Role of Hedgehog Signaling Pathways in Multipotent Mesenchymal Stem Cells Differentiation

The Hh Signaling Pathway Regulates Osteogenic Differentiation of MSCs

Osteoblasts develop from the MSCs through a process called osteogenesis. Osteogenesis is an important developmental event that results in bone formation. Bone-forming cells or osteoblasts develop from MSCs through a highly controlled process regulated by several signaling pathways, such as Hh, BMPs, Notch, neural epidermal growth factor-like 1 protein (NELL-1), and Wnt/β-catenin signaling ¹⁰³ . These signaling pathways individually as well as in coordination with other signaling molecules, regulate the osteogenic lineage commitment of MSCs by activating several osteo-lineage specific transcription factors ¹⁰³ . Among these factors, Hhs play an important role in bone formation and homeostasis, as their vital roles in modulating the osteogenesis of MSCs, and key functions both individually as well as in coordination with other signaling cascades in terms of Wnt, BMP, and parathyroid hormone-related protein (PTHrP) during skeletal development, bone repair, and bone regeneration^103–107. During embryonic development, SHh plays a critical role in skeletal development, participating in the shaping of the trunk, limbs, and facial bones ^108,109, while IHh has an important role in skeletal development and regulates endochondral bone formation, and intramembranous ossification ¹¹⁰ . In fact, disruption of the Hh signals can lead to bone-related diseases in terms of osteoarthritis, osteoporosis, bone defects, and severe skeletal deformities ¹¹¹ . Inhibition of Hh signaling using the Hh inhibitor cyclopamine blocks Hh signal and leads to a lack of peripheral cartilage ossification in the head ¹¹² .

Studies have shown that undifferentiated human MSCs have a basal level of Hh signaling, which may be maintained by autocrine secretion of SHh and IHh expressed in these cells ⁴⁹ . An early study has shown that recombinant N-terminal Sonic Hedgehog (rSHh-N) promotes proliferation and osteogenic differentiation of rat BM-MSCs, as evidenced by increased alkaline phosphatase (ALP) activity, upregulation of the osteogenesis-related genes collagen type I, core-binding factor alpha1 (Cbfa1), and osteocalcin (OCN), and increased matrix mineralization in differentiating BM-MSCs^113,114. Implantation of rSHh-N overexpressing MSCs significantly accelerates bone formation in vivo, and promotes significant improvements in scaffold revascularization at the bone defect reconstruction by enhancing the survival rate and differentiation of donor cells and revascularization of the bone defect site ¹¹⁵ . Therefore, the Hh signaling is essential for bone remodeling during postnatal life by directing the differentiation of MSCs into osteoblasts^116–118. In fact, there is still some controversy over the impact of Hh signaling on the osteogenic differentiation of MSCs. Some studies suggested that activation of the Hh signaling pathway reduces osteoblastic differentiation of human MSCs^49,119.

MSCs, give rise to osteo-chondro progenitors, which develop into osteo-precursors upon Runx2 (Runt-related transcription factor 2) activation or chondrocytes if Sox9 (SRY box transcription factor 9) is activated ¹²⁰ . SHh enhances osteogenic differentiation of MSCs in Runx2-dependent and manner and is accompanied by expression of bone progenitor cell markers and matrix mineralization of differentiated MSCs^{114,121–124}. Runx2, the master regulator of osteogenesis, has an important role in early osteogenic lineage commitment^125,126. And, it is a specific phenotype and key transcription factor that directs MSCs toward osteoblastic differentiation and inhibits them from differentiating into adipocytes and chondrocytes ¹²⁷ . The secreted molecule NELL-1 is another critical factor that binds with apoptosis-related protein 3 (APR3) ¹²⁸ and colocalizes with Cntnap4 on the cell surface to promote osteogenic differentiation of human AD-MSCs^62,129. NELL-1 is a transcriptional target of the transcription factor Runx2, where Runx2 increases NELL-1 transcription in a dose-dependent manner^103,118. Mechanistically, NELL-1 is preferentially expressed in osteoblasts, and its expression level is like that of Runx2, and is highest during skeletogenesis^130,131. Combined activation of Hh, NELL-1 signaling by SHh-N (SHh, N-terminus protein) and NELL-1 addition had an additive pro-osteogenic effect on human AD-MSCs ⁶² . NELL-1 promotes osteogenesis accompanied by activation of Hh signaling ⁶² . Therefore, Runx2 and NELL-1 are key players for Hh signaling to promote MSCs differentiation into osteoblasts.

In addition to SHh protein, some small molecule compounds (purmorphamine [PMA] and oxysterols) and drugs (simvastatin) can also promote the osteogenic differentiation of MSCs by enhancing Hh signaling. PMA and oxysterols are originally identified as effective osteogenic factors in MSCs and are identified as activators of the Hh pathway^132–136,94. They upregulated gene expression of the mediators of Hh pathway, SMO, PTCH1, GLI1, and GLI2, and increased the expression of several genes (eg, RUNX2 and BMPs) related to osteogenesis through the activation of Hh pathway ^89,92,94. Nevertheless, they activate the Hh signal through different mechanisms^134,136,137. PMA activates the Hh pathway by targeting the seven transmembrane pathway component Smoothened (SMO), a critical component of the Hh signaling pathway^89,92,94. Oxysterols stimulate Hh pathway activity by indirectly activating SMO^90,91. Simvastatin administration resulted in an enhanced osteogenic differentiation capacity, indicated by upregulated expression of COL1, ALP, and OCN, and increased ALP activity. More importantly, bone marrow mesenchymal stem/stromal cells (BMSCs) treated with simvastatin expressed higher levels of IHh and GLI1, and more nuclear translocation of GLI1 was observed ¹³⁸ .

Although several pathways individually influence the bone formation, osteogenesis is achieved by the coordinated action of multiple signaling pathways ¹⁰³ . For instance, Hh can coordinate other signaling pathways to promote osteogenic differentiation of MSCs. Bone morphogenetic protein 9 (BMP9) promotes osteogenic differentiation of mouse MSCs, and the Hh signaling pathway plays an important regulatory role in BMP9-induced MSCs osteogenic differentiation ¹³⁹ . Specifically, BMP9 induces the expression of early osteogenic markers (such as ALP) and late osteogenic markers (such as matrix mineralization) as well as osteopontin (OPN) and OCN in MSCs, but this effect is significantly inhibited by the Hh signaling inhibitor cyclopamine. Hh-induced osteoblastogenesis requires BMP signaling, and together they elicit a synergistic expression of alkaline phosphatase activity ¹⁴⁰ .

Micro-RNAs (miRNAs) are important regulators of gene expression involved in osteogenesis, and regulate the Hh signaling pathway in MSCs. The miR-324-5p regulates the Hh signaling pathway through different mechanisms in human BM-MSCs and mouse C3H10T1/2 cells (MSCs), thereby inhibiting osteogenic differentiation, miR-324-5p targets SMO and GLI1 in the human Hh signaling pathway, while targeting Glypican 1 (GPC1) in mice. MiR-196a can activate the Hh signaling pathway by targeting GNAS to reverse osteogenic differentiation in osteoporotic mice MSCs ¹⁴¹ , whereas miR-467g has been found to be an inhibitor of osteoblast differentiation, negatively regulating osteogenesis through the IHh/Runx2 signaling pathway ¹⁴² .

Additionally, in high glucose and oxidative stress conditions, partially inhibiting MSCs osteogenic differentiation through regulating the Hh signaling pathway is possible. High glucose inhibits SHh protein expression in rat BM-MSCs^143,144, while the activation of the SHh signaling pathway can upregulate the expression of bone morphogenetic protein 4 (BMP4), bone sialoprotein (BSP) and OPN in MSCs through transfection with lentivirus SHh vectors ¹⁴⁵ . Thus, SHh overexpression significantly increases the osteogenic ability in rat BMSCs in vitro and bone formation in vivo and reverses diabetes induced osteogenesis defects ¹⁴⁶ .

Oxidative stress inhibits the Hh signaling pathway and Hh-mediated osteogenic differentiation of MSC ¹⁴⁴ . The addition of exogenous H₂O₂ (125–500 mM) can decrease ALP activity in human BM-MSCs ¹⁴⁷ . Therefore, H₂O₂ can prevent SHh-mediated osteogenic differentiation of mice and other MSCs lines in vitro and decrease the expressions of ALP, Osterix (OSX) and BSP ¹⁴⁴ . GLI1 overexpression reverses oxidative stress-mediated reduction in osteogenic differentiation of MSCs ¹⁴⁴ . Pretreatment of MSCs with antioxidants may promote osteogenic differentiation of oxidative stress-damaged MSCs by modulating the Hh signaling pathway. Together, these studies suggest that although inhibiting the Hh signaling pathway is beneficial for osteogenic differentiation of MSCs, excessive inhibition of Hh signaling can impair MSCs’ osteogenic potential. The Hh signaling transduction affects early differentiation of osteoblasts ¹⁴⁸ and regulates mature osteoblasts ⁴⁹ . Thus, the Hh pathway may be a potential target for developing treatments for bone-related diseases, such as osteoporosis and fracture healing disorders.

The Hh Signaling Pathway Regulates Chondrogenic Differentiation of MSCs

The Hh signaling pathway plays an important role in the chondrogenic differentiation of MSCs^149–154 and in cartilage development in vivo. IHh and SHh have a synergistic effect on inducing chondrogenesis and cartilage formation in a microgravity environment, promoting the formation of cartilage matrix ¹⁵⁵ . Activation of the Hh signaling pathway promotes downstream target gene expression associated with cell proliferation by inducing GLI transcription factors, such as cyclin D1^{48,59,156,157}. Therefore, sustained activation of the Hh signaling pathway causes uncontrolled cell proliferation and may contribute to the development of chondrosarcoma^156,157. Regulation of the Hh signaling pathway suggests that it promotes MSCs proliferation and tends to chondrocyte differentiation. Three Hh signaling proteins (SHh, DHh, and IHh) contribute to the construction of tissue-engineered cartilage in vitro in a dose-dependent manner^150,158.

A study demonstrates that Hh, TGF-β1, and BMP-2 proteins are equivalent inducers of MSCs chondrogenesis ¹⁵⁴ . Nevertheless, compared with BMP, TGF, and other growth factors, SHh plays a stronger role in promoting human MSCs proliferation and chondrocyte differentiation. It enhances the expression of chondrocyte-specific markers collagen type II (Col II), chondroitin sulfate, Sox9, and CEP-68 ¹⁴⁹ . Reprogramming human MSCs to increase the activity of the SHh signaling pathway can promote MSCs chondrogenesis ¹⁵⁹ . Atractylenolide is enriched in atractylodis macrocephalae, which is commonly used to treat arthritis. One study has shown that adding atractylenolide induces GLI promoter and promotes chondrogenic differentiation of rat BM-MSCs. Compared with untreated BM-MSCs, the expression of chondrogenic markers Sox9, Col II, and sulfated proteoglycans is increased in the atractylenolide-treated group, suggesting that atractylenolide enhances MSCs chondrogenic differentiation by activating the SHh signaling pathway ¹⁵⁰ . Furthermore, when the Hh signaling pathway inhibitor cyclopamine was used to inhibit SHh signaling ¹⁵¹ , the effect of atractylenolides on promoting chondrogenic differentiation of MSCs was weakened, confirming that the promotion of chondrogenic differentiation by atractylenolide depends on the activation of the SHh signaling pathway. In addition, the SHh signaling pathway is an important regulatory factor in maintaining the chondrocyte phenotype. Under hypoxic conditions, SHh signal can stimulate the production of the transcription factor Nkx3.2, induce chondrogenesis, and inhibit the expression of another transcription factor Runx2 that induces osteogenesis, thereby maintaining the chondrocyte phenotype and promoting the secretion of cartilage matrix ¹⁶⁰ .

In addition to SHh, IHh is also considered to be involved in chondrocyte differentiation. IHh is mainly expressed in prehypertrophic chondrocytes and osteoblasts at puberty stages ¹⁶¹ . Genetic studies have shown that IHh signaling regulates the proliferation and differentiation of osteoblasts and chondrocytes during bone development and repair^162–166. IHh mainly regulates chondrocyte proliferation and differentiation through PTHrP^163,167. One study has found that SHh and IHh have the same effect on BMSCs in promoting chondrogenic differentiation. There is no significant difference in the expression levels of Sox9, ACAN, and Col II proteins in the IHh and SHh transfection groups ¹⁵² . These results may be because both IHh and SHh promote chondrogenesis by stimulating the Hh pathway^168,169. Moreover, several other signaling systems have been found to interact with IHh or SHh during chondrogenic differentiation^169–171. Previous studies have suggested that IHh is also important for maintaining endogenous TGF-β and BMP signaling, and these three signaling pathways collectively drive the optimal chondrogenesis in human BM-MSCs ¹⁵³ .

The Hh Signaling Pathway Regulates Adipogentic Differentiation of MSCs

Adipogenesis is a complex process that involves the coordinated regulation of multiple signaling pathways. The Hh signaling pathway has been widely reported as a negatively regulator of adipose tissue development^113,172. With most of its downstream targets having been shown to play important roles in adipose tissue development. Using genome-wide RNAi screening, 500 candidate obesity genes are identified, with many of them enriched in the Hh signaling pathway and having anti-adipogenic effects ¹⁷³ . Studies have shown that SHh is a key regulator of adipogenesis ¹⁷³ . Specifically, activation of the Hh signaling pathway in vivo and in vitro can block the differentiation of white adipocytes, but not brown adipocytes. Leptin promotes browning of white adipocytes by inhibiting the Hh signaling pathway, and thus reduces obesity ¹⁷⁴ . It has found that SHh and NELL-1 signals have a synergistic effect on the anti-adipogenic differentiation of human adipose-derived MSCs ⁶² . Targeting activation of the Hh signaling pathway through EVs secreted by AD-MSCs not only inhibits adipogenesis, but also suppresses lipogenesis^173,175. PMA is a highly efficient and specific activator of the Hh pathway, which directly activates the Hh pathway by acting on Smo, thereby impairing the adipogenic differentiation of human MSCs, reducing lipid accumulation, while maintaining the number of adipocytes ¹⁷⁶ . These studies confirm the anti-adipogenic potential of the Hh signaling pathway in MSCs.

Many studies have shown that there is a potential relationship between osteogenic and adipogenic differentiation of MSCs in rodents^{122,176–178}. Osteogenic and adipogenic differentiation are opposing biological processes^178–182. There is a balance between osteogenesis and adipogenesis, and promoting osteogenic differentiation while inhibiting adipogenic differentiation of MSCs is a key step for bone regeneration in tissue engineering ¹⁸³ . To support this relationship, numerous in vitro experiments using BM-MSCs have demonstrated that factors inducing adipogenesis inhibit osteoblast formation^184,185, whereas factors promoting osteoblast formation inhibit adipocyte differentiation ¹⁷⁸ . Oxysterols, which are activators of the Hh pathway, play a role in promoting osteogenic differentiation and inhibiting adipocyte formation in mouse MSCs ¹⁸⁶ . The molecular mechanism by which oxysterols inhibit adipogenesis is mainly mediated by activating the Hh pathway and is associated with inhibiting the expression of peroxisome proliferator-activated receptor-γ (PPARγ)^186,187. SHh, IHh, DHh, and GLI are highly expressed in MSCs ¹⁷⁶ . Characteristic of adipocyte differentiation in human MSCs is a decrease in Hh signaling, mainly due to decreased GLI expression ¹⁷⁶ . Conversely, activation of the Hh pathway interferes with adipocyte differentiation by reducing the induction of adipogenesis by CCAAT-enhancer-binding protein alpha and PPARγ, which are the master regulators of adipogenesis, as well as reducing the expression of adipocyte-specific markers, such as adipocyte fatty acid-binding protein, adipsin, CD36, adiponectin, and leptin, and significantly decreasing the lipid accumulation ¹⁷⁶ . This further confirms that the Hh signaling pathway is a negative regulatory factor in MSCs adipogenic differentiation.

Neuronal Differentiation of MSCs

In addition to possessing mesodermal tri-lineage differentiation potential^43,44, MSCs also have the ability to differentiate across embryonic layers into ectodermal cell lineages, such as nerve cells ⁴⁶ and lung epithelial cells^7,89–91. The differentiation of MSCs into neural cells is a complex process regulated by multiple factors, including transcription factors, growth factors, and signaling molecules. One of the most important signaling molecules in the process is SHh. Loss of neurons is a common feature of many neurodegenerative diseases (such as Alzheimer’s disease [AD], Parkinson’s disease, spinal cord injury, stroke, and Huntington’s disease [HD]), which are associated with function loss and disability^188,189. Because drugs cannot effectively improve damaged neurons in the brain^190,191, stem cell therapy is considered to be an effective therapeutic strategy for restoring lost cells in human nervous system diseases^92,192. Many sources of MSCs for neural differentiation can serve as alternative cell sources for therapy^193–195. MSCs from different sources, such as adipose, dental, and bone marrow can differentiate into various types of neural cells^{93–98,196–199}, including oligodendrocytes ⁹⁹ , motor neurons^{195,200–204}, dopaminergic-like cells, and cholinergic-like cells^94,205. These cells can express neural and cholinergic-specific markers at the protein level, including Nestin, SMI-32, Islet-1, and AChE, in the presence of Retinoic Acid (RA) and SHh^94–98,205. Several studies have also shown that MSCs isolated from adult teeth and shed deciduous teeth can differentiated into neurons and express neuronal markers in specific inducing culture media^206,207. MSCs from human periapical cysts (hPCy-MSCs) have higher neural differentiation potential and exhibit neural progenitor-like characteristics, spontaneously expressing neuron-specific proteins and genes following neural induction. Under appropriate neural stimulation conditions, hPCy-MSCs acquire neural morphology and significantly overexpress several neural markers ²⁰⁸ . In vivo studies have shown that MSCs from teeth can differentiate into active neurons under appropriate environmental stimulation ²⁰⁹ .

SHh is a secreted protein involved in the development and maintenance of the nervous system. It is known to play a critical role in MSCs differentiation into neurons ²¹⁰ . The therapeutic effects of MSCs can be mediated by the SHh pathway, which increases synaptic plasticity and synaptogenesis after MSCs treatment. SHh has been shown to promote the biological process of MSCs differentiation into neurons by upregulating the expression of transcription factors, such as Neurogenin-2 (Ngn2), NeuroD1, and Olig2^203,211, and by downregulating the expression of pluripotency- maintaining transcription factors Sox2 and Oct4^212,213. MSCs genetically modified with the SHh gene also readily differentiation into neurons and astrocytes ²¹² . It has been demonstrated that MSCs mediate an increase in tissue plasminogen activator through the SHh pathway, thereby promoting brain plasticity and neurological function ²¹⁰ . However, the SHh inhibitor cyclopamine can block the therapeutic effects of MSCs by inhibiting the SHh pathway ²¹⁰ .

Activation of the SHh pathway not only upregulates the expression and secretion of basic fibroblast growth factor and vascular endothelial growth factor in MSCs but also mediates their differentiation into neurons by upregulating downstream gene products of the SHh pathway, such as BDNF, NT-3, and GDNF^48,68,214. Chemical factors that control signaling pathways involved in neuronal development can enhance the neuronal differentiation of MSCs ²¹⁵ . For example, the SHh signaling pathway agonist PMA can improve neuronal differentiation by directly enhancing SMO receptor activation^136,216,217. Another study has shown that PMA promotes the differentiation of MSCs into motor neuron-like cells when cultured on polycaprolactone (PCL) scaffolds ⁴⁶ . Therefore, activation of the SHh signaling pathway is a necessary condition for MSCs to differentiation into neural cells.

Lung Epithelial Differentiation of MSCs

Lung disease is a growing global health concern and a major cause of death and disability worldwide. In 2017, 3.2 million people died from chronic obstructive pulmonary disease (COPD) ²¹⁸ (accounting for 5.7% of global deaths), and it is projected to become the third leading cause of death globally by 2030. ALI and its severe form, acute respiratory distress syndrome (ARDS), are also a concern for the global medical community ²¹⁹ , with mortality rates as high as 30%–40%. The pathogenesis of ALI/ARDS is mainly due to an imbalance of the body’s immune response, leading to severe acute hypoxic respiratory failure caused by diffuse alveolar epithelial and endothelial cell damage from excessive inflammation, which is characterized by decreased lung compliance and increased permeability leading to pulmonary edema, frequently progressing to multiple organ failure and pulmonary fibrosis^220,221. In addition, the critical condition caused by COVID in 2019 ²²² highlights the urgent need for innovative treatment strategies for ALI/ARDS.

More data support the potential and application prospects of MSCs in the treatment of pulmonary diseases ²²³ . Currently, phase I and II clinical trials have accumulated evidence confirming the safety and tolerability of these cells. Based on the anti-microbial, anti-inflammatory, regenerative, angiogenic, anti-fibrotic, anti-oxidative stress, and anti-apoptotic effects demonstrated by MSC therapy, these effects can block the physiopathological mechanisms of ALI/ARDS^222–224. In recent years, although more and more studies have shown that therapeutic potential of MSC-derived EVs appears to be comparable with that of MSC therapy, and can effectively simulate the therapeutic effects of MSCs in animal models of lung injury ³⁶ . The fact remains that using EVs alone cannot fully realize the potential of MSCs to regenerate and repair injured alveolar epithelial cells by cell differentiation potential and direct mitochondrial transfer, to save dying cells^39,89,90. Importantly, some studies have shown that MSC therapy is more effective than its EVs in alleviating lung injury, regardless of the etiology, because promoting MSCs differentiation into Alveolar Epithelial Type II (AT II) cells may be more favorable for accelerating re-epithelialization^36,37, ultimately repairing damaged alveolar structure and facilitating lung function improvement^7,89,90.

So far, studies have demonstrated that MSCs can differentiate into alveolar epithelial cells in vitro and in vivo, participating in the repair of alveolar epithelium in ALI/ARDS^{7,36,37,89,90}. Studies have confirmed that activation of the Wnt/β-catenin signaling pathway promotes MSCs differentiation into AT II cells^89,90,225, and Wnt proteins, which are important gene products of SHh pathway activation, have a profound effect on the activity of Wnt/β-catenin signaling pathway^68,79. MSCs secrete various types of cytokines through paracrine function or directly interact with immune cells and pulmonary epithelial cells (type I and II), regulating immune responses and repairing lung injuries ²²⁶ , suggesting that the interaction between MSCs and the pulmonary microenvironment has a broader therapeutic effect on lung diseases ²²⁴ . Therefore, we speculate that the SHh signaling pathway may play an important role in the differentiation of MSCs into AT II in the ALI microenvironment or in vitro differentiation induction system (this part of the experiment has been completed and confirmed this hypothesis).

Pancreatic Cell Differentiation of MSCs

MSCs are a cellular therapy approach for treating type I and II diabetes^45,86,227. In particular, the transplantation of artificial insulin-producing cells (IPCs) is a practical approach with permanent results in the treatment of type I diabetes mellitus^228–230. Numerous studies have shown that MSCs can successfully differentiate into functional pancreatic islet cells^45,86,87,227. In terms of differentiating into IPCs, MSCs can be obtained from multiple sources, with bone marrow and adipose tissue MSCs being the most extensively studied cells^45,88.

In humans, the Hh pathway plays an important role in the differentiation of endoderm to pancreatic β-cells in the embryonic gut^231,232. Studies have demonstrated that although during early stages of pancreas organogenesis, Hh signaling has been shown to inhibit tissue morphogenesis and cell differentiation, it is important for the reactivation of the SHh pathway during the late maturation of pancreatic β-cells ²³¹ , especially in maintaining the insulin secretion function of IPCs differentiated from MSCs^231–237. Therefore, manipulation of SHh signaling pathway can be used as reliable approach to improve the generation of functional IPCs from MSCs^238,239. Studies have found that inhibiting Hh activity in the early stages of induced differentiation is required for MSCs to successfully differentiate into IPCs^233,234,239. Dayer et al ²³⁹ also confirmed that early inactivation and late reactivation of the SHh signaling pathway during differentiation of AD-MSCs can beneficially affect the expression of functional and developmental pancreas-related genes (Pdx1, MAFA, Ngn3, Isl1, Nkx2.2, and Nkx6.1) and insulin secretion in differentiated cells.

Pancreatic and duodenal homeobox 1(Pdx1) is a key gene in pancreatic development and insulin regulation, and is required for β-cell maturation and maintenance of β-cell function^235,236, and plays a key role in the differentiation of MSCs into IPCs and maintenance of IPCs ²⁴⁰ . Thomas et al^233,237 demonstrated that the promoter of Pdx1 contains an Hh response element, and thus SHh affects Pdx1 expression thereby inducing insulin expression and secretion in mature pancreatic β-cells. Hashemi Tabar et al ²³⁸ proposed a novel scheme to optimize the differentiation of AD-MSCs to IPCs using Pdx1 overexpression and SHh manipulation (early repression late reactivation). These findings suggest that inhibition of the SHh signaling pathway during early MSCs differentiation and reactivation of this pathway in later stages are important biological processes for MSCs differentiation into IPCs. Because increasing evidence indicates that the differentiation state of IPCs is not permanent, and they lose their identity and functionality in response to a variety of signals, including changes in the transcriptional profile normally present in mature β-cells^241,242. Manipulation of the pancreatic signaling pathway (such as SHh pathway) is a practical approach to increase the efficiency of differentiation of MSCs to IPCs ²⁴³ . Although maintaining a certain level of SHh activity in β-cells and IPCs is important and necessary for optimal β-cell function ²⁴⁴ , excessive SHh activity can impair mature β-cells function ²³⁹ .

Hepatocyte Differentiation of MSCs

Liver disease is the 11th leading cause of death globally ²⁴⁵ . Regardless of the underlying etiology of the disease, patients will develop similar liver dysfunction in the later stages of the disease. Currently, only liver transplantation has a curative effect on late-stage disease, but the availability of donor liver is limited. The use of liver cell-like cells manufactured from MSCs for the treatment of liver disease is a hopeful strategy for late-stage liver disease patients, making MSCs of significant research value and having broad application prospects in liver diseases. Due to the ability of MSCs to promote liver regeneration and alleviate liver damage by differentiating into liver cells, inhibiting the release of inflammatory factors, and enhancing the proliferation of intrahepatic cells, MSC-based therapy has become a focus for promoting liver regeneration and repairing liver damage^64,246–248. Based on the demonstrated efficacy and safety of MSC therapy in preclinical studies in patients with no other treatment options, MSC-based therapy has become a promising approach for the treatment of liver disease^{64,82,245–248}.

Although numerous studies have demonstrated the MSCs transplantation can promote liver regeneration through differentiation into liver cells^{64,82–85,245,246}, reports on the role and mechanism of Hh pathway in MSCs differentiation into hepatocytes are rare. In the damaged liver, dying liver cells have been shown to release Hh ligands^249,250. These Hh ligands trigger the proliferation of Hh-responsive cells, such as liver progenitor cells (liver MSCs) and hepatic stellate cells (HSCs), and these cells also produce Hh ligands, amplify the activity of the Hh signal in these cells through autocrine and paracrine mehanisms^249–252. One study report that MSCs can promote liver regeneration by a mechanism that has a significant increase in miRNA-125b levels expressed by MSCs, leading to a reduction in Smo and inhibition of the Hh signaling, thereby reducing fibrosis, and ultimately promoting liver regeneration ⁸¹ . Although some studies have indicated that MSCs play an important role in liver regeneration by differentiating into liver-like cells^{64,82–85,245}, the exact mechanism of the Hh pathway in MSCs differentiation into hepatocytes requires further exploration.