Stem Cells and Signaling Pathways

Stem Cells and Signaling Pathways provides mechanistic insights into the role of stem cells to combat the COVID-19 outbreak and other pathologies where a cytokine storm is the cause of concern for e.g., radiation exposure, multiple organ failure and sepsis. The advent of SARS-CoV-2 resulted in a global pandemic, putting individuals with other comorbidities at a higher risk of infection. The whole world witnessed a massive shortage of medical and other essential supplies needed to combat the virus. That said, stem cell therapy has emerged as a potential treatment for viral diseases, including but not limited to COVID-19.

Interestingly, the clinical trials in the patients having COVID-19 complications depicted faster recovery in patients post mesenchymal stem cells therapy owing to the decreased cytokines levels, anti-viral effects and regeneration of the infected tissue.

• Provides a compilation of chapters dedicated to the study of the field with respect to properties of stem cells and their signaling pathways. It also provides detailed information on signaling pathways associated with stem cells.
• Provides a detailed and updated view of stem cell biology.
• Covers a wide range of areas within basic and translational research appropriate for scientists
and clinicians engaged in studying the treatments for diseases from interdisciplinary perspectives.
• Highlights the use of different models for stem cell research, significance of stem cell signaling in drug discovery.

• Language: English
• Publisher: Academic Press
• Release date: Sep 28, 2023
• ISBN: 9780443188015

Book preview

Preface

Surajit Pathak and Antara Banerjee

This book, Stem Cells and Signaling Pathways, deals with description of the role of various signaling pathways in tissues with a high cellular turnover and their stem cell populations, which are essential for the lifelong maintenance of organ function and prevent disease onset.

This book encompasses various topics dealing with stem cell signaling pathways that control stem cell maintenance and differentiation. The potency of stem cells, their self-renewal, and differentiation ability toward various lineages are key significant stem cell properties. These key features of stem cells are governed by a network of transcription factors, modulating kinase signaling pathways, and epigenetic factors. In stem cell research, the signals elaborated in stem cell programming are of major interest. The signaling mechanisms involved in regulating stem cell differentiation and reprogramming are the subject of intense study in the field of regenerative medicine and tissue engineering. The molecular interactions and signaling proteins related to stem cell differentiation are discussed vividly in this book.

Signals in stem cell are regulated both genetically and epigenetically by many signaling proteins. The programming of stem cell signaling is an imperative aspect of understanding the functioning and phenotype transition of stem cells. The differentiation process as well as inter- and intracellular signaling clues that govern the fate of stem cells is described in this book. The epigenetic regulation of these cells is also discussed. The primary objective of this book is to describe the latest developments on the implications of stem cell–associated signaling proteins in the pathogenesis of metabolic and autoimmune diseases and designing futuristic therapeutic strategies for various stem cell–based therapies. A major challenge still ahead will be the amalgamation of chief signaling pathways into networks of control mechanisms ultimately coupling extrinsic with intrinsic regulatory stem cell fate determinants.

The chapters are written by recognized regenerative medicine and stem cell biology specialists, medical scientists, and oncologists and cover the basics of stem cell–related signaling pathways. This book provides the latest developments to food scientists, biochemists, medical doctors, nutritionists, food technologists, students majoring in food science, and public health professionals.

We genuinely hope that researchers, practitioners, and industry partners in regenerative medicine and/or stem cells, students and instructors in regenerative medicine/stem cell courses around the globe that are seeking methods to improve their understanding of the crucial aspect of stem cell signaling pathways find this book helpful inspiration and possibly collaborative opportunities to enhance the well-being of the aged population.

We sincerely thank Elsevier for their help in publishing this book.

Chapter 1

Regulation of mesenchymal stem cell differentiation by key cell signaling pathways

Aishwarya Dhinekaran¹*, Mallela Lakshmi¹*, Hepzibah Graceline¹*, Amit Dey¹, Subhamay Adhikari¹, Satish Ramalingam², Ilangovan Ramachandran³, Atil Bisgin⁴,⁵, Ibrahim Boga⁴,⁵, Surajit Pathak¹ and Antara Banerjee¹,    ¹Medical Biotechnology Lab, Faculty of Allied Health Sciences, Chettinad Academy of Research and Education (CARE) (Deemed to be University), Chettinad Hospital and Research Institute (CHRI), Kelambakkam, Chennai, Tamil Nadu, India,    ²Department of Genetic Engineering, School of Bio-Engineering, SRM Institute of Science and Technology, Kattankulathur, Chengalpattu, Tamil Nadu, India,    ³Department of Endocrinology, Dr. ALM PG Institute of Basic Medical Sciences, University of Madras, Taramani Campus, Chennai, Tamil Nadu, India,    ⁴Department of Medical Faculty, Cukurova University AGENTEM (Adana Genetic Diseases, Diagnosis and Treatment Center) & Medical Genetics, Adana, Turkey,    ⁵InfoGenom R&D Laboratories, Cukurova Technopolis Adana, Adana, Turkey

Abstract

Mesenchymal stem cells (MSCs) are multipotent stem cells that have the ability to differentiate into osteocytes, chondrocytes, adipocytes, myoblasts, fibroblasts, and neuronal stem cells. MSCs are primarily derived from the bone marrow, adipose tissue, umbilical cord, and other sources such as menstrual blood and endometrium, peripheral blood, amniotic fluid, and dental pulp. MSCs are one of the most appealing choices in regenerative medicine for the development of numerous therapeutic applications due to their low immunogenicity and immunomodulatory function. Along with this, MSCs have an inherent ability to self-renew and undergo multilineage differentiation. Studies have revealed the various factors that influence the transdifferentiation of MSCs, and various signaling pathways that have been found to be involved in particular lineage commitments. MSC transdifferentiation is regulated by factors such as bone morphogenic protein (BMPs), TGF-β family proteins, RANKL, and signaling pathways such as Wnt/β-catenin signaling pathway, TGF-β/SMAD (suppressor of mothers against decapentaplegic) signaling pathway, Notch signaling pathway, Sonic Hedgehog signaling pathway, platelet-derived growth factor signaling pathway, FGF signaling pathway, JAK/STAT signaling pathway, and NELL-1 signaling pathway. Epigenetic mechanisms such as histone alteration, DNA methylation, and acetylation play a crucial role in the development of MSCs and also in their differentiation into specific lineages.

Keywords

Platelet-derived growth factor; TGF-β/SMAD; DNA methylation; reactive oxygen species; RANKL; Neural epidermal growth factor-like 1

1.1 Introduction

Our body is composed of a variety of cells of which one kind of cells called stem cells have the special ability to incessantly divide and differentiate into other types of cells such as muscle cells, skin cells, nerve cells, and fat cells. The stem cells are specialized cells that have a great disparity in repairing and regenerating potential of various cells and tissues. They also have the potential to proliferate and renew themselves and are blank cells that give rise to specialized cell types or tissues by differentiation.

Stem cell research is a relatively new field of science with techniques that aid in understanding the emergence and evolution of cells. This has led us to the discovery of novel and efficient treatments and cures for many diseases that often cannot be treated with drugs. It is, therefore, crucial for future medicine and public health development. Based on their differentiation potency, stem cells are of five types: (1) Totipotent, (2) Pluripotent, (3) Multipotent, (4) Unipotent, and (5) Oligopotent. With respect to their origin, they also can be classified as embryonic stem cells (ESCs) or adult stem cells (ASCs), which can be further classified as mesenchymal stem cells (MSCs), neural stem cells (NSCs), and hematopoietic stem cells.

MSCs have gained a lot of attention in the last 30 years due to their intriguing cell biology, extensive therapeutic value, and function as a crucial aspect in the rapidly expanding domain of tissue engineering. MSCs have inherent differentiating potentials that are not observed in other cells. MSCs can be easily expanded in a culture plate, and they can generate a significant number of beneficial cytokines and growth factors [1]. MSCs fall under the category of multipotent stem cells, also known as multipotent stromal cells, and can undergo adipogenic, chondrogenic, myogenic, fibrogenic, and osteogenic differentiation processes to form the adipose tissue, cartilage, muscle, tendon/ligament, and bone, respectively. They also have the ability to regenerate specific tissue types such as connective tissue, cartilage, and the circulatory system [2]. MSCs also have potent antiinflammatory, vasculoprotective, and immunomodulatory effects. These effects are primarily mediated by the soluble factors and extracellular vesicles (EVs) that can be released in their secretome [3]. In the past several years, various sources have already been used to isolate the MSCs, including the adipose tissue, placental tissue, lung, liver, spleen, pancreas, testes, peripheral blood, heart, umbilical cord (UC) blood (UCB), menstrual blood, amniotic fluid, dental pulp, and the dermis [2]. MSCs were first isolated from the bone marrow as a component of bone-marrow-derived cells, which have a distinct spindle-shaped morphology [4]. MSCs express various surface antigens, such as cluster of differentiation (CD) 44 (CD44), CD73, CD90, and CD105, and the absence of CD14, CD34, CD45, CD79a, and HLA-DR (human leukocyte antigen) expression [1]. MSCs differentiate based on specific factors including growth factors such as transforming growth factor-β (TGF-β) [TGF-β2, TGF-β3] fibroblast growth factors (FGFs) [FGF-2, FGF-4, and FGF-6] that are present in their surrounding milieu, and based on that, they differentiate into specific lineages. TGF-β and FGFs are responsible for differentiation of MSCs to chondrogenic lineage, dexamethasone along with ascorbic acid and β-glycerophosphate are responsible for osteogenic lineage, and insulin, dexamethasone and indomethacin along with 3-isobuty-l-methyl-xanthine are responsible for adipogenic lineage [5]. During the embryonic development period, TGF-β/BMP, Wnt/β-catenin pathway, Hedgehog (Hh), Notch, mitogen-activated protein kinase (MAPK), and other signaling pathways are substantial for stem cell maintenance and equivalent commensurate growth rate among all lineages, such as body patterning, cell fate determination, and organogenesis [6]. It is crucial to comprehend the signaling pathways that have a significant influence on MSCs development to develop the cell therapies for the replacement of damaged tissues. This will enable the integration of signal inputs and lineage-specific lineage initiation. The development of innovative treatments in the medical profession has benefited from extensive knowledge of signaling pathways [7]. This book chapter aims to shed light upon the importance of MSCs, their transdifferentiation, and the signaling pathways influencing the transdifferentiation mechanism.

1.2 Mesenchymal stem cells

MSCs or mesenchymal progenitor cells are a class of nonhematopoietic multipotent stromal cells primarily found within the bone marrow [8,9]. They have the ability of self-renewal and are capable of differentiating into cells of multiple lineages, unlike other classes of stem cells that follow single lineage differentiation. They also have immunomodulatory, antiinflammatory, and signaling properties. Under specialized conditions, MSCs can differentiate into various cell types including osteocytes, chondrocytes, adipocytes, myocytes, marrow stromal cells, and neural cells [8,10].

1.2.1 Sources of mesenchymal stem cells

MSCs are most readily found within the adult bone marrow; however, that is not the only source. The extraction of these cells from the bone marrow involves invasive procedures, which can be painful and ends up with a low cell turnover. Intending to curb and overcome these disadvantages, several studies conducted in recent years have found and identified alternative sources that have the potential for efficient and high-yield isolation of MSCs, namely the adipose tissue, UC tissue, menstrual blood, endometrium, amniotic fluid, and dental pulp [11].

Despite having the same basic features, that is, self-renewal and multilineage proliferation, MSCs derived from different sources are distinct at the genetic level and, thus, have varying proliferation capacity and differentiation properties [12]. For example, researchers have shown that fetal sources of MSCs can undergo many more divisions before senescence than MSCs derived from adult tissue [13]. This implies that fetal tissue-derived stem cells have a higher proliferative capability and therefore, are much more preferred in regenerative stem cell therapy.

1.2.1.1 Bone marrow

Following the discovery by Friedenstein and colleagues in 1976, bone marrow MSCs (BM-MSCs) were intensively studied and considered to be the primary source of MSCs in human adults. However, the process of isolating the BM-MSCs from bone marrow biopsy has more demerits than merits. The procedure involves the usage of high doses of anesthesia, it is highly invasive and is painful for the subject [14]. Moreover, the proliferative and differentiation capacity, cell count, and longevity of these stem cells decline as the subject’s age increases. A few advantages of BM-MSCs are that they are clinically proven to be much safer and more efficient to use, they have a greater differentiation potential, and their culture time is relatively shorter when compared with other cell types [15–17].

1.2.1.2 Adipose tissue

Adipose-tissue-derived MSCs (AD-MSCs) are most deemed to be the better alternative to BM-MSCs on basis of their accessibility and stability. Subcutaneous adipose tissue is abundant in the body and can easily be extracted through cosmetic or therapeutic liposuction, lipoplasty, or lipectomy [18]. AD-MSCs can remain stable throughout their long culture period and also have high differentiation potential, albeit not as high as BM-MSCs. Similar to BM-MSCs, the viability of AD-MSCs decreases with the donor’s age, but regardless of age, the differentiation capacity of these cells remains consistent, thus giving AD-MSCs another advantage over BM-MSCs.

1.2.1.3 Umbilical cord

The UC is a rich source of MSCs. UC-MSCs can be harvested noninvasively from the cord lining, cord blood, and perivascular region of the UC and from novel sources such as the UC matrix and Wharton’s jelly [19]. Fetal-related MSCs like UC-MSCs are more preferred in regenerative medicine for the very reason that they not only produce a high cell count in culture, but they are also not influenced or damaged by any acquired genetic mutations, unlike MSCs derived from adult tissue [18,20].

The viability of UC-MSCs, unlike the aforementioned MSCs, does not depend on the age of the donor; they are harvested from ethically obtained (easily discarded) tissue that has exceptionally high proliferative capacity. UC-MSCs secrete chemokines, cytokines, and growth factors that enhance cell repair systems, which in turn facilitate the immunomodulatory and antiinflammatory mechanisms of MSCs [21].

1.2.1.4 Amniotic fluid

Studies have shown that amniotic fluid is a rich source of MSCs that could be used for therapeutic purposes with less risk of tumor formation [22]. These cells are shown to exhibit ESC and ASC markers, and the characteristics of MSCs, thus being termed amniotic fluid-derived MSCs (AF-MSCs) [23]. AF-MSCs are generally isolated from amniotic fluid in the second or third trimester due to the abundance of the MSC population [24]. These multipotent cells have high proliferation and differentiation potential, having been reported to differentiate into osteocytes, chondrocytes, adipocytes, cardiomyocytes, and neuronal cells in vivo and in vitro [25–28].

1.2.1.5 Menstrual blood and endometrium

Menstrual blood MSCs (MB-MSCs) were first isolated and investigated by Meng et al. [29] and Cui et al. [30]. MB-MSCs have the proliferative and multilineage differentiation capacity even greater than BM-MSCs and are also easily accessible without any invasive procedure or ethical concern [31]. The endometrium has been found to be rich in MSCs and epithelial progenitor cells [32,33]. Endometrium MSCs (eMSCs) are also capable of differentiating into cells of all three germ layer lineages [34]. eMSCs can be isolated directly from the endometrium by hysterectomy or endometrial biopsy, which are invasive procedures or through noninvasive procedure by isolation from the menstrual blood.

1.2.1.6 Dental tissue

Dental MSCs are primarily obtained from the pulp of primary incisors and molar teeth (both exfoliated deciduous and permanent) [35]. They are capable of producing cells of all germ layer lineages, are easily accessible, and can be maintained in vitro for a long period [36]. Dental MSCs also exhibit immunomodulatory functions by secreting cytokines and the maintenance and repair of periodontal tissue [37,38].

1.3 Dedifferentiation versus metaplasia versus transdifferentiation

Multicellular organisms are composed of a variety of cell types, all of which have been derived from a single fertilized zygote that has undergone many cycles of cell division and differentiation [39]. As an organism develops, its cells continue to divide and mature into different phenotypes, progressively becoming restrained to only a fixed cell line. As the time progresses, the cells’ response to signals gradually decreases, and eventually they lose their ability to differentiate any further. At this final stage, the cells are considered to be terminally differentiated, and they cannot divide anymore.

Dedifferentiation is the process in which the terminally differentiated cells revert to their more primitive, less mature stage. The genes that determine the functioning of the mature cells are inhibited, and the genes that allow the cells to divide are reactivated, thereby allowing for the proliferation and generating more cells of that particular cell type. Dedifferentiation can be observed in the regeneration of limbs or tails in starfish and lizards and is a major mechanism that occurs and plays a role in tissue and wound repair [39].

Metaplasia and transdifferentiation, though similar in mechanism, were initially terms used to describe different events. Metaplasia was first used to describe the appearance of unexpected foreign tissue in ectopic cells and can now be defined as the transformation of one cell type to another that is not normally found in the observed tissue. This includes the interconversion between tissue-specific stem cells [40,41]. Metaplasia may occur when the original cells are not strong enough to survive in the cell environment (they are replaced with cells that seem better suited) or due to an abnormal stimulus [42].

Transdifferentiation was first described by [43] as the transformation of cuticle-producing cells to salt-secreting cells in silk moths during metamorphosis. Transdifferentiation is an irreversible switch of one type of differentiated cells to another type of differentiated cells, with or without the accompaniment of cell division. However, metaplasia is the reversible substitution of one type of differentiated cells to another type in a tissue [41,44]. Eguchi and Kodama defined two important criteria that define transdifferentiation: (1) there must be a well-defined morphological and biochemical/molecular difference between the original and transdifferentiated cell types and (2) there must be evidence of the cell lineage relationship between the two cell types [44,45]. Transdifferentiation is a mechanism of intrigue in regenerative medicine as transdifferentiated cells are already in their mature stage, thus alleviating concerns about possible rejection or a change in cell fate following their transplantation into a patient. Hence, it is essential to know the process of transdifferentiation and understand its significance as a new target for regenerative therapy, as will be discussed in the upcoming sections of this chapter.

1.4 Transdifferentiation of mesenchymal stem cells

Stem cells can be obtained from all tissues in the body; however, the yield of those stem cells is too low to be useful, or it is impractical to obtain them from a particular organ, such as the brain or heart. This is where the process of transdifferentiation receives attention. As mentioned in the previous sections, transdifferentiation is the conversion from one type of differentiated cell to another, and MSCs are cells that have the capacity for multilineage differentiation. It is this ability of MSCs to form different cell types that is exploited to induce transdifferentiation under different conditions and produce the desired type of cells. So far, mesenchymal transdifferentiation has been studied in adipocytes, osteoblasts, chondrocytes, hepatocytes, fibroblasts, dental pulp tissue, cardiac adipose tissue, UC tissue, body fluids (cord blood, menstrual blood, amniotic fluid), endometrium epithelial cells, alveolar epithelial cells, pancreatic cells, and retinal pigment cells [46,47]. Nevertheless, a better understanding of the mechanisms underlying MSC transdifferentiation is necessary to determine the full potential of using ASCs in treating various diseases.

1.4.1 Transdifferentiation potential of hMSCs from adipose tissue to neuronal cells

Adipose tissue develops from the mesoderm in humans. Adipose-derived stem cells (ADSCs) show multilineage and multipotent differentiation, capable of producing osteoblasts, chondrocytes, adipocytes, and neurocytes. Conventionally, nervous system tissue is limited in its regenerative ability because mature neural cells do not differentiate. The identification of specific cells isolated from the adipose stromal vascular portion provides evidence of neuronal differentiation. ADSCs are capable of differentiation into cells of multilineage origin [48]. ADSCs can transdifferentiate into NSCs, which can develop into neurons, Schwann cells [49] in the peripheral nervous system, and oligodendrocytes [50] in the central nervous system. These two types of cells play a crucial role in the myelination of neurons.

1.4.2 Mesenchymal stem cells for cardiac cell regeneration

Heart failure is a debilitating and deadly illness. In the past decade, adequate advancement in the medical field has been built to cure heart failure following myocardial infarction and reasonably extend the human life span [51]. By using a variety of MSCs, specifically those derived from UCBMSCs and cardiac adipose-tissue-derived MSCs (cATMSCs), addressing the challenge of cardiac healing after myocardial infarction has become a possibility [52]. The cardiovascular potential of UCBMSCs and cATMSCs has been broadly estimated as an alternative therapy that eases reversed or attenuated adverse rejuvenation, self-repair, and long-term functional stability that improves heart function [53]. MSCs integrate into the injury sites and release trophic and immunostimulatory factors, which have either tissue reformatory or pharmacological activities. UCBMSCs are significant in cardiovascular regeneration as they are a plentiful reservoir of multipotent progenitor cells, which can differentiate into mesenchymal cell lineages and are involved in immune-modulatory activity [54,55]. Previous studies have exposed UCBMSCs’ molecular mechanisms that participated in angiogenesis regulation [56] and triggered vascular growth in vivo [57].

cATMSCs are found in the adipose tissue present around the heart [58]. They can be extracted from the base of the heart and around the aortic arch. cATMSCs, which show an MSC-like pattern of cell surface antigen expression, play a vital role in heart homeostasis involved in the regeneration of myocardial tissue.

1.4.3 Epigenetic modulation for transdifferentiation of adipose-derived stem cells

Transdifferentiation causes changes in a cell’s gene activity. Transdifferentiation of ADSCs into neural cells comprises two crucial perspectives: (1) interruption requires genetic modification that influences the gene expression without changing the DNA sequence and (2) the essential stability of neural cells, which are derived from the ADSCs because they are circumspect by epigenetic factors. ADSC transdifferentiation mainly involves three systems: (1) histone alteration, (2) DNA methylation, and (3) posttranscriptional RNA regulation [59].

1.4.3.1 Histone alteration

Histones are the main protein components present around the DNA forming a thread-like structure (chromatin), which plays a role in gene expression. Posttranslational modifications of histone proteins include methylation, acetylation, phosphorylation, and ubiquitylation occurring at the site of tail regions of core histones [50].

Histone acetylation is a major step in the transdifferentiation of ADSCs into neural cells. It involves the addition of an acetyl group to histone tails by histone acetyltransferase, the transcriptional activator of histone molecules. It weakens the interaction between histone and DNA and results in the occurrence of transcription. Histone deacetylation occurs when acetyl groups from histone tails are removed by the enzyme histone deacetylases (HDACs), repressing the transcription by increasing the interaction between DNA and histone. The HDAC inhibitors could potentially induce the neurogenic differentiation of human ADSCs (hADSCs) by activating the canonical or noncanonical Wnt signaling pathways [60].

In histone methylation, histone methyltransferase, histone lysine methyltransferase, and protein arginine methyltransferase are involved. Histone methylation may lead to the expression and suppression of genes. The trimethylation of histone H3 at lysine 4 (H3K4) activates transcription, whereas dimethylation of histone H3 at lysine 9 (H3K9) may signal the inhibition of transcription. At the time of neural cell proliferation from the ADSCs, H3K4, H3K9, and H3K27 play a major role in histone methylation [61]. In the transdifferentiation of ADSCs into neural components, both acetylation/deacetylation and methylation/demethylation processes occur simultaneously and regulate the mechanism of histone alteration.

1.4.3.2 DNA methylation

Methyl groups consist of one methylated carbon along with three hydrogen atoms that have the effective tendency to bind to the other molecules. The methylation process involves allowing methyl groups (having an affinity toward the cytosine) to bind to DNA. The methyl group is added by DNA methyltransferases (DNMTs) [DNMT1, DNMT3a, and DNMT3b] at CG dinucleotide (CpG) sites in the DNA sequence [62]. It has been found that DNA methylation and acetylation have a great impact on the differentiation of MSCs into NSCs, which can further form neuronal cells and glial cells [63]. It has been reported that demethylation and remethylation of Nestin protein in the neural-specific enhancer region occur during reprogramming and upon neurogenic differentiation, respectively [64]. Furthermore, the DNA methylation patterns in the promoter region of adipogenic and nonadipogenic genes of ADSCs such as peroxisome proliferator-activated receptor-2 (PPAR-2), leptin, fatty acid binding protein 4, and lipoprotein lipase appear to be generally hypomethylated, whereas in the case of myogenic and endothelial cell regulatory areas, they often have higher levels of methylation [65].

1.4.4 Factors influencing transdifferentiation of mesenchymal stem cells

Transdifferentiation of adult MSCs into mesodermal-derived cells, ectoderm-derived cells, and endodermal-derived cells requires specific factors in their differentiation environment to commit the specific lineages. Several factors including activin, nodal, BMPs, and other TGF-superfamily proteins, as well as epidermal growth factor, FGF, platelet-derived growth factor (PDGF), and receptor activator of nuclear factor Kappa-B ligand (RANKL), all play important roles in the lineage specification of MSCs.

1.4.4.1 Growth factors

Various growth factors influence the MSCs in both autocrine and paracrine manner and their receptors expressed on the surface of these cells. The TGF-β family encompasses three members: TGF-β1, TGF-β2, and TGF-β3. Among these, TGF-β1 is the most important growth factor that influences the MSCs. TGF-β1 can induce the migration of MSCs to the sites of bone remodeling where bone resorption coupled with bone formation takes place. The effect of TGF-β1 is mediated by canonical signaling pathway through SMADs along with the noncanonical pathway involving AKT, ERK1/2, FAK, and p38 [66,67].

TGF-β1 can induce the switch of adipogenesis to osteogenesis when the cells are exposed to the adipogenic medium under in vitro condition [68]. Moreover, TGF-β1 is reported to decrease the number of osteoprogenitor cells and expansion of terminal differentiation, suggesting that the effects of TGF-β1 on MSCs depend on the state of the cells [69–71].

1.4.4.2 Bone morphogenetic proteins

The BMP family consists of at least 15 members, and they generally utilize synergistic effects with TGFβs and regulate SMAD transcription factors and expression of certain genes (PPARγ, RUNX2, or SOX9). BMPs can promote the differentiation of MSCs to the adipogenic (e.g., BMP2, BMP4, and BMP7), osteogenic (e.g., BMP2, BMP6, and BMP9), or chondrogenic (e.g., BMP2 and BMP7) lineage, depending on their type and concentration [72,73].

1.4.4.3 Receptor activator of nuclear factor kappa-B ligand (RANKL)

Receptor activator of nuclear factor kappa-B ligand (RANKL) is an essential factor influencing the MSCs for the production of osteoblasts and osteocytes [74,75]. RANKL may have anabolic effects on the bone when low doses of cytokines are injected into the ovariectomized mice [76,77]. In a group of ovariectomized mice that are RANKL-deficient, there is limited osteogenic differentiation, which is enhanced by restoring the production of the RANKL in the soluble form. These indicate that RANKL might contribute to the direct regulation of MSCs to osteoblastic lineage in an autocrine/paracrine manner [78].

1.5 Signaling pathways involved in transdifferentiation of mesenchymal stem cells

Transdifferentiation of ADSCs into NSCs can be stimulated by specific transcription factors correlating to certain key signaling pathways. ADSCs differentiation shows their greater potential and effectiveness in therapies to treat neuronal disorders. It is similar to the adult neurogenesis process wherein active ADSCs are formed into immature neuron-like cells. The cells have the same neural marker expression required to produce NSCs, including the switch-on mechanism of neurogenesis factors, namely Tbr1, Tbr2, Ngn2, NeuroD1, Pax6, and Mash1; and their motif of articulation is similar to the intermediate stages of neuronal differentiation [79].

1.5.1 Wnt/β-catenin pathway

Wingless-related integration site (Wnt) signaling pathway controls the signals passed through the cell surface protein receptors. Wnt proteins are highly conserved glycoproteins that are involved in cell differentiation and regulate the cell migration, cell polarity, neural patterning, and cell fate determination [80,81]. The activation of Wnt/β-catenin signaling causes the expression of Wnt proteins, which are crucial in promoting the transdifferentiation of adipocyte lineage MSCs [82]. Wnt/β-catenin signaling is involved in the activation of neuronal development [83]. The regulation of adipocytes is promoted by signaling that suppresses the CCAAT enhancer binding protein-α (CEBP-α) and peroxisome proliferator-activated receptor (PPAR)-γ’ regulators [84].

Wnt5a protein promotes hADSC transdifferentiation when it binds to Frizzled receptors (Fz3/Fz5) by activating the c-Jun N-terminal kinase (JNK) signaling pathway [83]. The early differentiation is associated with an increase in the expression of genes such as cyclin D1 and signal transducer and activator of transcription 3 (STAT3) and a reduction in the expression of BMP2 and BMP4 [57,83].

During the activation of Wnt/β-catenin pathway, the nonphosphorylated β-catenin that accumulates in the neural cells’ cytoplasm gets translocated into the nucleus, which then along with the transcription factors lymphoid enhancer factor (LEF)/T-cell factor (TCF) binds to the gene and initiates the transcription of the genes especially Neurod1 and PROX1, which are involved in the neuronal differentiation. The activation of Wnt/β-catenin signaling pathway plays a major role in neural cell fate [85]. The schematic representation of the Wnt signaling pathway is depicted in Fig. 1.1.

Figure 1.1 Gene expression of NeuroD1 and Prox1 through Wnt/β-catenin pathway. APC, Adenomatous polyposis coli gene; AXIN, axis inhibition; DVL, disheveled; GSK-3β, Glycogen synthase kinase 3β; Lrp, lipoprotein receptor-related protein; TCF, T-cell factor/LEF, lymphoid enhancer factor; Wnt, Wingless-related integration site.

1.5.2 Sonic hedgehog pathway

Sonic Hedgehog (SHH) pathway plays a key role in the embryonic development as well as tumorigenesis. The key components of the SHH pathway include 12-transmembrane receptor patched homolog (PTCH), 7-transmembrane receptor smoothened homolog (SMO), suppressor of fused homolog (SUFU), and glioma-associated oncogene homolog (GLI) family of transcription factors. In ADSC transdifferentiation, SHH controls the proliferation and development of neural cells [85]. During the neurogenesis process, the Hh pathway becomes activated to induce the gene expression involved in neural induction. After the neural induction, these genes such as neuron-specific enolase (NSE), microtubule-associated protein-2 (MAP-2), growth associated protein-43 (GAP-43), neural cell adhesion molecule (NCAM), and synapsin-1 (SYN-1) are inhibited [86].

SHH ligands inactivate the PTCH receptor that leads to the activation of SMO protein. The SMO activates the GLI transcriptional factors (GLI1, GLI2, GLI3), which stimulate the transdifferentiation of neural cells. SHH plays a crucial role in the chondrogenic differentiation of BM-MSCs by stimulating the Hh pathway. Thus, PTCH, SMO, and GLI1 are the central regulators of chondrogenic differentiation of BM-MSCs [87]. SHH plays an important role in the differentiation of neuronal stem cells by activating various homeodomain proteins such as NK2 homeobox 2 (NKx2.2) and paired box protein-6 (Pax6) via sequence-specific binding to DNA [88]. Fig. 1.2 depicts the SHH pathway.

Figure 1.2 Sonic Hedgehog signaling pathway and its role in the neuronal differentiation of MSCs. HES, hairy and enhancer of split; PTCH, Protein patched homolog 1; SHH, Sonic hedgehog; SMO, Smoothened.

1.5.3 Notch signaling pathway

Notch signaling regulates the cell pluripotency and transdifferentiation by inducing the expression of specific target genes to create the necessary cell environment [89]. In the Notch signaling pathway, a decrease in the number of receptors present on the surface of the target cells enhances the ADSC transdifferentiation into neural cells [90]. Notch signaling is the main regulator of cell transdifferentiation and the development of cells in both in vitro and in vivo environments [91].

The Notch receptor activates specific genes such as hairy and enhancer of Split-3 (HES-3) and SHH. It regulates the rapid activation of cytoplasmic signals such as Akt and (STAT3), which promote the neural cell genesis [91]. One of the most potent osteogenic BMPs is BMP-9, which mediates the Notch signaling pathway that is essential for the osteogenic differentiation of MSCs [92]. It has been found that BM-MSCs express an enhanced level of jagged1 on their surface, which is a major Notch ligand, whereas Sca-1+ BM-MSC-driven dendritic cells (DCs) (sBM-DCs) derived from hemopoietic progenitor cells (HPCs) that express an enhanced level of Notch1. This Jagged1 activates the Notch signaling, which allows the sBM-DCs to retain their immunomodulatory properties [93]. The Notch signaling cascade is depicted in Fig. 1.3.

Figure 1.3 Notch signaling pathway and its role in activating the HES-3 and SHH gene expression. CSL-CBF1, Suppressor of hairless, lag-1; MAML, mastermind-like protein; NECD, notch extracellular domain; NICD, notch intracellular domain.

1.5.4 Transforming growth factor-β/SMAD signaling

TGF-β1, β2, and β3, activin, nodal, inhibin, myostatin, growth and differentiation factors (GDFs), and BMPs are important TGF-superfamily ligands that play a dual role in cell proliferation and differentiation [93]. TGF-β/SMAD signaling pathway has two types of serine-threonine kinase receptors, type I and type II. Binding of these ligands to the receptor activates both SMAD-dependent and SMAD-independent signaling pathways (including ERK, JNK, and the p38 MAPK kinase signaling pathway). TGF-β/SMAD signaling plays a dual role in the commitment of MSCs to adipocyte differentiation to form mature adipocyte tissue, with BMPs signaling promoting adipocyte commitment and TGF-β signaling inhibiting it. It has been reported that TGF-β and myostatin ligands phosphorylate SMAD2/3 to regulate the adipocyte commitment of C3H10 MSCs, whereas BMP2/4/7 ligands phosphorylate SMAD1/5/8 to control the adipocyte commitment of MSCs [93]. Moreover, BMP2 and myostatin can induce osteogenesis while inhibiting the myogenic differentiation. TGF-β superfamily proteins regulate the pivotal steps in hADSC and NSC transdifferentiation, proliferation, migration, and apoptosis by turning on the growth factor production and activity [93]. The BMP signaling monitors the NSC generation, but it varies from one neural cell type to another. Therefore, the role of BMP2 expression is to increase the transcription prevalence in type 1 and type 2 astrocytes; however, it has no role in the regulation of neurons and oligodendrocytes [94]. BMP5 and BMP7 regulate the transdifferentiation of NSC into midbrain dopaminergic (mDA) neurons in the brain [95], and they also interact with Smad 1 and Smad 5 proteins regulated by calcineurin for neural induction.

1.5.5 Janus kinase/signal transducer and activator of transcription pathway

The Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway is a well-controlled and effective mechanism that primarily controls the gene expression and entails the stimulation of JAK in cell membranes by the action of various polypeptides such as growth factors, hormones, or cytokines on cell membrane receptors [96,97]. It is a kind of conserved signal transduction pathway, which accounts for normal development, physiology, and regenerative responses during infection or injury [98]. The pathway is enabled by altering the receptors binding to extracellular ligand and made available for the intracellular JAKs to be linked to phosphorylate one another. The receptor and STATs are among the downstream substrates that are phosphorylated by trans-phosphorylated JAKs. In the nucleus, activated STATs interact with distinct enhancer regions in the target genes as dimers or more complex oligomers to control the gene transcription [99]. There are four JAK family members and seven STATs in mammals. Depending on their tissue specificity and the receptors involved in the signaling event, several JAKs and STATs are recruited. However, parts of other signaling pathways, such as those requiring the ERK, MAP kinase, PI 3-kinase (PI3K), control or are governed by elements of the conventional JAK/STAT pathway. Additionally, noncanonical JAK and STAT actions modify the chromatin structure to have an impact on the overall transcriptional activity [100]. Various developmental and physiological processes, such as stem cell maintenance, organismal growth, hematopoiesis, development of immune cell and mammary gland, rely on JAK/STAT signaling. The JAK-STAT pathway has been shown to be important for astrocytic differentiation and to promote the proliferation and differentiation of NSCs and neural progenitor cells (NPCs) [101,102]. A number of tissues, such as the gonads, gut, and limbs, undergo regenerative processes that are controlled by this pathway, it also controls vital homeostatic activities in germline and somatic stem cells [103]. A study conducted using a rat model reported that BM-MSCs have the ability to regenerate a vascular arterial wall where recombinant human BMP2 (Rh-BMP2) plays an important role in enhancing the expression of hypoxia inducing factor-1α (HIF-1α) and inhibitor of DNA binding (Id1) through, at least in part, the stimulation of JAK2, STAT3, and STAT5 signaling pathways. This Rh-BMP2 aids in mimicking the embryological condition required for BM-MSCs vascular differentiation [104]. Fig. 1.4 depicts the JAK/STAT signaling pathway.

Figure 1.4 Role of JAK/STAT pathway in mesenchymal stem cell’s differentiation into vascular, astrocyte, and neuronal lineage. JAK, Janus kinase; STAT, signal transducer and activator of transcription.

1.5.6 Platelet-derived growth factor pathway

PDGF is one of the key growth factors that control cell division and proliferation. It plays a crucial role in angiogenesis. PDGF can also activate VEGF-independent mechanism to stimulate the angiogenesis. PDGFA/PDGF receptor (PDGFR) signaling axis is essential for the mesenchymal progenitor cell proliferation and lineage commitment throughout the organogenesis and embryogenesis [105,106]. Moreover, PDGF signaling is important for the adipogenesis and chondrogenesis. The PDGFR, which is found on the outer surface of cell membrane, recognizes PDGF. When PDGF binds to its receptor, a signaling transduction cascade is induced, prompting ERK1/2 to produce cytosolic phospholipase 2 (cPLA2). Arachidonic acid synthesis is increased by cPLA2. It causes the production of reactive oxygen species (ROS) through NADPH oxidase and protein kinase C (PKC). Thus, the accumulation of arachidonic acid in the cells is necessary for NADH-induced ROS production. To promote the cell proliferation, ROS is implicated in MAPK activation. The PDGF signaling pathway also involves a number of intermediary pathways [107]. Importantly, it has been reported that the PDGF signaling pathway can regulate the osteogenic differentiation of MSCs [108]. Upon binding of PDGF A, B, C, and D ligands synthesized from osteoblasts, chondrocytes, and MSCs, to these receptors, they have the ability to accelerate the bone fracture repair process via the recruitment of MSCs to the injury site [109].

1.5.7 Neural epidermal growth factor-like 1 pathway

Neural epidermal growth factor-like 1 (NELL-1) is one type of glycoprotein that is found in secreted form and acts as a potent osteoinductive factor, and thus it regulates the skeletal ossification through rheostatic mechanisms. Upregulation of these proteins during early bone development in sporadic coronal craniosynostosis in humans led to the initial discovery of its osteoinductive characteristics. Both endochondral and intramembranous bone developments are accompanied by the expression of NELL-1. The NELL-1 upregulation preferentially boosts the osteoblast development and mineralization and is largely specialized to the osteochondral lineage [110]. The condition seen in humans is replicated in transgenic mice overexpressing NELL-1, which also exhibited untimely cranial suture fusion and bone overgrowth. It’s fascinating to note that nontissue-specific overexpression of NELL-1 in mice only caused calvarial bone abnormalities to appear. This finding implies that NELL-1 signaling has a relative osteo-specific impact [111]. Additionally, total deletion of NELL-1 in mice causes a notable drop in the mineralization of calvarial bones and a diminished rate of osteoblastogenesis and has been demonstrated to play a crucial part in the differentiation and development of bone in the craniofacial region [112]. It stimulates the osteogenesis followed by stimulation of canonical Wnt, HH, and MAPK signaling. It also stimulates both ERK1/2 and JNK-MAPK pathways in SaOS-2 osteosarcoma cell type [72,113]. Emerging evidence demonstrates that NELL-1 also has antiadipogenic properties. Both the preadipocyte cell line 3T3-L1 cells and primary adipose-derived MSCs showed these properties [114]. For the treatment of osteoporosis, one novel therapeutic strategy has been discovered where NELL-1 can be given synergistically with other osteogenic factors such as BMP2 and 9 [115]. It has been reported that NELL-1 signaling in osteogenesis is mediated by binding to the integrin 1 receptor on the cell surface and then activating the canonical WNT-catenin pathway. When NELL-1 binds to integrin 1, an intracellular signaling cascade is triggered; this encourages cell adhesion, growth, and differentiation into osteogenic tissue. Furthermore, inhibiting Wnt signaling interferes with NELL-1 osteogenic actions [116]. Through promoting the production of Runx2, a key transcription factor involved in the regulation of osteogenesis, Wnt and consequently NELL-1 signaling increase osteoblast differentiation and skeletal development (Table 1.1). Runx2 then controls NELL-1 expression upstream. Various studies have demonstrated that Runx2 and NELL-1 are intertwined, and Runx2 has a binding site in the promoter region of NELL-1 [110,117,118]. Runx2 causes the upregulation of NELL-1 levels by binding to the osteoblast-specific cis-acting element2 region in the promoter. Additionally, NELL-1 can activate Runx2 by phosphorylation via induction of the MAPK pathway