Stem cells are distinguished from other cell types by their self-renewal capabilities and developmental capacity. Stem cells can differentiate into an array of specific lineage progenies. These properties make stem cells an attractive avenue for potential treatments for cancer, and age-related illness, as well as in regenerative medicine. Based on their source of origin, stem cells can be categorized into three classes. Table 1 summarizes different classes of stem cells.

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Figure 1. Schematic outline of mouse development illustrating the relationship of the early cell populations to the primary germ layers (endoderm, mesoderm, ectoderm).

Embryonic stem (ES) cells are derived from the inner cell mass of the blastocyst of an embryo (Figure 1) and are defined by indefinite self-renewal and pluripotency [3]. They can be propagated in culture as populations of undifferentiated cells for extended periods and retain normal karyotypes following extensive passaging [14]. The derivation of human ES cells in 1998 markedly raised interest in the cell therapy aspect of ES cells, and moved this concept one step closer to reality [15]. One such a line is the H1 cell line from WiCell Research Institute [16]. G Dixon et al investigate the role of QSER1 in DNA methylation in H1 and HUES8 cell lines [17]. L Pellegrini et al, for example, used H1 and H9 lines from WiCell to develop choroid plexus organoids [18]. Haploid embryonic stem cells from human and other species have been derived, and considered to be useful tools for genetic deletion screening [19].

Adult stem cells can originate from an array of adult tissues such as skin [20], bone marrow, blood vessels, skin, muscle and bone. They have multipotent differentiation capability, indicative of their tissues of origin. For example, hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs) exist in the bone marrow [21] whereas neural stem cells (NSCs) abide in the subventricular zone and hippocampus [22], although the presence of neural stem cells in human adults has been questioned [23]. Leng Z et al injected mesenchymal stem cells into patients to treat COVID-19 [24]. Ouadah Y et al identified a rare group of pulmonary neuroendocrine cells which could deprogram and become stem cells [25]. Cancer stem cells, for example, those in liver [26], represent a small sub-population of cells that retain stem-like properties and possess the capacity to induce tumour formation when engrafted in an experimental model.

Induced pluripotent stem cells (iPSCs) are generated by genetic reprogramming of differentiated somatic cells (i.e., fibroblasts) into de-differentiated stem cells using ectopic expression of a combination of transcriptional factors [27, 28]. Disease-specific iPSCs can be generated as well, for example, hiPSC cell lines with familial Alzheimer's disease gene mutations [29]. Reprogramming of fibroblasts to pluripotent stem cells through episomal expression of OCT3/4, SOX2 and KLF4 can be routinely achieved [30, 31]. The commonly used iPSC line EC11 from Lonza was derived from primary human umbilical vein endothelial cells [16]. Commercial kits are available to accomplish the reprogramming, such as Thermo Fisher Scientific CytoTune-iPS 2.0 Sendai Reprogramming Kit [32, 33] or the retroviral STEMCCA polycistronic reprogramming system from MilliporeSigma (SCR548) [34]. Induced pluripotent stem cell lines are also available from commercial and non-profit organizations such as Coriell Institute (Iso-E and Iso-T [32] or mEGFP-tagged CTNNB1 WTC iPS cell line AICS-0058-067 [35] ), WiCell [18, 32], RUCDR, Rutgers University (cell line NCRM1 [35] ), European Bank For Induced Pluripotent Stem Cells (EBiSC) [36]. L Pellegrini et al, for example, obtained iPS cells (IMR90-4) from WiCell to develop choroid plexus organoids [18].

Cultured ES cells derived from human, mouse and other species epitomize one of the major breakthroughs in genetics, developmental biology, and biomedical research. Maintenance of ES cells in an undifferentiated and pluripotent state in culture requires a combination of growth components. Below are described several factors that contribute to sustaining self-renewal of stem cells. Thermo Fisher Essential media (A1517001) is a popular media [29]. For instance, G Dixon et al maintained HUES8 and H1 cells in E8 condition on polystyrene plates coated with vitronectin (Thermo Fisher A14700) [17]. Gifford CA et al maintained hiPSC cells in feeder free culture on Corning Matrigel (BD, #354277) using Essential 8 Medium [31]. Tao Y et al cultured H1 hESCs on Geltrex-coated plates in StemFlex basal medium from Gibco, passaging these cells with StemFlex medium supplemented with RevitaCell and induced differentiation with the treatment of GHIR99021 [37].

ES cells were initially grown on feeder cells, a layer of mitotically inactive fibroblasts that support and promote ES cell growth, in growth media supplemented with fetal calf serum (FCS). To create a more refined system, exogenous factors were explored for their potential to support ES cell growth in vitro. The major attachment factors in serum are fibronectin and vitronectin. Feeder cells release fibronectin, collagen types I and IV, and laminin. With regard to mouse ES cells, the feeder layer can be easily substituted by adding bone morphogenetic proteins (BMPs), which are also found in serum, and leukaemia inhibitory factor (LIF), which inhibits differentiation. Thermo Fisher provides embryonic stem cell-qualified FBS, as, for example, used in E14TG2a mouse ES cell culture by Yasuda S et al [38].

Initially, human ES cells were sustained on mouse embryonic fibroblast (MEF) feeder layers [15] in the presence of FCS. Developments in the field lead to most labs adopting new culture conditions, whereby human ES cells are maintained in knockout serum replacement (KSR) and basic fibroblast growth factor (FGF2), instead of FCS, but culturing in the presence of single MEF feeder cell layers, for example, EmbryoMax PMEF from Millipore [39]. This helps to lower variability between cultures and limit differentiation, but still requires animal components to be part of the culture. This combination contains xenomaterials and causes a significant drawback for clinical application. Thus, there has been a move towards generating so-called “xeno-free” culture conditions [40], as well as the generation of more defined culture conditions that rely less on exogenous signals [41].

Leukaemia inhibitory factor (LIF) is applied to sustain self-renewal of mouse ES cells and inhibit their differentiation. LIF elicits the formation of a heterodimer between the LIF receptor and glycoprotein 130 (gp130), which activates the tyrosine kinase Jak. The activated Jak phosphorylates several proteins including the signal transducer and activator of transcription 3 (Stat3) [42], causing its translocation to the nucleus where it can upregulate pluripotency genes. In line with this, the culture of mESCs can be carried out in the absence of feeder cells if the media is supplemented with LIF.

In contrast, human ES cells do not depend on the LIF/Stat3 to sustain pluripotency but require Activin/Nodal and FGF signaling pathways [4]. This variation is related to the developmental stages from which these two cell types were derived.

The stem cell microenvironment, also known as the niche which harbours stem cells, has a critical function in stem cell self-renewal and differentiation processes. The niche consists of stem cells attached to supporting cells and soluble factors fixed in an extracellular matrix (ECM) [43].The matrix is composed of the circuitry of fibrous structural proteins (i.e., collagens, fibronectin, laminin, elastin, and vitronectin) and polysaccharides (i.e., glycosaminoglycan) and provides mechanical support and storage for cellular signalling molecules. Cells network with ECM components through receptors known as integrins and interact with neighbouring cells through receptors known as cadherins. Cytokines such as wingless-related (Wnts) and hedgehog proteins, fibroblast growth factors (FGFs), and bone morphogenetic proteins (BMPs) are soluble factors that also contribute to stem cell function [44, 45]. Biomaterial has been applied to grow ES cells in vitro. For example, mouse ES cells were grown on a polyamide-based 3D nano-fibrillar porous matrix (Ultra-Web) in a LIF based medium [46].

Human ES and iPSC cells can also be maintained in the absence of feeder culture medium, abolishing the concerns about xenogenic substances. Human ESCs can be cultured on dishes coated with active components such as vitronectin (A14700 from Thermo Fisher [17, 39] ), Matrigel [34, 47], or Geltrex (A1413301) from Life Technologies [48]. Matrigel™ is a commercial extract of natural basement membrane that contains laminin, collagen type IV, entactin, heparan sulfate proteoglycans and several other components [49].

An enhanced understanding of the transcription factor networks involved in the establishment and maintenance of pluripotent state has led to the generation of modified culture systems which select for cells in the naïve state of pluripotency. ES cells possess an intrinsic capability to support pluripotency without requesting exogenous stimulation [50]. This is achieved by culturing cells without FCS or feeder cells, in the presence of two small molecule inhibitors (2i) of mitogen-activated protein kinase (MAPK)/extracellular-signal-regulated kinase (ERK) kinase (MEK), and glycogen synthase kinase 3 (Gsk3) [50]. PD0325901,for example, from MilliporeSigma [51] interferes with FGF/ERK signalling by inhibiting MEK. CHIR99021, for example, from Axon Medchem [51], augments Wnt signalling by inhibiting Gsk3. This blocks differentiation signals and promotes the pluripotency network, securing the mouse ES cells in a truly naïve state of pluripotency. ROCK inhibitors, such as Y27632 from Selleck Chemicals [17], ApexBio [47], Santa Cruz (sc-281642A) [52], thiazovivin from Selleck Chemicals (S1459) [48], can enhance or ensure the survival of human stem cells during passaging.

The ground state of human ES cell pluripotency is more complex to comprehensively test owing to the inability to carry out chimera formation [53]. However, human ESCs can recapitulate some (but not all) key features of the in vivo blastocyst using the 5i/L/A system, which involves an additional 3 kinase inhibitors and 2 growth factors on top of the original 2i media components [41].

Modulating the transcription network is crucial in order to maintain self-renewal and pluripotency of ES cells. A strong understanding of the players is key in realizing the therapeutic potential of these cells [54]. Reprogramming fibroblasts to pluripotent stem cells was originally carried out in vitro by the ectopic expression of Oct4, Sox2, c-Myc, and Klf4, the minimal set of factors required for reprogramming [27]. However, reprogramming using these factors is not entirely efficient. These experiments were initially carried out in mouse, but the factors were later found to also reprogram human fibroblasts to induced pluripotent stem cells [28]. Additional so-called reprogramming enhancers’ were used in combination with these four factors to improve the efficiency of reprogramming [55]. These factors include pluripotency genes such as TBX3, UTF1 and SALL4, cell cycle genes such as REM2 or cyclin D1, and epigenetic modifiers such as inhibitors of EZH2 and Suv39h1.

Some of the key pluripotency factors are discussed below and also listed in Table 2.

Oct4 (Octamer-binding transcription factor 4, also known as Oct 3) is a mammalian POU transcription factor. in vivo , Oct4 is highly expressed in inner cell mass of the blastocyst, but is excluded from the trophectoderm [56]. It’s expression decreases during development. After implantation, it is thought only to be expressed in primordial germ cells [57]. Expectedly, Oct4 is expressed in cultured ES cells. It coordinates the expression of multiple target genes including the gene encoding fibroblast growth factor-4 (FGF4). Importantly, mouse embryos deficient of FGF4 create blastocysts but do not advance beyond implantation. Silencing Oct4 in embryos and ES cells causes impaired pluripotency and spontaneous differentiation into trophoblast lineages [58].

Nanog is a critical factor implicated in ES cell self-renewal and pluripotency and is present in the morula and the inner cell mass of the blastocyst. It supports ES cell self-renewal independently of LIF/Stat3. Nanog-deficient cells fail to maintain pluripotency and differentiate into extraembryonic endoderm lineage [42]. Interestingly though, Nanog-mutant embryos can be derived and cultured, suggesting that Nanog is dispensable for the maintenance of ESCs [59].

Sox2 is an essential factor the self-renewal of ES cells. in vivo , Sox2 is initiated early during development, and is expressed in embryonic and extra-embryonic (ectoderm) lineages. Sox2 expression is not restricted to early development; this transcription factor is more globally expressed in adult tissues, and therefore appears to be required for tissue regeneration or cell survival post-development [60]. Sox+ neuroprogenitors in killifish, for example, enable its fast development [61]. Sox2 and Oct4 work in concert with Nanog in both human and mouse ES cells to promote pluripotency [62]. In human ES cells, SOX2 has 1279 binding sites and Oct4 has 623 binding sites. A total of 404 are in common and 87% (353) of these sites also coincide with Nanog (1687 binding sites) targets [63].

Klf4 (Krüppel-like factor 4) is a transcription factor present in several tissues. Forced induction of Klf4 in ES cells leads to the inhibition of differentiation into erythroid progenitors [65]. Its exact role in the reprogramming process is not completely clear, but it has been shown to bind to enhancers and could potentially modulate gene expression programmes to favor pluripotency [66]. During the process of reprogramming, KLF4 can be replaced with members of the Klf family (i.e., Klf2 and Klf5) as well as other transcriptional factors such as Nanog and Lin28 [67].

Lin28, also known as zinc finger CCHC domain-containing protein 1 in mammals, is a conserved RNA-binding protein [68]. Lin-28 is highly expressed in mouse and human embryonic stem cells [69] and is employed to enhance the efficiency of the formation of induced pluripotent stem cells from human fibroblasts [67].

c-Myc is an additional key transcription factor that is involved in stem cell self-renewal and differentiation. It has a specific role in the interplay between stem cells and the local microenvironment [70]. c-Myc is also involved in several additional cellular functions, including cell-cycle regulation, proliferation, growth, differentiation and metabolism.

Having robust methods to characterise stem cell populations properly is crucial in studying their behaviour. Stem cells can be characterized by using 1) cell-surface marker profiling, 2) gene expression analysis, 3) biological assays of differentiation potential, and 4) genetic integrity examination. Flow cytometry [71, 72] provides quantitative data related to the ratio of cells in the culture that express surface markers and pluripotent transcription factors, while immunofluorescence allows cellular localization of the markers [73]. Gene expression levels of pluripotency markers can be detected by quantitative real-time polymerase chain reaction (qRT-PCR) and RNA sequencing. Utilizing the qRT-PCR method, the expression of a single gene can be detected while RNA-seq analysis can be employed to analyse global gene expression [74].

An additional tool that researchers can take advantage of to follow stem cell populations is reporter genes. For example, a reporter gene that expresses a fluorescent protein (i.e., enhanced green fluorescence protein (eGFP)) can be stably integrated into the cell, tagged on to a pluripotency marker such as Rex1 [75]. The gene is only induced (reports) when cells are in an undifferentiated state, and is completed inactivated as the cells differentiated. Once the gene is turned on, the cells are fluorescent. This fluorescence can be measured and analysed using flow cytometry. Cells of interested can also be sorted from the rest of the population, allowing researchers to analyse specific sub-groups of stem cells.

Stem cell populations can be isolated using techniques such as fluorophore efflux and cell surface markers. Fluorophore efflux (Rhodamine 123 alone or in conjunction with Hoechst 33342) can be used, for instance, to define different bone marrow populations; the cell fractions that contain greater stem cell activity display lower levels of fluorescence dye staining than the rest of the bone marrow [76]. Fluorescent activated cell sorting (FACS) can be used to analyse and sort cells tagged with fluorescent markers. In this technique, a fluorochrome conjugated to a signaling molecule or antibody proffers specific affinity and binding specificity to a lineage-specific marker. Various fluorescent probes are applicable that produce diverse colours and intensities [77]. For instance, several antibodies have been used to detect for the enrichment of HSC against hematopoietic lineage antigens such as: 1) T cell-associated antigen (Thy-1), 2) Lineage antibody cocktail (Lin-), 3) Stem cell antigen-1 (Sca-1), 4) Tyrosine kinase receptor c-kit [78, 79]. Furthermore, cancer stem cells (CSCs) from a broad spectrum of human cancers have been identified by employing an amalgam of markers. Table 3 summarizes CSC cell surface markers, among which, SSEA3, SSEA4, TRA-1-60, TRA-1-81, SSEA1, CD133, CD90, CD326, Cripto-1, PODXL-1, ABCG2, CD24, CD49f, Notch2, CD146, CD10, CD117, CD26, are expressed in human ESCs but not in normal tissues; CXCR4, CD34, CD271, CD13, CD56, CD105, LGR5, CD114, CD54, CXCR1, TIM-3, CD55, DLL4, CD20, CD96, are expressed in adult stem cells but not in normal tissues; and CD29, CD9, CD166, CD44, ABCB5, Notch3, CD123 are expressed in both human embryonic stem cells and adult stem cells, and also in normal tissues [80].

ES cells can be induced to differentiate into different cell lineages utilizing different approaches including: 1) aggregation and formation of 3D colonies known as embryoid bodies (EBs) [81], 2) differentiated in contact with stromal cells (i.e., OP9 stromal cell line) [82], 3) differentiated in a monolayer on extracellular matrix proteins (e.g., cadherin) [83].

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Figure 2. Micrographs [1] from mouse ES cells illustrating A) Embryoid bodies (EBs) after removal of LIF. Aggregates are round and have a well-defined border. They maintain their aggregate morphology and are distinguished from the feeder layer. B) EBs in the absence of LIF and feeder layers. They lose their aggregate morphology and migrate to the periphery as they continue to differentiate.

The formation of embryoid bodies (EBs) is the initial process for differentiation of ES cells [84]. Briefly, in the absence of LIF or feeder cells, ES cells differentiate spontaneously and form 3D aggregates called embryoid bodies. These structures contain cells from ectodermal, mesodermal and endodermal lineages. Figure 2 illustrates micrographs from mouse ES cells forming EBs in the absence of A) LIF and B) LIF and feeder layer [1].

EBs can be differentiated by culture with growth factors, non-coated plates or suspension culture. Table 4 outlines several methods used to induce EBs configuration such as dissociated suspension culture, methylcellulose culture, hanging drop (HD) culture, spinner flask, bioreactor culture, and round microwell plates [85].

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Figure 3. Schematic illustration of a protocol [2] used for differentiation of ES cells into cardiomyocytes.

EBs offer the advantage of supporting a three-dimensional structure that strengthens cell-cell interactions that may be significant for certain developmental stages. However, the disadvantage of using the EB system is that the presence of cytokines and inducing factors within these structures can complicate interpretations of the signaling pathways involved in the generation and maintenance of specific lineages. Figure 3 illustrates a schematic outline of a protocol used for a spontaneous differentiation of ES cells into cardiomyocytes [2]. The generation of cardiomyocytes can also be achieved through the treatment of ES cells with small molecule inhibitors. Lee A et al treated HES3 human ES cells with CHIR99021 (C-6556) from LC laboratories and Wnt-C59 (S7037) from Selleck Chemicals to generate cardiomyocytes [48].

ES cells have the potential to differentiate, spontaneously under appropriate conditions, into cells representing all three germ layers [3] (Figure 1). For instance, organs such as the liver and pancreas are originated from the endoderm and can be the major focus of cell-based therapies. Activation of the activin / Nodal pathway promotes endoderm induction in mouse ES cultures [4]. Subpopulations of the mesoderm layer give rise to the hematopoietic, vascular, cardiac, and skeletal muscle lineages. The induction of the ectoderm leads to the neural lineages and skin.

In ES cells, the ectoderm formation is also known as the “default” pathway, since the neuroectoderm readily originates in cultures in the absence of serum. Stimulation of signaling pathways by BMP, Wnt and Activin/Nodal prevents the generation of neuroectoderm [5] (Figure 4). The promise of therapeutic lineage reprogramming (transdifferentiation) has been explored in several areas of regenerative medicine. Cell types from all three germ layers have been reprogrammed successfully to induced pluripotent cells using combinations of transcription factors. Here, cell types that are of significant clinical interest are described.

Epigenetic differences between human induced pluripotent stem cells (hiPS cells) and human embryonic stem were studied by genome-wide DNA methylation profiling throughout reprogramming of somatic cells to hiPS cells [86]. The epigenetic memory in hiPS cells was found to be located in the repressive chromatin marked by H3K9me3, lamin-B1 and aberrant CpH methylation. The transient-naive-treatment (TNT) reprogramming developed in this study modifies these domains to an hES cell-like phenotype and does not impair genomic imprinting. Overall, the authors showed that TNT reprogramming can repair the transposable element overexpression and differential gene expression in hiPS cells and stimulates the differentiation of hiPS cells developed from multiple cell types.

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Figure 4. A scheme of ES cell differentiation depicting the development of the primary germ layers (endoderm, mesoderm, ectoderm) [3] and the role of specific signaling processes. BMP4 induces the formation of mesoderm and skin while Activin/Nodal [4] signaling induces endoderm formation. FGF leads to neuroectoderm induction, whereas Activin, BMP and Wnt [5] are all implicated as inhibitors of this pathway.

Various cell types have displayed potential for transdifferentiation into pancreatic β cells both in vitro and in vivo. The pancreatic and duodenal homeobox factor-1 (Pdx1), a homeodomain-containing transcription factor, is involved in pancreatic development and insulin gene transcription. Transduction with the Pdx1 protein facilitated the differentiation of ductal progenitor cells into insulin-producing cells [6]. Moreover, downstream signaling events intermediated by triggering the glucagon-like peptide-1 (GLP1) receptors precipitated the Pdx1-mediated effects [87]. Using exendin-4, a GLP1 receptor ligand, in conjunction with Pdx1 overexpression in vitro , hepatocytes were directed to transdifferentiate into insulin-producing β cells [7].

It has been demonstrated in vivo that pancreatic exocrine cells can be induced to transform to pancreatic endocrine β cells following transfection with an adenovirus that expresses genes for a set of three transcription factors: Pdx1, Ngn3 (neurogenin3), and MafA (v-maf musculoaponeurotic fibrosarcoma oncogene family, protein A) [8]. This approach for insulin-producing β cells by direct lineage reprogramming paves a novel potential avenue for treating type 1 diabetes. Figure 5 presents a list of examples of in vitro cell reprogramming by the lineage-instructive approach.

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Figure 5. Examples [6], [7], [8], [9], [10], [11], [12], [13] of in vitro cell reprogramming (transdifferentiation) by the lineage-instructive approach.

The development of the cardiac lineage in ES cell differentiation advances through distinctive phases that are analogous to the progression of the in vivo lineage. An induction of arranged paradigm of cardiac genes is expressed in the differentiation cultures, with expression of the transcription factors Gata-4 (binds to the DNA sequence "GATA") and Nkx2.5 (homeobox protein that interacts with Gata4) that are necessary for lineage development leading to the activation of genes that are characteristic of developmental stages in vivo : ANP (atrial natriuretic protein), MLC (myosin light chain-2v), myosin heavy chains, and connexin 43 [88, 89]. Studies have suggested that lineage reprogramming could be implemented for cardiomyocyte regeneration, and importantly, mouse fibroblast cells were transdifferentiated into cardiac cells following transduction with a combination of three cardiac transcriptional factors: Gata4, Tbx5 (T-box transcription factor), and Mef2c (myocyte-specific enhancer factor 2C, also known as MADS box transcription enhancer factor 2) [9]. This transdifferentiation of fibroblast cells into cardiac cells has important implications in deciphering heart developmental biology and might provide a potential therapeutic approach in human cardiovascular diseases.

Studies have shown that lineage-reprogramming intervention could also be applied for neuronal regeneration. The various approaches include (1) culture of serum-stimulated EBs with retinoic acid [90], (2) treatment of EBs in serum followed by serum-free medium to promote differentiation down the neural lineage [91], (3) differentiation of ES cells as a monolayer in serum-free medium and enhanced by inhibiting the transforming growth factor β (TGFβ) related signalling pathway [92], and (4) differentiation of ES cells directly under the influence of stromal cells in the absence of serum and retinoic acid [93]. Commercial kits, such as StemCell Technologies STEMdiff Neural Induction Medium (#05831) can also be used to induce neural induction of human stem cells [94].

Several studies provide proof of principle that specific subtypes of induced neuronal cells can be produced from human somatic cells by transcription factor-mediated reprogramming. Mouse fibroblasts can be reprogrammed into functional neuronal cells in vitro following transfection with lentivirus expressing genes of a set of nervous system transcriptional factors: Ascl1 (achaete-scute homolog 1), Brn2 (POU domain-containing transcription), and Myt1l (myelin transcription factor 1-like) [10]. It has also been shown that adult astrocytes can be reprogrammed into neurons in vitro by induction of the following transcriptional factors: Pax6, Ngn2 (neurogenin-2), Mash1 (mammalian achaete schute homolog 1) [11]. This pathway is able to reprogram astroglial cells towards neuronal lineages, and promotes neuronal features such as intrinsic excitability and action potentials. Therefore, the transcriptional network initiated by Pax6 in astrocytes represents an avenue for therapeutic intervention. Examples of in vitro reprogramming by the lineage-instructive approach are shown in Figure 5. Neurogenin-2 can also be introduced via doxycycline-inducible vectors to enable neuronal differentiation from iPS cells [95].

Induced dopaminergic neurons, a cell type lost in Parkinson’s disease, can be generated from mouse and human fibroblasts by the following two approaches: 1) combining an amalgam of transcriptional factors (Ascl1, Brn2, and Myt1l) and neural conversion factors: Lmx1a (LIM homeobox transcription factor 1 alpha) and FoxA2 (forkhead box A2) [12] and 2) a combination of three transcription factors: Mash1, Nurr1 (nuclear receptor related 1 protein) and Lmx1a (LIM homeobox transcription factor 1, alpha) [13]. These neurons present gene expression and electrophysiological traits in accordance with midbrain dopaminergic neurons, including automatic dopamine release. Generation of induced dopaminergic neurons cells from somatic cells might have important implications for understanding critical processes for neuronal development, as well as cell replacement therapies in disease which these cells have been depleted.

A recent study has described a method for isolating distinct neural stem and progenitor cell NSPC lineages from the developing brain using specific surface markers [96]. The authors enriched CD24-THY1-/lo cells for glial cell subsets, which were engrafted and developed into three neural cell lineages in the mouse brain. THY1hi cells marked oligodendrocyte lineage precursors, while CD24+THY1-/lo cells marked excitatory and suppressor neuronal cell subsets. The study detected a THY1hiEGFRhiPDGFRA- bipotent glial progenitor cell, which differentiate into astrocytes and oligodendrocytes.

The main functions of stem cell niches include maintenance of the stem cell pool and control of their activities by cellular interactions and by secretion of regulatory factors. Hematopoietic stem cells (HSC) are located in the bone marrow in HSC niches which consist of the endosteal and vascular niches. The endosteal HSC niche is found between bone tissue and bone marrow. The cellular components of this niche include osteoblasts, osteoclasts, macrophages and endothelial cells [97]. In contrast, the vascular HSC niche contains vascular sinuses formed by endothelial and reticular cells [98]. With regard to growth factors produced by the cells in the stem cell niche, stem cell factor (SCF) [99] and C-X-C motif chemokine 12 (CXCL12) are crucial for the regulation of stem cells. In particular, SCF binds to its specific receptor KIT, which is expressed by HSCs and is involved in their maintenance [100]. Additionally, homing and maintenance of HSCs are regulated by interactions of CXCL12 with its receptor CXCR4 [101]. Hepatic thrombopoietin is required for bone marrow hematopoietic stem cell maintenance [102, 103]. Other regulatory factors include angiogenin [104], and pleiotrophin [105].

Different cells forming the microenvironment of HSC niches may regulate the growth and survival of HSCs. In particular, osteolineage cells were shown to maintain the population of HSCs [106]. Moreover, CXCL12-abundant reticular cells produce a chemokine CXCL12 and express leptin receptor LepR [107]. Vascular endothelial cells stimulate the proliferation and homing of HSCs by the production of SCF and CXCL12 [107]. Expression of L-selectin by endothelial cells activated the growth of HSCs [108]. With regard to the effects of the nervous system, signals from sympathetic nerves inhibit the activity of stem cell niche [109] and stimulate the development of HSCs from the supporting cells via cytokine production [110]. In immune cells, CD169⁺ macrophages stimulate the production of CXCL12 by stromal cells, which activates homing of HSCs to the bone marrow [111].

A fundamental mechanism describing interactions between niche cells to regulate their activity and frame the signaling environment of the resident stem cell niche has recently been reported [112]. Cells of the hematopoietic niche interact via gap junctions (GJs) and generate a signaling network, which is vital for exchanging Ca2+ signals. Impaired Ca2+ signaling in the stem cells interfered with differentiation of HSCs and related progenitor lineages.

Regarding the plethora of factors regulating HSCs growth and differentiation, angiopoietin-like protein 2 (ANGPTL2), which stimulates the expansion of HSCs via human leukocyte immunoglobulin-like receptor B2, was found to support repopulation activity and quiescent status of HSC and niche localization [113]. The study used an Angptl2-flox/flox transgenic mouse cells lacking Angptl2 expression in several niche cells, including endothelial cells, mesenchymal stem cells, and megakaryocytes. ANGPTL2 was shown to activate the expression of peroxisome-proliferator-activated receptor D (PPARD) to sustain the perinuclear localization of nucleolin and prevent HSCs from undergoing the cell cycle.

Human skin is formed by epidermis and dermis separated by a basal membrane (BM). Stem cells are closely attached to BM. Epidermal stem cells are located in three niche loci: intefollicular epidermis, hair follicle and the sebaceous gland (Table 5). The variety of skin stem cells are represented by keratinocytes, follicular stem cells, sebaceous stem cells, hematopoietic stem cells, nerve progenitor cells and mesenchymal stem cells. The proliferating cells of follicular epidermis are crucial for skin regeneration.

Epidermal stem cells become active during the transformed cell phase, divide fast and participate in skin regeneration [114]. These cells differentiate into epidermal cells. The markers of epidermal stem cells include integrins, K15, p63 and nestin. The epidermal stem cells are connected to laminin proteins through integrins.

In the hair follicle, the stem cells are located in the bulge. These cells are involved in wound healing mechanisms, including reorganization of the epithelium and regeneration of hair follicle. Bulge stem cells demonstrate the slower cell cycle and express alpha 6-integrin and CD34. Notably, expression of CD34, a marker for HSCs, has been found in the protrusion region of follicles. Y Shwartz et al isolated mouse hair follicle stem cells as CD45^-, integrin alpha 6⁺, CD34⁺ and Sca-1^- [115]. In addition, these stem cell subsets express K15, K19, Sox9, Lgr5, CD200 and PHLDA1. The cells of the isthmus, an area between bulge and the sebaceous gland, express Gli1, MTS24 and Lgr6 and are involved in the maintenance of homeostasis and regenerative processes following a trauma. The interactions of the bulge stem cells with neighboring cells in the niche are mediated via Wnt pathway.

The sweat glands represent another area containing cells with high ability to proliferate. Outer myoepithelial cells are characterized by the detection of K5, K14 and actin. In contrast, inner luminal cells express K8, K18 and K19. With regard to the sebaceous gland, bulge stem cells are crucial for the regeneration of gland tissue [116]. B lymphocyte-induced maturation protein (Blimp1) is considered to be a specific marker of sebocytes and Blimp1+ cells differentiate into the sebum-secreting cell subset [117].

With regard to specific regulation of Flightless I (Flii), a cytoskeletal protein and inhibitor of wound healing, was shown to act a suppressor of epidermal stem cell activation by interfering with Wnt/β-Cathenine signaling [118].

Melanocyte stem cells (McSC), another stem cell lineage present in skin primarily residing in an undifferentiated stage in the hair follicle niche, were shown to switch between transit-amplifying and stem cell phenotypes for both renewal and maturation [119]. Both live imaging and single-cell RNA sequencing indicated that McSCs migrate between hair follicle stem cells and transit-amplifying compartments. McSC system is carried out by reverted McSCs as opposed to reserved stem cells that are not capable of reversible transformations. In addition, aging process was reported to correlate with expansion of stranded McSCs, which do not contribute to the melanocyte line regeneration.

Intestinal stem cell niche is located in the intestinal epithelium and is formed by different cell populations, mainly stem cells, stromal cells and Paneth cells [127] (Table 6). A population of proliferating stem cells is located in epithelial cavities and maintain the continuous renewal of the intestinal epithelium. The actively dividing Lgr5+ stem cells are responsible for the continual regeneration of intestinal epithelium. These cells give rise to transit amplifying cells, which differentiate into tuft and goblet cells and enterocytes. The proliferation of stem cells is mainly regulated by the interaction between classical Wnt/β-catenin, Hedgehog, Notch, BMP and growth factor pathways. In particular, the intestinal stem cells express NOTCH1, which binds to DLL1 and DLL4 on Paneth cells. This interaction results in the stimulation of Notch signalling followed by the increased proliferation of the stem cells [128].

Among other cells of the intestinal stem cell niche, Paneth cells are the main source of Wnt and EGF and therefore they act as vital regulators of intestinal stem cells [129]. Strictly specialized secretory Paneth cells are located in the small intestinal crypts. The granules secreted by Paneth cells contain immunoregulatory molecules, which regulate the spectrum of intestinal flora. In addition, Paneth cells secrete pro-angiogenic factors, thus enhancing intestinal angiogenesis.

Recent studies have shown that Krüppel-like zinc-finger transcription factor 5 (KLF5) might be the main regulatory factor controlling the maintenance of stem cells by influencing Wnt and Notch signalling pathways [130]. In particular, KLF5 controls access to chromatin and histone modification.

Intestinal stromal cells control the stem cell niche by producing various regulatory factors. In particular, stromal PdgfRα+ cells develop close connections with intestinal epithelial cells. Recent studies have shown that PdgfRα+ cells regulate the maintenance of alveolar type 2 stem cells in the lungs [131]. In line with this finding, PdgfRα+FoxL1+Gli+ cells most probably act similarly in the intestines and control the homeostasis of the niche and tissue regeneration by producing Wnts and R-spondins [132, 133]. Also, stromal cells secrete bone morphogenic proteins (BMPs), while epithelial cells produce Hedgehog pathway ligands. Various fibroblast markers, such as PDGFRα, CD34, FOXL1, and GLI1 are involved in Wnt signalling and maintenance of the stem cells.

Discs large 1 (Dlg1) has been found to be essential for ISC survival related to increased Wnt signaling [134]. RNA sequencing and genetic mouse models have shown that DLG1 controls the cellular response to elevated canonical Wnt ligands via Arhgap31, a GTPase-activating protein, which inhibits CDC42, an activator of the non-canonical Wnt pathway. Thus, stem cell response to activated Wnt signaling is regulated by the DLG1-ARHGAP31-CDC42 pathway.

Regarding the stem cell niche morphology, the mouse intestinal stem cell niche was revealed to contain both mesenchymal and smooth muscle cell subsets [135]. The specialized muscle layer begins to form during postnatal crypt morphogenesis and supplements neighboring RSPO and BMPi sources. The in vivo ablation of mouse postnatal smooth muscle was shown to stimulate BMP signaling activity by restricting limiting a burst of crypt fission. The microenvironment formed by the specialized mesenchymal cells is essential for promoting crypt formation and maintaining adult intestinal stem cell niche.

Landmark developments have been made over the past decade in the stem cell field. With a greater understanding of the networks involved in the establishment and maintenance of pluripotent populations, there has been a surge in efforts made to harness the cells in these precious states.

The potential of ES cells to give rise to essentially any differentiated cell type has advanced the development of regenerative medicine. To fully explore the treatment potential of pluripotent stem cells, it is imperative that researchers are able to command ES cell lineage differentiation, and to instruct these cells towards specific lineages. Ethical issues associated with the use of ES cells might be reconciled by current research into adult stem cell plasticity, which presents with a possible source of cells for regenerative medicine and the competency to control immune-rejection issues. Cell reprogramming, in which a differentiated cell is directed to alter its fate, is also an innovative field with important aspects for biotechnology and medicine. As new research advances are unravelling, the stem cell field is rapidly evolving and becoming one of the most galvanized areas of biomedical research.

The article was updated by Dr. Kathryn McLaughlin in Oct 2018. Dr. Konstantin Yakimchuk added sections on Skin stem cell niche and Intestinal stem cell niche in May 2020.