietic lineage, including somatic mutations and myeloid neoplasms [108].

Overall, these findings suggest that under an inflammatory microenvironment or specific signaling of acute inflammation, HSCs actively cooperate with downstream hematopoietic progenitors, mature cells, and environmental stromal cells as frontline responders to preserve blood homeostasis [79]. However, their ability to respond deftly through self-renewal and differentiation at times brings about detrimental consequences, namely in the case of chronic inflammation. In this sense, a deeper understanding of how different inflammatory microenvironments and signaling factors can affect and influence the HSC biology, in terms of heterogeneity, functionality, adaptability, survival, mutations, and cell death, could clarify in which phase of the regenerative process HSPCs are more appropriate for therapeutic use.

Exploring the plasticity potential of HSCs

In the last decades, some data have demonstrated the ability of HSCs to transdifferentiate into cells types that do not belong to the hematopoietic lineage tree, such as central nervous, skeletal muscle, endothelial, heart, and liver cells under restrictive and specific conditions [78, 84, 86, 109, 110]. Despite several controversies, the plasticity potential of single HSCs has emerged as a new concept to be explored in the field of HSC-based therapy for regenerative medicine.

For example, the culture of CD34⁺ cells, also defined as HSPCs, on a surface of fibrin and activated platelets promoted the maturation of CD34⁺ cells toward a mature endothelial cell phenotype [111]. The endothelial phenotype was then correlated with significant upregulation of CD31, CD105, and VEGF receptor-2 (VEGFR-2) expression, confirming the ability of CD34⁺ cells to convert into endothelial cells. Although, activated platelets secrete CXCL12, which has been shown to mobilize and activate integrins on the surface of stem cells, including HSCs, the molecular mechanisms by which platelets could induce HSC transdifferentiation needs to be addressed both in vitro and in vivo.

Furthermore, to evaluate the HSC potential to differentiate into liver cells, an in vitro combination of FGF-4 and HGF was used to stimulate HSCs from UCB for 14 days [86]. After stimuli, the cells demonstrated high expression of genes related to hepatocytes [tryptophan 2,3-dioxygenase (TO), glucose 6-phosphate (G6P), cytokeratin 18 (CK 18) albumin, and α-fetoprotein (AFP)]. According to the authors, this transdifferentiation potential of HSCs into hepatocytes could be an interesting alternative for cell replacement in case of liver diseases. Translating to in vivo models, Zhou et al. [78] used immune-deficient mouse models with liver damage to evaluate both the capacity of HSCs to migrate to the damaged tissue and their subsequent contribution to liver repair. Notably, transplanted human HSCs isolated from BM or UCB did not robustly become hepatocytes, but some hepatic transdifferentiation was documented. However, injected HSCs did home to the injured liver, contributing to liver repair not by hepatocyte transdifferentiation, but by the local release of paracrine factors.

Regarding the hematopoietic capacity to transdifferentiate into cardiomyocytes, it was demonstrated that not only HSCs-derived progenitors but also mature blood cells, namely macrophages, in response to a cocktail growth factors and ILs treatment or in co-culture with cardiac explants demonstrated ability to cardiac differentiation and integration into contractile heart tissue [110]. Considering that the time-course for cardiac differentiation of HSPCs was only 4 days, there is a lack of information about for example the viability and functionality of these differentiated cells over time. Some in vivo experimentation should also be performed to corroborate the in vitro results.

Together, these findings suggest that the plasticity of adult HSCs, even if it is rare, can be correlated with specific microenvironments/tissues or due to restricted stimulation which reprograms the fate of HSCs to differentiate into non-hematological cells. Additionally, these data highlight further and intensive investigation to elucidate specific conditions and mechanisms by which these HSCs and their progeny can directly or indirectly differentiate into non-hematological cells.