TGFβ family signaling in human stem cell self-renewal and differentiation

Abstract

Human stem cells are undifferentiated cells with the capacity for self-renewal and differentiation into distinct cell lineages, playing important role in the development and maintenance of diverse tissues and organs. The microenvironment of stem cell provides crucial factors and components that exert significant influence over the determination of cell fate. Among these factors, cytokines from the transforming growth factor β (TGFβ) superfamily, including TGFβ, bone morphogenic protein (BMP), Activin and Nodal, have been identified as important regulators governing stem cell maintenance and differentiation. In this review, we present a comprehensive overview of the pivotal roles played by TGFβ superfamily signaling in governing human embryonic stem cells, somatic stem cells, induced pluripotent stem cells, and cancer stem cells. Furthermore, we summarize the latest research and advancements of TGFβ family in various cancer stem cells and stem cell-based therapy, discussing their potential clinical applications in cancer therapy and regeneration medicine.

Background

Human stem cells are present in embryonic, postnatal, and adult tissues (Fuchs and Segre 2000). Based on their differentiation potential, stem cells can be classified into five types: totipotent, pluripotent, multipotent, oligopotent, and unipotent stem cells (Fig. 1A) (Essawy et al. 2020; Apostolou et al. 2023). Totipotent stem cells exhibit the highest capacity for proliferation and differentiation, as demonstrated by the zygote's ability to generate all embryonic and extraembryonic structures (Baker and Pera 2018; Xu et al. 2022). Pluripotent stem cells, such as embryonic stem cells (ESCs), have a lower potency compared to totipotent cells and can differentiate only into various embryonic tissues (Donovan and Gearhart 2001; Du and Wu 2024). Somatic stem cells, also known as adult stem cells or tissue-specific stem cells, are multipotent stem cells. They have the ability to differentiate into cell types specific to their tissue or organ of origin, such as hematopoietic stem cells and neural stem cells (Ferraro et al. 2010; Zakrzewski et al. 2019). Oligopotent stem cells are more restricted in their differentiation potential but can still produce specific cell types within certain tissues, such as the common lymphoid progenitor, which generates T lymphocytes, B lymphocytes, and natural killer cells (Bryder et al. 2006; Warner et al. 2012). Furthermore, unipotent stem cells exhibit the highest degree of lineage restriction, giving rise exclusively to cells within a specific lineage, such as spermatogonial stem cells (de Rooij 2017, Yu et al. 2022).

Fig. 1.

Schematic illustration of different types of human stem cells. A Stem cells can be categorized into totipotent, pluripotent, multipotent, oligopotent, and unipotent stem cells based on their varying degrees of differentiation potential. B Induced pluripotent stem cells are generated through the reprogramming of somatic cells into a pluripotent state. C Cancer stem cells constitute a minor subset of undifferentiated cancer cells that possess characteristics resembling those of stem cells and are responsible for tumor differentiation and metastasis

In addition to the presence of human endogenous stem cells, there exists another category of pluripotent stem cells known as induced pluripotent stem cells (iPSCs), which are derived from somatic cells and can be reprogrammed to regain their pluripotent state (Fig. 1B) (Nishikawa et al. 2008). This groundbreaking technique of somatic cell reprogramming has facilitated the generation of various differentiated cell types with defined functions, such as cardiomyocytes, hematopoietic cells, islet cells, and neural cells by directly reprogramming somatic cells using lineage-specific master genes (Maherali and Hochedlinger 2008).

Moreover, cancer stem cells (CSCs) are a rare subpopulation of cells identified in tumors originating from diverse organs and tissues (Reya et al. 2001). These CSCs possess stem cell properties, such as self-renewal capacity and multipotency, and are essential for both tumor formation and maintenance (Fig. 1C). CSC also rely on functional local microenvironments for support, and reciprocal interactions are observed between CSC and their microenvironment (Lane et al. 2009).

TGFβ superfamily signaling are important for maintaining the self-renewal and differentiation of human stem cells (James et al. 2005; Pauklin and Vallier 2015; Mullen and Wrana 2017). TGFβ superfamily signaling also plays pivotal role in maintaining the pluripotency of iPSCs, and its modulation can effectively guide lineage-specific differentiation (Fei and Chen 2010; Wu et al. 2023). CSCs are regulated by TGFβ family signaling, which can be provided either through autocrine mechanisms or by factors present in their residing microenvironment (Nishimura et al. 2010; Scheel et al. 2011). While TGFβ family signaling governs the stemness and differentiation of normal and neoplastic stem cells, its effects exhibit diversity contingent upon cell types, microenvironmental factors, and physiological states (Watabe and Miyazono 2009). This review will primarily focus on elucidating the multifaceted roles of TGFβ superfamily signaling in governing various functions and properties of ESCs, somatic stem cells, iPSCs, and CSCs.

TGFβ family signaling pathway

The TGFβ family signaling play a crucial role in a wide range of biological processes (Massagué and Sheppard 2023). This superfamily consists of a diverse group of growth factors, including TGFβs, BMPs, Activins and Nodal (Fig. 2). These ligands bind to specific cell surface receptors, initiating a complex signaling cascade. There are two main types of receptors: type I and type II serine/threonine kinase receptors on cell surface (James et al. 2005; Fei and Chen 2010). When ligand binds to the receptor complex, it triggers a cascade of intracellular signaling events. The activated receptors phosphorylate and activate SMAD proteins, which are the main intracellular mediators of TGFβ superfamily signaling (Hata and Chen 2016). There are three classes of SMADs: receptor-regulated SMADs (SMAD2 and SMAD3 for TGFβ/Activin/Nodal signaling; SMAD1, SMAD5 and SMAD8 for BMP signaling), common mediator SMAD (SMAD4), and inhibitory SMADs (SMAD6/SMAD7) (Wrana 2000). R-SMADs are phosphorylated by the activated receptors and form complexes with Co-SMAD (SMAD4) (Wrana 2009). Inside the nucleus, SMAD complexes interact with other transcription factors and co-regulators to modulate the expression of genes that control stem cell fate (Massagué and Xi 2012; Beyer et al. 2013). Meanwhile, the signaling cascade induces inhibitory SMADs (SMAD6/SMAD7) provide negative feedback on these pathways (Nakao et al. 1997; Attisano and Wrana 2002; Avery et al. 2010).

Fig. 2.

TGFβ family signaling pathway. TGFβ superfamily including TGFβ, Activin, Nodal, and BMP ligands, transmits signals through intrinsic type I and type II serine/threonine kinase receptors on the stem cell surface. Upon ligand stimulation, the type I receptor phosphorylates SMAD proteins (SMAD2 and SMAD3 for TGFβ/Activin/Nodal signaling; SMAD1, SMAD5, and SMAD8 for BMP signaling) and accumulate in the nucleus form complexes with a co-SMAD (SMAD4). Meanwhile, the signaling cascade induces inhibitory SMADs (SMAD6/SMAD7) to provide negative feedback on these pathways

TGFβ family signaling in pluripotent stem cells

TGFβ superfamily signaling regulates the self-renewal and differentiation of pluripotent stem cells including ESCs and iPSCs (Fig. 3) (Li et al. 2016b). In pluripotent stem cells, TGFβ signaling activates a set of transcriptional factors that are crucial for self-renewal and the prevention of premature differentiation. The pluripotency is maintained through the expression of transcription factors including OCT4, NANOG and SOX2 (Boyer et al. 2005). TGFβ/Activin and fibroblast growth factor signaling pathways synergistically collaborate to suppress BMP signaling, sustain the expression of pluripotency-associated genes including OCT4, NANOG and SOX2, and facilitate long-term undifferentiated proliferation of ESCs (Xu et al. 2008; Greber et al. 2010). Both TGFβ and BMP-responsive SMADs exhibit binding affinity towards the NANOG proximal promoter. TGFβ signaling enhances NANOG promoter activity, while BMP signaling attenuates it (Suzuki et al. 2006; Xu et al. 2008; Galvin-Burgess et al. 2013). Nuclear accumulation of activated SMAD2 induced by TGFβ, Activin or Nodal was observed in undifferentiated ESCs, and exhibited a decline during early differentiation (James et al. 2005; Vallier et al. 2009; Sakaki-Yumoto et al. 2013a, b; Itoh et al. 2014). When ESCs initiate differentiation, the level of TGFβ/Activin/Nodal signaling decreases (Xiao et al. 2006; Xu et al. 2008). Inhibition of kinase activities of type I receptors for Activin-ALK4, TGFβ-ALK5, and Nodal-ALK7 using chemical kinase inhibitors such as SB431542 led to a reduction in the expression levels of pluripotency markers in human embryonic stem cells (Inman et al. 2002; James et al. 2005; Madhu et al. 2016). TGFβ signaling is essential for maintaining the pluripotency of human naïve pluripotent stem cells; however, inhibition of TGFβ signaling in naive human PSCs results in the downregulation of genes targeted by SMAD2/3 and the departure from pluripotency (Osnato et al. 2021). The generation of human ground state naive embryonic stem cells from embryos involves employing exogenous TGFβ and chemical inhibitors targeting ALK4/7, which induce a specific differentiation state in the resulting pluripotent stem cells (Gafni et al. 2013; Theunissen et al. 2014). Additionally, the downstream inhibition of DNA binding (ID) proteins, induced by BMP signaling, has been reported to maintain the self-renewal capacity of embryonic stem cells (Ying et al. 2003; Lasorella et al. 2014).

Fig. 3.

Role of TGFβ family signaling in human pluripotent stem cells. The signaling pathway mediated by TGFβ/Activin/Nodal occupies a pivotal role in the regulation of self-renewal in human embryonic stem cells and induced pluripotent stem cells. NANOG serves as a downstream target of the TGFβ/Activin/Nodal signaling cascade. Both TGFβ and BMP signaling inhibit ectoderm differentiation, while activating BMP/Activin/Nodal signaling mainly induces differentiation towards mesoderm, endoderm, and germ cells

Pluripotent stem cells exhibit an extraordinary capacity to undergo differentiation into all three germ layers: ectoderm, mesoderm, and endoderm (Thomson et al. 1998). The initiation of the primitive streak (PS), which serves as the source of mesodermal and endodermal cells, constitutes the initial stage in ESC differentiation (Tam and Behringer 1997; Gadue et al. 2006). BMP4 can induce PS-like cells characterized by robust expression of the mesodermal marker Bra as well as posterior PS markers Msdp1 and Hoxb1 (Nostro et al. 2008). Multiple studies in the fields of development and stem cells have shown that Nodal/Activin signaling serves as the principal driver of definitive endoderm formation (Tiedemann et al. 2001; Kubo et al. 2004; D'Amour et al. 2005; Parashurama et al. 2007; Brown et al. 2011; Lee et al. 2015). Progression of the anterior PS population to definitive endoderm requires sustained activation of TGFβ/Nodal/Activin signals, consistent with the observation that Nodal/Activin signals are required for development of definitive endoderm in embryo (Gadue et al. 2006). Neural lineages are derived from ectoderm, BMP4 acts as a suppressor for neural differentiation induced in the PA6 stromal cells co-culture system or under serum-free culture conditions (Schulz et al. 2004). The induction of neural commitment is facilitated by inhibiting BMP signaling through Noggin or the small molecule BMP receptor kinase inhibitor dorsomorphin (Pera et al. 2004; Zhou et al. 2010). Simultaneous inhibition of both BMP and TGFβ/Activin/Nodal signaling using Noggin/SB431542 or dorsomorphin/SB431542 synergistically augmented the efficacy of neural differentiation (Chambers et al. 2009; Morizane et al. 2011). Human embryonic stem cells also exhibited the potential to differentiate into primordial germ cells upon stimulation with BMP. The addition of BMP4 to differentiating human embryoid bodies induced the expression of germ-cell specific marker genes, while the combined use of BMP7 and BMP8b showed additive effects with BMP4, resulting in a remarkable enhancement of germ cells (Kee et al. 2006; Panula et al. 2011). Moreover, Activin/Nodal showed promotion function in germ cell differentiation of human embryonic stem cells (Duggal et al. 2015; Jørgensen et al. 2018; Mishra et al. 2021).

TGFβ family signaling in somatic stem cells

The somatic stem cells undergo symmetric or asymmetric cell divisions to generate both new stem cells and differentiated cell types, thereby replenishing dying cells and facilitating tissue regeneration (Miettinen et al. 1994; Shah and Khan 2021). The stemness of somatic stem cells is regulated by their intrinsic properties and external features that are maintained by their local cellular microenvironment (Spradling et al. 2001; Bendall et al. 2007; Ferraro et al. 2010, Voog and Jones 2010, Snippert and Clevers 2011). TGFβ family signaling pathways have been implicated in the maintenance and differentiation of various types of somatic stem cells (Watabe and Miyazono 2009; Sakaki-Yumoto et al. 2013a, b). This section focuses on elucidating the distinct roles of TGFβ family signaling in various stem cell populations, including hematopoietic stem cells, neural stem cells, hair follicle stem cells, intestinal stem cells and mesenchymal stem cells.

TGFβ family signaling in hematopoietic stem cells

Hematopoietic stem cells (HSCs) are primarily localized within the bone marrow and serve as the progenitors for erythrocytes, thrombocytes, and leukocytes encompassing both lymphoid and myeloid lineages (Dykstra et al. 2007; Wang and Wagers 2011). The maintenance of the HSC state is regulated by signals originating from the microenvironment, including mesenchymal stem cells, endothelial cells, osteoblasts, and sympathetic nerve fibers (Morrison and Scadden 2014). TGFβ family signaling in the hematopoietic system regulates the maintenance of quiescence and self-renewal capabilities in HSCs (Fig. 4) (Blank and Karlsson 2015). Endothelial cells are responsible for the production of stem cell factor (SCF), while mesenchymal stem cells (MSCs) produce both SCF and CXCL12, and HSCs generate TGFβ; all these factors are essential for HSC maintenance (Sugiyama et al. 2006; Yamazaki et al. 2009; Ding et al. 2012; Greenbaum et al. 2013). TGFβ serves as a potential signaling molecule in the bone marrow microenvironment to induce quiescence of hematopoietic stem cells (Yamazaki et al. 2009). Non-myelinating Schwann cells were found to contact a significant proportion of HSCs in the bone marrow and facilitate TGFβ activation through integrin αvβ8 expression, which directs metalloproteinases to cleave latent TGFβ (Fig. 4) (Yamazaki et al. 2011). TGFβ elicits highly variable biological responses and exerts both positive and negative effects on cellular proliferation, differentiation, and apoptosis (Larsson and Karlsson 2005; Ruscetti et al. 2005; Söderberg et al. 2009; Blank and Karlsson 2015). The response of HSCs to TGFβ stimulation demonstrates a biphasic pattern, whereby low concentrations of TGFβ induce stimulatory effects on HSC proliferation and differentiation, while high concentrations exert inhibitory effects (Kale and Vaidya 2004). BMP plays a crucial role in promoting the specification and expansion of HSCs during gastrulation in vertebrates, as well as being essential for their maintenance and proliferation in vitro (Bhatia et al. 1999; Kang et al. 2004). BMP9 has also been demonstrated as a potent synergistic factor in the generation and formation of hematopoietic progenitor cells (Ploemacher et al. 1999). BMP4, in conjunction with cytokines, facilitate hematopoietic specification, differentiation, and proliferation of human embryonic stem cells (Park et al. 2004).

Fig. 4.

Role of TGFβ family signaling in hematopoietic stem cells. Hematopoietic stem cells (HSCs) primarily reside in the bone marrow and give rise to erythrocytes, thrombocytes, and leukocytes of both lymphoid and myeloid lineages. The maintenance of HSCs relies on TGFβ, CXCL12 and stem cell factor (SCF) provided by HSCs, mesenchymal stem cells (MSCs) and endothelial cells. The latent TGFβ produced by HSCs is activated by integrin αvβ8 in nonmyelinating Schwann cells, which binds to and cleaves the complex. BMP4 and low levels of TGFβ promote HSCs differentiation, while high levels of TGFβ inhibit it

TGFβ family signaling in neural stem cells

The adult brain contains two populations of neural stem cells (NSCs) located in the subgranular zone (SGZ) of the hippocampal dentate gyrus and the subventricular zone (SVZ) lining the lateral ventricles (Alvarez-Buylla et al. 2002; Kriegstein and Alvarez-Buylla 2009). The NSCs have the ability to differentiate into intermediate progenitor cells (IPCs), also known as transit-amplifying or neural progenitor cells, which subsequently generate neurons, astrocytes, and oligodendrocytes (Reynolds and Weiss 1992). NSCs can be cultured in vitro as neurospheres and differentiated into both neuronal and glial cells; however, it has been suggested that this differentiation behavior is influenced by the specific culture conditions employed (Marshall, Laywell et al. 2006). TGFβ signaling pathway plays pivotal roles in the maintenance and proliferation of neural stem cells (Falk et al. 2008; Seuntjens et al. 2009). While the effects of TGFβ family signaling in NSCs are highly context-dependent and can vary depending on the developmental stage and specific signaling components involved. The canonical TGFβ pathway in midbrain development acts as a suppressor of Wnt-induced proliferation and expansion of neuroepithelial cells, which are the NSCs involved in early brain development (Fig. 5) (Falk et al. 2008). ALK5-dependent TGFβ signaling plays a crucial role in regulating the later stages of adult hippocampal neurogenesis, facilitating stem cell quiescence and promoting neurogenesis within the adult neurogenic niche (He et al. 2014; Kandasamy et al. 2014). Considering the heightened levels of TGFβ1 observed in the context of aging and neurodegenerative disorders, targeting TGFβ1 signaling emerges as a promising molecular strategy for future interventions in such pathological conditions (Kandasamy et al. 2014).

Fig. 5.

Role of TGFβ family signaling in neural stem cells. BMP signaling activates NSCs in the subgranular zone (SGZ), while Noggin produced by quiescent NSCs inhibits BMP signaling. Inhibition of BMP signaling leads to the formation of intermediate progenitor cells (IPCs), which differentiate into neuroblasts and astrocytes in vivo. The NSCs in the subventricular zone (SVZ), known as type B cells, are maintained as slowly cycling NSCs through BMP and fibroblast growth factor (FGF) signaling. The production of Noggin by ependymal cells hinders the maintenance of NSC state regulated by BMP signaling. Upon initiation of differentiation, BMP signaling pathway facilitates astrocyte differentiation while repressing neural and oligodendrocyte lineages. Later in development, TGFβ facilitates the differentiation and lineage expansion of established progenitors

BMPs induce various biological responses in embryonic neural stem cells, with BMP2/4 playing a crucial role in promoting neuroepithelial proliferation during early stages of embryonic central nervous system development (Panchision et al. 2001). During later stages of development, BMPs stimulate the differentiation of neural stem cells into neurons and astrocytes (Gross et al. 1996). BMP signaling pathway plays a crucial role in regulating the multipotency of neural stem cells in both SGZ and SVZ; however, divergent mechanisms have emerged regarding its utilization by these two distinct stem cell populations (Alvarez-Buylla and Lim 2004; Chen and Panchision 2007). The NSCs in the SVZ, known as B cells, express BMP2/4/7 and are located in the ventricular walls. The Noggin secreted by the adjacent ependymal cells acts as an inhibitory factor for BMP in B cells (Lim et al. 2000; Bonaguidi et al. 2008). The BMP signaling pathway helps maintain the B-cell state, but once differentiation begins, it promotes astrocyte differentiation while suppressing neural and oligodendrocyte fates (Lim et al. 2000). The downstream targets of BMP signaling, ID1/3, are essential for the synchronization of stemness and anchorage to the neural stem cell niche (Niola et al. 2012).

TGFβ family signaling in hair follicle stem cells

Throughout adulthood, hair follicle stem cells (HFSCs) undergo dynamic and synchronized cycles of telogen, anagen and catagen (Millar 2002; Schmidt-Ullrich and Paus 2005). In the telogen phase, HFSCs are quiescent and reside in a specialized microenvironment called the hair bulge, which is directly adjacent to the underlying dermal papilla (DP), a signaling center for HFSCs (Cotsarelis et al. 1990). The follicle is maintained in telogen primarily by BMP secretion from surrounding cell populations (Fig. 6A). Adipocytes express BMP2, dermal fibroblasts express BMP4, and keratin 6 + inner bulge cells express BMP6, suppressing HFSCs proliferation (Plikus et al. 2008; Hsu et al. 2011; Oshimori and Fuchs 2012). The transition from telogen to anagen is characterized by the suppression of BMP signaling, leading to the proliferation and differentiation of HFSCs. The inactivation of the BMPR1A gene in postnatal skin epithelium triggers the activation and proliferation of quiescent HFSCs, resulting in the depletion of slowly proliferating cells (Zhang et al. 2006; Kobielak et al. 2007). Consistent with this observation, BMP inhibitory factors secreted by neighboring niche cells have been implicated in the initiation of anagen (Kulessa et al. 2000; Botchkarev et al. 2001). TGFβ2 isoform plays a distinct role, unique among other TGFβ isoforms, in the induction of hair follicle morphogenesis and is both essential and adequate for facilitating this process (Foitzik et al. 1999). Additionally, TGFβ2 antagonizes BMP signaling in HFSCs by upregulating the expression of TMEFF1, a potent antagonist of the BMP pathway. This mechanism effectively restricts and attenuates the responsiveness of HFSCs to BMP signals within their niche (Oshimori and Fuchs 2012). Mesenchymal DP cells transmit paracrine TGFβ2 to epidermal HFSCs prior to their activation, thereby initiating TGFβ/SMAD2/3 signaling cascade that stimulates HFSCs and promotes tissue regeneration (Oshimori and Fuchs 2012). TGFβ1 plays a crucial role in regulating catagen induction in vivo by inhibiting keratinocyte proliferation and promoting apoptosis, suggesting it as a potential target for treating hair growth disorders caused by premature or delayed catagen development (Foitzik et al. 2000; Soma et al. 2003; Mesa et al. 2015).

Fig. 6.

Role of TGFβ family signaling in hair follicle, intestinal and mesenchymal stem cells. A BMP2/4/6 effectively inhibits hair follicle stem cell (HFSC) proliferation and maintains them in a quiescent state. The expression of TGFβ2 and Noggin by dermal papilla cells inhibit BMP signaling and promotes HFSC proliferation in an activation state. B Wnt signaling promotes intestinal stem cells (ISCs) proliferation in the crypts, while BMP signaling inhibits stem cells proliferation. The inhibitors of BMP signaling, such as Noggin, Gremlin 1/2, and Chordin-like 1, promote the maintenance of ISCs. C TGFβ1 and BMP3 promotes mesenchymal stem cells (MSCs) maintenance, while other BMPs promote adipocyte and osteocyte differentiation. TGFβ1/3 and BMP2 work together to promote chondrocyte differentiation, whereas TGFβ inhibit myoblast differentiation

TGFβ family signaling in intestinal stem cells

The intestinal stem cells (ISCs) are located within the crypts of both the small intestine and colon, providing a continuous supply of intestinal epithelial cells that subsequently migrate to the villi (Fig. 6B) (Leblond and Stevens 1948; Creamer et al. 1961; Flier and Clevers 2009). Intestinal crypts contain two types of stem cells: Lgr5 + stem cells, which proliferate rapidly and produce intestinal epithelial cells, and Bmi1 + stem cells, which are quiescent and play a crucial role in regenerating the intestinal epithelium after injury (Barker et al. 2007; Sangiorgi and Capecchi 2008). The proliferation and differentiation of ISCs are regulated by factors secreted from the underlying mesenchymal layer, including fibroblasts, enteric neurons, blood vessels, and extracellular matrix components (Takeda et al. 2011). Wnt signaling promotes the proliferation of stem cells and transiently amplifying cells in the crypts, while BMP signaling functions as a negative regulator (Gregorieff and Clevers 2005; Gregorieff et al. 2005). The intravillus mesenchyme shows high expression of BMP4, and phosphorylation and nuclear localization of SMADs have been observed in differentiated villus epithelial cells and ISCs (Haramis et al. 2004; He et al. 2004).The BMP antagonists Noggin, Gremlin1 and 2, and Chordin-like 1 exhibit elevated expression levels in the crypts or their underlying mesenchyme (He et al. 2004; Kosinski et al. 2007). The loss of BMP signaling in the intestinal epithelium leads to an expansion of crypts, indicating that BMP signaling suppresses crypt formation and inhibits the expansion of the stem-cell niche (Haramis et al. 2004; He et al. 2004; Davis et al. 2015). BMP signaling is crucial for maintaining the quiescence of Bmi1 + stem cells and promoting terminal differentiation of intestinal epithelial villi, potentially by antagonizing Wnt signaling, which stimulates the proliferation of the Lgr5 + stem cell population (He et al. 2004).

TGFβ family signaling in mesenchymal stem cells

The mesenchymal stem cells (MSCs) can be achieved from various adult tissues, encompassing connective tissue, adipose tissue, muscle, bone marrow, blood, placenta and umbilical cord (Pittenger et al. 1999; Shi et al. 2005; Phinney and Prockop 2007). MSCs are of clinical significance due to their easy accessibility from adult patients, remarkable capacity for self-renewal, multipotency, and potent immunomodulatory effects on immune cells (Caplan 2000; Chen et al. 2022).The differentiation potential of MSCs is influenced by the specific tissue environment from which they are derived, and it is determined by their localization and microenvironment in vivo (Fig. 6C) (Roelen and Dijke 2003). Bone marrow-derived MSCs possess the capacity to undergo differentiation into osteoblasts, adipocytes, chondrocytes, myocytes, and stromal cells that support hematopoiesis (Pittenger et al. 1999). Muscle-derived MSCs have the capacity to differentiate into myogenic, osteogenic, chondrogenic, and adipogenic lineages (Williams et al. 1999). MSCs derived from adipose tissue possess the capacity to undergo differentiation into osteocytes, chondrocytes, adipocytes, and myocytes (Zuk et al. 2002).The proliferation of human MSCs is stimulated by Wnt or TGFβ signaling pathways (Boland et al. 2004; Jian et al. 2006). Transcriptomic analysis suggests that Wnt signaling plays a crucial role in maintaining the self-renewal and proliferation of primitive MSCs, whereas TGFβ signaling is implicated in the gradual decline of self-renewal capacity and initiation of senescence (Mazzella et al. 2023). TGFβ1 induces SMAD3-dependent nuclear accumulation of β-catenin in MSCs, promoting their proliferation. Conversely, BMP2 inhibits WNT3A signaling and MSC proliferation by interacting with Dishevelled-1, a component of the Wnt pathway (Liu et al. 2006). BMP3 signaling enhances MSC proliferation through the TGFβ/Activin pathway by inducing SMAD2 phosphorylation (Stewart et al. 2010). TGFβ signaling has also been implicated in orchestrating the differentiation fate of MSCs, promoting chondroblast differentiation during early stages while subsequently inhibiting osteoblast maturation (Roelen and Dijke 2003; Grafe et al. 2018). Pharmacological inhibition of TGFβ signaling significantly enhances the maturation process of osteoblasts (Maeda et al. 2004). The presence of BMPs has been demonstrated to facilitate the differentiation of MSCs into osteocytes, while also promoting the development of adipocytes and chondrocytes (Ahrens et al. 1993; Luu et al. 2007). The signaling of BMP2/4/7 promotes adipocyte differentiation by inducing the expression of PPARγ, whereas TGFβ and Activin A exert inhibitory effects on adipocyte development (Choy et al. 2000; Lee et al. 2000; Jin et al. 2006). The adipocytes can be divided into two categories: white adipocytes synthesize white fat to store triglycerides, while brown adipocytes promote energy expenditure and develop brown fat (Cristancho and Lazar 2011). BMP4 is widely acknowledged as a protein that facilitates the differentiation of white adipocytes, whereas BMP7 has emerged as a specific regulator of brown adipogenesis, suggesting that targeting BMP7 could represent a potential therapeutic approach for obesity (Tseng et al. 2008; Zamani and Brown 2011). TGFβ is also known for its role in driving the differentiation of MSCs into myofibroblasts, which contribute to excessive collagen deposition and fibrosis (Popova et al. 2010). Manipulating TGFβ family signaling pathways can enhance MSC functionality for tissue repair and tissue remodeling, with significant implications for MSC-based therapies and regenerative medicine.

TGFβ family signaling in human iPSC reprogramming

Reprogramming of human somatic cells into iPSCs can be achieved by overexpressing specific transcription factors including OCT4, SOX2, KLF4, and CMYC (Takahashi et al. 2007; Li et al. 2016a, b; Guo et al. 2019). The molecular profiling identified three distinct transcriptional phases during reprogramming: essential early phase of mesenchymal-to-epithelial transition (MET) initiation, intermediate maturation phase acquires pluripotent competency, and stabilized pluripotent state with full pluripotency network (Samavarchi-Tehrani et al. 2010; Mullen and Wrana 2017).

A large number of experimental studies have proven that the TGFβ signaling pathway is a key signaling pathway in iPSC reprogramming (Li et al. 2016a, b; Fan et al. 2024). TGFβ facilitates the induction of epithelial-to-mesenchymal transition (EMT) in cells, exerting inhibitory effects on iPSC reprogramming (Lin et al. 2009; Li et al. 2010). Inhibiting TGFβ/Activin/Nodal type I receptor kinases enhances iPSC induction and eliminates the need for introducing SOX2 expression (Ichida et al. 2009). Additionally, treatment of partially reprogrammed iPSCs with TGFβ receptor inhibitor significantly induces NANOG expression, promoting full reprogramming (Ichida et al. 2009; Maherali and Hochedlinger 2009). Reprogramming efficiency of iPSCs can be attenuated by treatment with TGFβ or introduction of an activated type I TGFβ receptor, whereas enhancement of iPSCs reprogramming processes can be achieved through inhibition of TGFβ type II receptor expression by miRNAs (Li et al. 2011; Miyoshi et al. 2011; Subramanyam et al. 2011). BMP signaling promotes iPSCs reprogramming by inducing MET and antagonizing TGFβ stimulation in specific contexts (Li et al. 2010; Samavarchi-Tehrani et al. 2010).

TGFβ signaling is also essential in maintaining the self-renewal and pluripotency of human iPSCs similar like ESCs (Fig. 3), and its modulation can direct lineage-specific differentiation (Robinton and Daley 2012; Moradi et al. 2019). The optogenetic modulation of TGFβ signaling can precisely control the differentiation of human iPSCs into the mesenchymal lineage (Wu et al. 2023).The synergistic action of two inhibitors of SMAD signaling (Noggin and SB431542) is sufficient to induce iPSCs differentiate to neural tissue and are patternable to dopaminergic neurons and motoneurons (Chambers et al. 2009). Inhibition of TGFβ signaling pathway promotes differentiation of human iPSC-derived brain microvascular endothelial-like cells (Yamashita et al. 2020). A8301, a TGFβ signaling inhibitor, is sufficient to switch the cell fate from iPSCs into neural progenitor cells in OCT4/SOX2/KLF4/MYC-mediated human urine cells reprogramming (Wang et al. 2016a, b). BMPs are commonly combined with other growth factors (e.g., FGF, Wnt, or VEGF) and/or pathway inhibitors like small molecule inhibitors targeting TGFβ or Wnt signaling (SB431542 and CHIR99021), to induce iPSC differentiation towards specific cell types (Sánchez-Duffhues and Hiepen 2023). By manipulating these pathways, researchers can control iPSC behavior and enhance their utility in regenerative medicine and therapeutic applications.

TGFβ family signaling in cancer stem cells

Cancer stem cells (CSCs) represent a sub-population of cells within a tumor that have the ability to self-renew and give rise to the heterogeneous cell types found in the tumor (Ayob and Ramasamy 2018). CSCs play a pivotal role in tumorigenesis, progression, metastasis, relapse, and resistance mechanisms, exerting a profound impact on the aggressiveness of cancer (Reya et al. 2001; Graham et al. 2002; Bao et al. 2006; Nunes et al. 2018; Huang et al. 2020). TGFβ signaling can have a dual role in relation to CSCs. Initially, TGFβ can act as a tumor suppressor (Gu and Feng 2018). In normal and pre-malignant stem cells, it can maintain genomic stability and inhibit excessive self- renewal (Futakuchi et al. 2019). For example, TGFβ signaling initially restricts their proliferation in intestinal stem cells, but as tumors progress, cancer cells can hijack the TGFβ signaling pathway, where mutations can lead to constitutive activation of downstream pathways, and this abnormal activation promotes the self-renewal of CSCs, enabling them to continuously generate more cancer cells and contribute to tumor growth, metastasis, and drug resistance (Chen et al. 2024).

EMT process leads to an augmentation in the population of cells exhibiting stem cell properties, commonly referred to as CSCs (Dongre and Weinberg 2019, Celià-Terrassa and Jolly 2020). During cancer progression, TGFβ signaling pathway plays an important role in orchestrating EMT, facilitating the generation of stem cells, development of resistance to anti-cancer drugs, induction of genomic instability, and establishment of localized immunosuppression (Katsuno et al. 2013; Moses et al. 2016; Katsuno and Derynck 2021). TGFβ is a major driver in promoting the differentiation of mesenchymal stem cells derived from bone marrow and adipose tissue into cancer-associated fibroblasts (Jotzu et al. 2010; Yang et al. 2012). TGFβ can also induce the differentiation of mesenchymal stem cells into endothelial cells, promoting a pro-angiogenic microenvironment and facilitating tumor angiogenesis (Li et al. 2016; Batlle et al. 2019). In breast cancer, TGFβ-induced activation of the SMAD pathway can up-regulate genes that enhance the self-renewal capacity of CSCs (Wang et al. 2023). TGFβ superfamily signaling pathway regulates the state of pluripotency in human stem cells and their capacity to generate well-structured telencephalic organoids, thereby enhancing the formation of sophisticated brain disease models (Watanabe et al. 2022).

In cancer therapy, hematopoietic stem cell gene therapy targeting TGFβ augmented the efficacy of irradiation therapy in a preclinical glioblastoma model (Andreou et al. 2021). Inhibition of TGFβ enhances the functionality of the hematopoietic stem cell niche and mitigates cancer-related anemia (Wang et al. 2021). In a preclinical glioblastoma model, researchers demonstrated that targeting the αv integrin/TGFβ axis enhances natural killer cell functionality against glioblastoma stem cells (Shaim et al. 2021). Inhibitory effect of TGFβ1-blocking peptides P17 and P144 on EMT in CSCs was demonstrated, leading to the suppression of liver metastasis in a preclinical model of advanced colorectal cancer (Zubeldia et al. 2013). In Table 1, we summarize the studies that demonstrate the significant involvement of TGFβ superfamily signaling in the maintenance and differentiation of various cancer stem cells, offering promising clinical applications for targeting TGFβ family signaling to effectively impede cancer progression (Aigner and Bogdahn 2008; Watabe and Miyazono 2009; Caja et al. 2012; Sakaki-Yumoto et al. 2013a, b; Caja et al. 2015).

Table 1.

Roles of TGFβ family signaling pathway in various cancer stem cells

Cancer	Function
Glioma	TGFβ increases glioma-initiating cell self-renewal through the induction of LIF in human glioblastoma (Peñuelas et al. 2009)
	TGFβ promotes Sox4 and SOX2 expression and maintains stemness of glioma-initiating cells (Ikushima et al. 2009)
	BMP4 suppresses glioblastoma stem cell proliferation and promotes differentiation (Piccirillo et al. 2006)
	TβR2 serves as a novel regulator governing the properties of glioblastoma stem cells (Narushima et al. 2016)
	BMP signaling orchestrates the quiescence of glioma stem cells and confers resistance to radiation and temozolomide chemotherapy in glioblastoma (Sachdeva et al. 2019)
	NOX4 regulates TGFβ signaling to promote the proliferation and self-renewal of glioblastoma stem cells (García-Gómez et al. 2022)
	BMPRIB/ALK6 promotes terminal differentiation of brain cancer stem cells (Lee et al. 2008)
Leukemia	TGFβ controls Foxo3a localization and maintains stem cell-like properties of leukemia initiating cells (Naka et al. 2010)
	SMAD4 protects Hoxa9-induced transformation of normal hematopoietic stem cells and inhibits leukemia initiation (Quéré et al. 2011)
Breast cancer	TGFβ reduces the size of the putative cancer stem cell population and inhibits tumor formation (Tang et al. 2007)
	BMP2/7 suppresses breast cancer stem cell subpopulation and bone metastases formation (Buijs et al. 2012)
	TGFβ regulates the ILEI/LIFR signaling axis and promote self-renewal of breast cancer stem cells (Woosley et al. 2019)
	Treatment with TGFβ1 at a low concentration for a short time may induce the auto-induction of TGF β1 in breast cancer stem cells (Hariyanto et al. 2021)
Diffuse-type gastric cancer	TGFβ inhibits ATP binding cassette subfamily G member 2 (ABCG2) transcription and decreases the cancer-initiating cell populations within the cancer (Ehata et al. 2011)
	TGFβ reduces the expression of ALDH1 and REG4 to suppress cancer-initiating cell populations and tumorigenicity (Katsuno et al. 2012)
Pancreatic cancer	Nodal/Activin signaling promotes self-renewal and tumorigenicity in pancreatic cancer stem cells (Lonardo et al. 2011)
	Bromodomain containing 9 controls the TGFβ/Activin/Nodal pathway to regulate self-renewal, differentiation of human embryonic stem cells and cancer cell progression (Wang et al. 2023)
Colorectal cancer	BMP4 promotes colorectal cancer stem cell differentiation and enhances their chemo-sensitization (Lombardo et al. 2011)
	The downstream targets ID1/3 of BMP signaling regulate the self-renewal capacity of human colon cancer-initiating cells (O'Brien et al. 2012)
	Activin promotes self-renewal of colorectal cancer stem cells and facilitates tumor progression (Liu et al. 2016)
	BMP2 enhances radiosensitivity in colorectal cancer stem cells (Mahmoudi et al. 2024)
Ovarian cancer	TGFβ-induced transglutaminase 2 promotes ovarian tumor metastasis by inducing EMT and a cancer stem cell phenotype (Cao et al. 2012)
	TGFβ-mediated LEFTY/Akt/GSK-3β/Snail axis modulates EMT and cancer stem cell properties in ovarian clear cell carcinomas (Matsumoto et al. 2018)
Prostate cancer	BMP7 inhibits prostate cancer stem-like cell growth by activating p38 and increasing p21/NDRG1 expression (Kobayashi et al. 2011)
Squamous cell carcinomas	TGFβ signaling pathway limits the self-renewal and proliferation of cancer stem cells (Schober and Fuchs 2011)
Esophageal cancer	TGFβ1 inhibitors can reduce the migration and invasion abilities of esophageal cancer stem cells (Yue et al. 2015)
Liver cancer	TGFβ1 regulates liver cancer cells by modulating the expression of liver cancer stem cell markers such as CD133 and EpCAM (Wang et al. 2016a, b)

TGFβ family signaling in stem cell therapy

Stem cell therapy, represents a regenerative medicine approach employed for the investigation and treatment of various human diseases (De Luca et al. 2019). Recent progress in stem cell technology has opened up a new avenue for patients afflicted with diseases and disorders that remain untreated (Hoang et al. 2022). TGFβ family signaling have been investigated in the context of stem cell therapy for modulating various diseases and offering promising therapeutic approaches. Targeting these pathways offers potential to enhance therapeutic outcomes by directing stem cell behavior more precisely.

In the context of neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease, TGFβ signaling in NSCs is disrupted (Krieglstein 2013) (von Bernhardi et al. 2015; Karampetsou et al. 2022). Normally, TGFβ is involved in the self-renewal and maintenance of NSCs, but in the diseased brain, altered TGFβ levels are evident as in Alzheimer's disease where amyloid-beta plaques and tau tangles interfere with TGFβ signaling in NSCs, and reduced TGFβ signaling may lead to a decrease in the self-renewal capacity of NSCs, impairing the brain's ability to regenerate neurons and repair damaged neural tissue and contributing to the progressive loss of cognitive function (Meyers and Kessler 2017). Photoactivation of the TGFβ/SMAD signaling pathway improves neurogenesis in neural stem cells within an Alzheimer's disease model (Wu et al. 2021).

In fibrotic diseases such as liver fibrosis and pulmonary fibrosis, TGFβ signaling plays a central role in the behavior of mesenchymal stem cells (MSCs) (Taherian et al. 2024). In fibrotic conditions, MSCs are recruited to the injury site where TGFβ from damaged and inflammatory cells activates them and induces differentiation into myofibroblasts, whose enhanced self-renewal due to TGFβ signaling leads to excessive collagen production that disrupts normal tissue architecture and function (Qin et al. 2023). In liver fibrosis, the abnormal TGFβ-mediated self-renewal of activated stellate cells (a type of MSC-like cell) results in the deposition of fibrotic tissue and the progressive loss of liver function (Dewidar et al. 2019).

In type I diabetes, abnormal TGFβ signaling disrupts the cues for islet stem cell differentiation, high levels of TGFβ inhibit the differentiation of islet stem cells into functional β-cells by modulating crucial transcription factors and signaling molecules, impairing the body's ability to regenerate insulin-producing cells and contributing to the persistence of hyperglycemia (Lee et al. 2021). By manipulating TGFβ signaling pathways, researchers may be able to enhance the efficacy of stem cell therapies and promote the differentiation of stem cells into β-cells to improve insulin production and manage hyperglycemia (Brown and Schneyer 2021).

In the field of stem cell-based therapies, the immunomodulatory properties of TGFβ are of great importance (Akhurst and Hata 2012). MSCs secrete TGFβ which can act on immune cells like T-lymphocytes, B-lymphocytes, and NK cells to down-regulate their activation and proliferation and inhibit the production of pro-inflammatory cytokines such as IL-2 and IFN-γ by T-cells, thereby reducing the inflammatory environment (Atiya et al. 2020). Disruption of the TGFβ signaling is necessary for effective killing of hepatocellular carcinoma by human iPSC-derived NK cells (Thangaraj et al. 2024). Combined blockade of TGFβ and PD-L1 facilitates the expansion and differentiation of CD8 T cells with stem cell-like properties in immune-excluded tumors (Castiglioni et al. 2023).

TGFβ family signaling in tissue regeneration

Regenerative medicine is a novel and promising mode of therapy for patients who have limited or no alternative treatment options for their illness (Mousaei Ghasroldasht et al. 2022). TGFβ superfamilies are important mediators of tissue repair and regeneration as TGFβ secreted by stem cells or other cells in the damaged tissue microenvironment during tissue repair processes helps in recruiting other cells to the injury site (Xu et al. 2018). Researchers showed that MSCs therapy decreased fibrosis in a murine full thickness wound healing model, which correlated with a reduced TGFβ1/TGFβ3 ratio from wound lysates (Qi et al. 2014). TGFβ3 has been reported promote scarless healing in the fetus and reduced scarring in adults, which offer a scar-reducing therapy for acute and chronic wounds and fibrosing disorders (Lichtman et al. 2016). Periodontal ligament stem cells and gingival mesenchymal stem cells encapsulated in TGFβ3-loaded Arg-Gly-Asp modified alginate microspheres are promising candidates for tendon tissue regeneration (Moshaverinia et al. 2014). Exogenous addition of TGFβ protein in vivo during muscle regeneration results in a loss of muscle function while inhibition of TGFβR2 induces the formation of giant myofibers (Girardi et al. 2021). BMP7 has been reported significantly enhanced the odontoblastic differentiation and mineralized nodule formation in dental pulp stem cells, which are crucial for dental pulp regeneration (Suzuki et al. 2011; Liang et al. 2021). Inhibition of TGFβ signaling via suppressing muscle segment homeobox2 enhances hematopoietic differentiation of human ESCs, providing valuable insights into the efficient generation of functional blood cells from pluripotent stem cells for regenerative medicine (Wang et al. 2020).

Conclusions and perspectives

Recent advancements in stem cell research have paved the way for promising therapeutic approaches to address a wide range of diseases, including diverse types of cancer as well as metabolic and neurodegenerative disorders. The development of human induced pluripotent stem cells offers transformative potential for medical practice, promising to revolutionize therapeutic approaches. To effectively harness the therapeutic potential of stem cells, a comprehensive understanding of both extrinsic and intrinsic factors governing self-renewal and differentiation processes is imperative. Previous studies have demonstrated the crucial roles of the TGFβ family in governing stem cell self-renewal and differentiation across various developmental stages and diseases. One of the major challenges in using TGFβ family signaling in stem cell therapy is achieving precise control of the signaling pathways. Since these pathways are complex and involved in multiple cellular processes, over-or under-activation can lead to unwanted outcomes.

TGFβ family signaling often crosstalk with other signaling pathways in stem cells, such as Wnt, Notch, and Hedgehog pathways. Future research needs to focus on deciphering these complex crosstalk mechanisms to better design stem cell-based therapies. The investigation of dysregulated regulation in TGFβ family signaling pathways and the crosstalk between other signaling pathways governing the proliferation and differentiation of cancer stem cells holds significant interest for future research to identify suitable targets for personalized cancer therapies. Insights gained from these investigations are anticipated to facilitate the development of innovative therapeutic and prognostic modalities for addressing a range of cancers.

Although signaling within the TGFβ family has demonstrated substantial potential in pre-clinical studies related to stem cell therapy and tissue regeneration, there remain significant obstacles in clinical translation. The delivery of TGFβ family factors or modulators to the target tissue in a safe and effective manner also needs to be addressed. This could include the use of nanoparticles, gene therapy vectors, or engineered stem cells that can secrete these factors in a controlled manner at the target site. Additionally, long-term safety and efficacy evaluations in human patients are required to ensure the successful application of TGFβ family signaling in stem cell-based therapy and tissue regeneration. In conclusion, the elucidation of the complex regulatory networks involving TGFβ family signaling in stem cells is of utmost importance for future study to uncover the full therapeutic potential of stem cells in both regenerative medicine and cancer therapy.

Abbreviations

ABCG2

ATP Binding Cassette Subfamily G Member 2

ALDH

Aldehyde dehydrogenases

BMP

Bone morphogenetic protein

CSC

Cancer stem cell

DP

Dermal papilla

EMT

Epithelial-mesenchymal transition

ESC

Embryonic stem cell

FGF

Fibroblast growth factor

HFSC

Hair follicle stem cell

HSC

Hematopoietic stem cell

ICM

Inner cell mass

ID

Inhibition of DNA binding protein

ILEI

Interleukin-like EMT inducer

IPC

Intermediate progenitor cell

iPSC

Induced pluripotent stem cell

ISC

Intestinal stem cell

LIF

Leukemia inhibitory factor

MSC

Mesenchymal stem cell

MET

Mesenchymal-to-epithelial transition

NDRG1

N-Myc Downstream Regulated 1

NOX4

NADPH Oxidase 4

NSC

Neural stem cell

PS

Primitive streak

REG4

Regenerating Family Member 4

SCF

Stem-cell factor

SGZ

Subgranular zone

Sox

SRY-box transcription factor

SVZ

Subventricular zone

TGFβ

Transforming growth factor β

Authors’ contributions

S.L. and J.R. wrote the manuscript and created the figurers. Y.H. revised the manuscript and figures. L.Z. and F.Z. provided valuable discussion. All authors have read and approved the article.

Funding

This work was supported by National Natural Science Foundation of China (31925013, U20A20393, 32200575, W2411011 and 32460164), and the National Key R&D Program of China (2021YFA1101000).

Data availability

Not applicable.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.