Culturing human intestinal stem cells for regenerative applications in the treatment of inflammatory bowel disease
2017; Springer Nature; Volume: 9; Issue: 5 Linguagem: Inglês
10.15252/emmm.201607260
ISSN1757-4684
AutoresFredrik Eo Holmberg, Jakob Benedict Seidelin, Xiaolei Yin, Ben Mead, Zhixiang Tong, Yuan Li, Jeffrey M. Karp, Ole Haagen Nielsen,
Tópico(s)Digestive system and related health
ResumoReview10 March 2017Open Access Culturing human intestinal stem cells for regenerative applications in the treatment of inflammatory bowel disease Fredrik EO Holmberg Fredrik EO Holmberg Department of Gastroenterology, Herlev Hospital, University of Copenhagen, Herlev, Denmark Search for more papers by this author Jakob B Seidelin Jakob B Seidelin Department of Gastroenterology, Herlev Hospital, University of Copenhagen, Herlev, Denmark Search for more papers by this author Xiaolei Yin Xiaolei Yin Division of BioEngineering in Medicine, Department of Medicine, Center for Regenerative Therapeutics, Brigham and Women's Hospital, Cambridge, MA, USA Harvard Medical School, Boston, MA, USA Harvard Stem Cell Institute, Cambridge, MA, USA Harvard - MIT Division of Health Sciences and Technology, Cambridge, MA, USA David H. Koch Institute for Integrative Cancer Research, MIT, Cambridge, MA, USA Search for more papers by this author Benjamin E Mead Benjamin E Mead Division of BioEngineering in Medicine, Department of Medicine, Center for Regenerative Therapeutics, Brigham and Women's Hospital, Cambridge, MA, USA Harvard Medical School, Boston, MA, USA Harvard Stem Cell Institute, Cambridge, MA, USA Harvard - MIT Division of Health Sciences and Technology, Cambridge, MA, USA David H. Koch Institute for Integrative Cancer Research, MIT, Cambridge, MA, USA Broad Institute of Harvard and MIT, Cambridge, MA, USA Search for more papers by this author Zhixiang Tong Zhixiang Tong Division of BioEngineering in Medicine, Department of Medicine, Center for Regenerative Therapeutics, Brigham and Women's Hospital, Cambridge, MA, USA Harvard Medical School, Boston, MA, USA Harvard Stem Cell Institute, Cambridge, MA, USA Harvard - MIT Division of Health Sciences and Technology, Cambridge, MA, USA Search for more papers by this author Yuan Li Yuan Li Department of Gastroenterology, Herlev Hospital, University of Copenhagen, Herlev, Denmark Search for more papers by this author Jeffrey M Karp Corresponding Author Jeffrey M Karp [email protected] Division of BioEngineering in Medicine, Department of Medicine, Center for Regenerative Therapeutics, Brigham and Women's Hospital, Cambridge, MA, USA Harvard Medical School, Boston, MA, USA Harvard Stem Cell Institute, Cambridge, MA, USA Harvard - MIT Division of Health Sciences and Technology, Cambridge, MA, USA David H. Koch Institute for Integrative Cancer Research, MIT, Cambridge, MA, USA Broad Institute of Harvard and MIT, Cambridge, MA, USA Search for more papers by this author Ole H Nielsen Corresponding Author Ole H Nielsen [email protected] orcid.org/0000-0003-4612-8635 Department of Gastroenterology, Herlev Hospital, University of Copenhagen, Herlev, Denmark Search for more papers by this author Fredrik EO Holmberg Fredrik EO Holmberg Department of Gastroenterology, Herlev Hospital, University of Copenhagen, Herlev, Denmark Search for more papers by this author Jakob B Seidelin Jakob B Seidelin Department of Gastroenterology, Herlev Hospital, University of Copenhagen, Herlev, Denmark Search for more papers by this author Xiaolei Yin Xiaolei Yin Division of BioEngineering in Medicine, Department of Medicine, Center for Regenerative Therapeutics, Brigham and Women's Hospital, Cambridge, MA, USA Harvard Medical School, Boston, MA, USA Harvard Stem Cell Institute, Cambridge, MA, USA Harvard - MIT Division of Health Sciences and Technology, Cambridge, MA, USA David H. Koch Institute for Integrative Cancer Research, MIT, Cambridge, MA, USA Search for more papers by this author Benjamin E Mead Benjamin E Mead Division of BioEngineering in Medicine, Department of Medicine, Center for Regenerative Therapeutics, Brigham and Women's Hospital, Cambridge, MA, USA Harvard Medical School, Boston, MA, USA Harvard Stem Cell Institute, Cambridge, MA, USA Harvard - MIT Division of Health Sciences and Technology, Cambridge, MA, USA David H. Koch Institute for Integrative Cancer Research, MIT, Cambridge, MA, USA Broad Institute of Harvard and MIT, Cambridge, MA, USA Search for more papers by this author Zhixiang Tong Zhixiang Tong Division of BioEngineering in Medicine, Department of Medicine, Center for Regenerative Therapeutics, Brigham and Women's Hospital, Cambridge, MA, USA Harvard Medical School, Boston, MA, USA Harvard Stem Cell Institute, Cambridge, MA, USA Harvard - MIT Division of Health Sciences and Technology, Cambridge, MA, USA Search for more papers by this author Yuan Li Yuan Li Department of Gastroenterology, Herlev Hospital, University of Copenhagen, Herlev, Denmark Search for more papers by this author Jeffrey M Karp Corresponding Author Jeffrey M Karp [email protected] Division of BioEngineering in Medicine, Department of Medicine, Center for Regenerative Therapeutics, Brigham and Women's Hospital, Cambridge, MA, USA Harvard Medical School, Boston, MA, USA Harvard Stem Cell Institute, Cambridge, MA, USA Harvard - MIT Division of Health Sciences and Technology, Cambridge, MA, USA David H. Koch Institute for Integrative Cancer Research, MIT, Cambridge, MA, USA Broad Institute of Harvard and MIT, Cambridge, MA, USA Search for more papers by this author Ole H Nielsen Corresponding Author Ole H Nielsen [email protected] orcid.org/0000-0003-4612-8635 Department of Gastroenterology, Herlev Hospital, University of Copenhagen, Herlev, Denmark Search for more papers by this author Author Information Fredrik EO Holmberg1, Jakob B Seidelin1, Xiaolei Yin2,3,4,5,6, Benjamin E Mead2,3,4,5,6,7, Zhixiang Tong2,3,4,5, Yuan Li1, Jeffrey M Karp *,2,3,4,5,6,7 and Ole H Nielsen *,1 1Department of Gastroenterology, Herlev Hospital, University of Copenhagen, Herlev, Denmark 2Division of BioEngineering in Medicine, Department of Medicine, Center for Regenerative Therapeutics, Brigham and Women's Hospital, Cambridge, MA, USA 3Harvard Medical School, Boston, MA, USA 4Harvard Stem Cell Institute, Cambridge, MA, USA 5Harvard - MIT Division of Health Sciences and Technology, Cambridge, MA, USA 6David H. Koch Institute for Integrative Cancer Research, MIT, Cambridge, MA, USA 7Broad Institute of Harvard and MIT, Cambridge, MA, USA *Corresponding author. Tel: +1 617 817 9174; E-mail: [email protected] *Corresponding author. Tel: +45 3868 3621; E-mail: [email protected] EMBO Mol Med (2017)9:558-570https://doi.org/10.15252/emmm.201607260 See the Glossary for abbreviations used in this article. PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Both the incidence and prevalence of inflammatory bowel disease (IBD) is increasing globally; in the industrialized world up to 0.5% of the population are affected and around 4.2 million individuals suffer from IBD in Europe and North America combined. Successful engraftment in experimental colitis models suggests that intestinal stem cell transplantation could constitute a novel treatment strategy to re-establish mucosal barrier function in patients with severe disease. Intestinal stem cells can be grown in vitro in organoid structures, though only a fraction of the cells contained are stem cells with regenerative capabilities. Hence, techniques to enrich stem cell populations are being pursued through the development of multiple two-dimensional and three-dimensional culture protocols, as well as co-culture techniques and multiple growth medium compositions. Moreover, research in support matrices allowing for efficient clinical application is in progress. In vitro culture is accomplished by modulating the signaling pathways fundamental for the stem cell niche with a suitable culture matrix to provide additional contact-dependent stimuli and structural support. The aim of this review was to discuss medium compositions and support matrices for optimal intestinal stem cell culture, as well as potential modifications to advance clinical use in IBD. Glossary Anoikis Dissociation-induced apoptosis occurring when anchorage-dependent cells, such as epithelial cells, detach from the underlying extracellular basement membrane. Cell–cell contact can sometimes prevent anoikis from occurring. Inflammatory bowel disease (IBD) A group of chronic remitting inflammatory conditions localized to the intestine, often debuting in adolescence. The two major subtypes are ulcerative colitis and Crohn's disease, but it also includes microscopic colitis and diversion colitis. Crohn's disease can affect segments of the entire gastrointestinal tract, while ulcerative colitis is restricted to the colon. Symptoms include abdominal pain, diarrhea, anemia, rectal bleeding, and weight loss. However, the condition is often complicated by extra-intestinal symptoms, commonly affecting skin, joints, or eyes. IBD is frequently treated with anti-inflammatory and immunomodulatory drugs, although surgical bowel resection may be required in severe disease. Intestinal organoid A three-dimensional organlike structure grown in vitro, consisting of intestinal epithelial cells. The nomenclature varies and is also referred to as a mini-gut. It has been suggested that the term organoid should be reserved for structures containing both epithelial and mesenchymal components. In turn, enteroids may be used for structures consisting solely of epithelial components. Intestinal stem cell niche A specific microenvironment which dynamically regulates stem cell renewal and differentiation. It consists of an intricate signaling system of chemical mediators and mechanical cues derived from epithelial and mesenchymal sources, as well as from the extracellular matrix. Introduction Inflammatory bowel disease (IBD) of which Crohn's disease (CD) and ulcerative colitis (UC) are the two most prevalent entities, constitute a chronic remitting disorder with increasing incidence worldwide, reported in the range of up to 50 per 100,000 in the Western population (Molodecky et al, 2012). IBD causes lifelong morbidity, including extra-intestinal complications (Larsen et al, 2010), and can greatly impair quality of life of affected individuals. It also constitutes a considerable economic burden for society in terms of direct medical costs (Burisch et al, 2013), and indirect costs arising from impaired work performance, including sick leave (Hoivik et al, 2013). Mucosal healing is associated with a more favorable prognosis for patients with IBD, including lower relapse and hospitalization rates, as well as a diminished risk for surgery (Peyrin-Biroulet et al, 2011; Shah et al, 2016). Successful transplantation of intestinal stem cells (ISCs), which are responsible for tissue homeostasis and injury response, has been achieved in murine models of experimental colitis, demonstrating that they adhere to and become an integrated part of the epithelium, thereby improving mucosal healing (Yui et al, 2012; Fordham et al, 2013; Fukuda et al, 2014). Hence, ISC transplantation might constitute an appealing therapeutic approach to re-establish the epithelial barrier in IBD. ISCs are located at the base of the intestinal crypts where they renew the epithelium through differentiation to multiple epithelial progenies (Bjerknes & Cheng, 2006), and drive mucosal regeneration. Several genes mark the ISC population, including LGR5 (Barker et al, 2007), olfactomedin 4 (OLFM4) (van der Flier et al, 2009a), and ASCL2 (van der Flier et al, 2009b). ISCs can be cultured in vitro, giving rise to three-dimensional self-organizing structures called organoids (Sato et al, 2009). Organoids resemble the intestinal epithelium in vivo, possessing crypt and villus domains that contain multiple epithelial cell types derived from the ISCs (Sato et al, 2011b). Since intestinal stemness is determined by extrinsic signals, multiple culture protocols exist to emulate the in vivo ISC niche, and to sustain them in vitro. Protocols for human cell culture are based on a coordinated stimulation of wingless-type mouse mammary tumor virus integration site (WNT) signaling, epidermal growth factor (EGF), as well as inhibition of bone morphogenic protein (BMP), transforming growth factor-β (TGF-β) signaling, and p38 signaling (Jung et al, 2011; Sato et al, 2011a). The primary distinguishing factors between protocols are the growth medium constituents and the support matrices applied, resulting in differences in cellular composition. Nevertheless, most culture protocols for human intestinal organoids are unable to efficiently increase the frequency of ISCs within organoid structures, as only a few percent of the cells contained are self-renewing and multipotent stem cells (Jung et al, 2011). This raises the need for devising improved culture techniques to yield a purer population of ISCs, applicable for clinical transplantation strategies. This review provides an updated overview of current growth protocols for human ISCs in vitro, seeking to pinpoint obstacles in stem cell enrichment and matrix support, which should be addressed to allow for regenerative application of ISCs in IBD. Growth medium The basal medium for culturing ISCs often contains Advanced Dulbecco's Modified Eagle Medium/F12, supplemented with Glutamax, B-27, N-2, HEPES, acetylcysteine, and penicillin/streptomycin, though human colonic organoids can be sustained without N-2 supplement (Fujii et al, 2015). It is also possible to replace B-27, N-2, and acetylcysteine with serum (Van Dussen et al, 2015), but this approach may pose other challenges for clinical applications, as discussed in the subsequent section. The basal medium prevents bacterial contamination and provides buffering capacity, necessary amino acids, vitamins, antioxidants, hormones as well as inorganic compounds. Apart from the basic components, the growth media applied may vary according to the type or composition of growth factors and small molecules, either in the form of conditioned media, or high-purity recombinant proteins. Frequently used growth media constituents, their working mechanisms and effects, as well as applications are summarized in Table 1. Table 1. Frequently used growth media constituents, their working mechanisms and effects, as well as applications Growth medium constituents Working mechanism in ISCs Effect on ISCs and application WNT3aa Activates canonical WNT signaling (Clevers & Nusse, 2012) Stimulates crypt cells proliferation and maintains the stem cell state (Clevers & Nusse, 2012; Farin et al, 2012; Krausova & Korinek, 2014) R-spondin 1a Augments WNT/β-catenin signaling (de Lau et al, 2014) Stimulates crypt cell proliferation and maintains stem cell state (Farin et al, 2012; Krausova & Korinek, 2014; de Lau et al, 2014) CHIR99021 Stimulates canonical WNT signaling (Yin et al, 2014) Stimulates stem cell proliferation and can be used in combination with VPA, when growing single mouse ISCs in absence of Paneth cells (Yin et al, 2014) Valproic acid Inhibits histone deacetylase and activates Notch signaling (Yin et al, 2014) Maintains proliferative crypts and blocks secretory differentiation (Sato et al, 2011b). Can be used in combination with CHIR99021 when growing single mouse ISCs in absence of Paneth cells (Yin et al, 2014) Noggina Inhibits BMP signaling (Haramis et al, 2004) Stimulates crypt formation (Haramis et al, 2004) Jagged-1 Activates Notch signaling (Sato et al, 2009) Maintains the stem cell state, and promotes proliferation, while blocking secretory differentiation, thereby maintaining proliferative crypts (Stanger et al, 2005; Van Dussen et al, 2012) Used in the early phase of single-cell cultures in absence of Notch signaling from adjacent supportive cells (Sato et al, 2009; Grabinger et al, 2014) EGFa Activates RAS/RAF/MEK/ERK signaling pathway (Suzuki et al, 2010; Date & Sato, 2015) Stimulates stem cell migration, proliferation, and inhibits apoptosis (Frey et al, 2004; Suzuki et al, 2010) PGE2 Enhances canonical WNT signaling (Buchanan & DuBois, 2006) Prevents anoikis as well as promotes stem cell survival and proliferation, thereby improving culture efficiency. Stimulates spheroid morphology (Cohn et al, 1997; Joseph et al, 2005) Nicotinamide Inhibits the activity of sirtuins (Denu, 2005) Improves ISC maintenance when cultured > 1 week (Sato et al, 2011a). Often used for long-term human intestinal organoid cultures (Sato et al, 2011a), but can be omitted (Fujii et al, 2015) Gastrin-17 Not decisively concluded Marginally increases culture efficiency (Sato et al, 2011a) A83-01 or SB431542a Inhibits TGF-β signaling (Sato et al, 2011a) Inhibits differentiation and allows human intestinal stem cell cultures to be sustained in the long term (Sato et al, 2011a) SB202190a Inhibits P38 MAPK (Sato et al, 2011a) Inhibits secretory differentiation, increases plating efficiency, and decreases degradation of the EGF receptor (Frey et al, 2006; Sato et al, 2011a; Date & Sato, 2015). Allows human intestinal stem cell cultures to be sustained in the long term (Sato et al, 2011a) Y-27632 or thiazovivin Inhibition of caspase-3 (Wu et al, 2015) Prevents anoikis after single-cell dissociation (Watanabe et al, 2007). Used in the early phase of single-cell cultures IL-22 JAK/STAT signaling (Lindemans et al, 2015) ISC proliferation and organoid growth. Can potentially further increase ISC expansion and make EGF redundant (Lindemans et al, 2015) a Mandatory growth medium components for long-term culturing human intestinal stem cells as organoids. WNT/R-spondin signaling WNT signaling plays a crucial role in tissue development and homeostasis, though over-activity is associated with tumorigenesis (Krausova & Korinek, 2014). Two primary branches of WNT signaling exist: canonical and non-canonical. Non-canonical signaling is implicated in the establishment of cell polarity and migration, as well as inflammation and cancer development (Kumawat & Gosens, 2016), and has been less implicated in sustaining ISCs. The canonical WNT pathway is β-catenin dependent, and it is best studied owing to its essential role in preserving the undifferentiated stem cell state and promoting proliferation (van de Wetering et al, 2002). The canonical WNT pathway is activated by binding of a WNT ligand to the Frizzled receptor and its co-receptor complex low-density lipoprotein receptor-related protein 5/6 (LRP5/6). This leads to stabilization of β-catenin that translocates to the nucleus where it interacts with T-cell factor/lymphoid enhancer factor (TCF/LEF), thereby activating downstream target genes such as c-MYC, Cyclin D1, and Axin2 (Mah et al, 2016). In the absence of WNT activation, β-catenin is subject to proteosomal degradation promoted by the Axin/APC/GSK3β complex-mediated phosphorylation. WNT signaling can in turn be augmented by binding of R-spondins (RSPOs) to the LGR5 receptor, which suppresses internalization and degradation of Frizzled by neutralizing transmembrane ligases RNF43/ZNRF3 (Li et al, 2012). Several other signaling pathways, for example, BMP, Notch, EGF, and prostaglandin E2 (PGE2), have been suggested to interact with the canonical WNT pathway as summarized in Fig 1. Figure 1. Suggested downstream effects of growth medium components on canonical WNT signalingActivation of the WNT pathway inhibits phosphorylation-induced degradation of β-catenin mediated by Axin/APC/GSK3β, which precipitates nuclear translocation of β-catenin and activation of target genes. BMP inhibition and EGF activation increase nuclear β-catenin levels, due to phosphorylation and inactivation of GSK3β or phosphorylation of β-catenin itself. Similarly, CHIR99021 can increase WNT signaling by inactivation of GSK3β. PGE2 can promote β-catenin stability through suppression of GSK3β, but perhaps also through interaction between PGE2-R subunits and Axin, activation of cAMP/PKA and PI3K/PIP3/AKT activity. SB202190 inhibits p38, thereby decreasing ligand-driven degradation of the EGF receptor. Delta like canonical Notch ligand 1/4 (DLL1/4) can activate membrane-bound Notch, and the adaptor protein NUMB can associate with unphosphorylated β-catenin, precipitating its lysosomal degradation, thereby dampening WNT activity. Download figure Download PowerPoint To culture human intestinal organoids, the growth medium needs to be supplemented with a WNT ligand, and conditioned medium is often applied. The use of conditioned media is generally more cost-effective than recombinant proteins, though conditioned media contains serum for the purpose of protein stabilization, and includes the inherent risk for xenogeneic and pathogenic contamination, although presumably quite small (Tekkatte et al, 2011). Serum also contains undefined components and demonstrates batch-to-batch variability that hampers standardization. Nonetheless, mesenchymal stem cells cultured in serum have already been used in human trials without issues (Panes et al, 2016). Nevertheless, serum substitutes have successfully been applied to circumvent potential issues when culturing human mesenchymal stem cells (Kim et al, 2013). Human recombinant WNT3a is commercially available, but substituting conditioned medium with recombinant WNT3a reduces the growth efficiency of intestinal organoids (Fujii et al, 2015). WNT proteins are palmitoylated, which is crucial for interactions with the Frizzled receptor, though this is difficult to express and to purify (Willert et al, 2003). Impurities can activate mediators of TGF-β and BMP signaling, which is undesirable when culturing ISCs (Carthy et al, 2016). Even though human high-purity recombinant WNT3a has become commercially available, it is unlikely to be a fitting substitute for WNT3a conditioned medium, since purified WNT proteins rapidly lose their biologic activity, presumably due to hydrophobic aggregation (Dhamdhere et al, 2014). However, it was recently shown that the serum glycoprotein afamin stabilizes WNT proteins by forming water-soluble complexes, thereby preventing aggregation while at the same time maintaining their biologic activity (Mihara et al, 2016). This is reflected in the EC50 value that is estimated to be 5–10 times lower for afamin/WNT3a versus purified WNT3a. Hence, afamin/WNT3a complex might be a better means to accomplish WNT activation in ISC-derived organoids for clinical applications. Small molecules such as the GSK3β inhibitor CHIR99021, which prevents β-catenin degradation, can further activate the WNT pathway (Yin et al, 2014). Augmentation of WNT signaling with RSPO1 is most commonly used, either in the form of conditioned media or as a recombinant protein, with similar efficacy in human organoid growth (Fujii et al, 2015). BMP and TGF-β signaling BMP signaling gradients promote spatially arranged differentiation of ISCs, in part by suppressing WNT signaling, thereby regulating the number of stem cells in vivo (He et al, 2007; Krausova & Korinek, 2014). BMP signaling is activated by ligand binding to a multi-component receptor complex and incorporates several complex pathways, for example, activation of the SMAD cascade (SMAD 1, 5, and 8), and MAPK, as well as positive regulation of PTEN (He et al, 2007; Katagiri & Watabe, 2016). In turn, PTEN negatively regulates the phosphatidylinositol 3-kinase (PI3K)/phosphatidylinositol triphosphate (PIP3)/AKT cascade, which has several downstream substrates, including GSK3β and β-catenin (He et al, 2007). Thus, AKT interacts with the canonical WNT pathway by increasing β-catenin levels in the nucleus due to phosphorylation and inactivation of GSK3β or phosphorylation of β-catenin itself (Fig 1). Hence, active BMP signaling suppresses the β-catenin/WNT pathway, thereby counteracting the proliferative effects of WNT activation. Noggin is a BMP antagonist, and as such, the addition of recombinant Noggin or conditioned medium, combined with exogenous WNT activation, leads to preservation and proliferation of ISCs. Without Noggin, intestinal organoids cannot be maintained in culture (Sato et al, 2009). The TGF-β pathway activates the SMAD 2/3 cascade, but it clearly demonstrates context dependency (Hata & Chen, 2016), and is capable of activating several other pathways, including the MAPK pathway. The exact mechanism of action in ISCs remains obscure, but TGF-β appears not to affect ISC proliferation, although it controls clone expansion and extinction, as well as modulates the differentiation of secretory lineage precursors (Fischer et al, 2016). TGF-β receptor inhibitors, like A83-01 or SB431542, increase plating efficiency and are necessary for long-term culture of intestinal organoids by maintaining the undifferentiated stem cell state (Sato et al, 2011a). EGF EGF is an important regulator of intestinal epithelial cell migration and proliferation (Suzuki et al, 2010). Binding of EGF to its receptor results in induction of tyrosine kinase activity, with subsequent activation of the RAS/RAF/MEK/ERK signaling as well as the PI3K/PIP3/AKT cascades, inducing organoid growth (Date & Sato, 2015). The PI3K/PIP3/AKT pathway overlaps with the EGF and the BMP pathways, and provides a link to the canonical WNT pathway, as shown in Fig 1. EGF in the form of recombinant protein is essential for culturing human intestinal organoids, and lack of EGF or addition of an inhibitor of the EGF receptor causes decreased organoid formation and survival (Matano et al, 2015). Yet, human intestinal organoids have been cultured without EGF when large amounts of serum were used, in the form of conditioned medium containing WNT, RSPO3, and Noggin (Van Dussen et al, 2015). EGF signaling in vivo is partly regulated by a negative feedback system, constituted by the p38 MAPK pathway that affects EGF receptor (Frey et al, 2006). This pathway regulates numerous cell responses, including inflammation, apoptosis, cell cycle, differentiation, proliferation, and tumorigenesis (Zarubin & Han, 2005). In the intestinal epithelium, p38 determines whether EGF stimulation results in migration or in proliferation (Frey et al, 2004). Pharmacological inhibition of p38 decreases ligand-driven degradation of the EGF receptor, without affecting its internalization (Frey et al, 2006), resulting in increased proliferation. Similarly, deletion of p38 in intestinal epithelial cells results in increased proliferation, but also in a decreased goblet cell differentiation (Otsuka et al, 2010). Hence, a p38 inhibitor, such as SB202190, should be added to the growth medium of intestinal organoids to stimulate proliferation and long-term maintenance of human ISCs. IGF-1 can, similarly to EGF, stimulate PI3K/PIP3/AKT and RAS/RAF/MEK/ERK signaling, resulting in growth of intestinal organoids. However, EGF tends to more efficiently induce budding, corresponding to crypt formation and organoid expansion (Reynolds et al, 2014). Notch signaling Notch is essential to maintain the ISC pool by controlling stem cell self-renewal, as well as the balance between absorptive and secretory cell lineage specification (Demitrack & Samuelson, 2016). Pathway inhibition reduces ISCs proliferation and induces secretory lineage differentiation, thereby diminishing the ISC population (van Es et al, 2005; Van Dussen et al, 2012). Conversely, activation of the Notch pathway maintains stem cell multipotency and promotes stem cell proliferation, while directing progenitors toward an absorptive, rather than a secretory fate (Stanger et al, 2005; Demitrack & Samuelson, 2016). When a Notch ligand binds to the receptor, the Notch intracellular domain (NICD) is separated through proteolytic cleavage, initiating nuclear translocation and activation of target genes (Date & Sato, 2015). However, in some cases, ligand binding is insufficient to cause cleavage and receptor activation. The process requires both ligand stabilization and mechanical force, inducing conformational changes of the receptor (Varnum-Finney et al, 2000; Musse et al, 2012). Thus, direct activation of Notch pathway using recombinant Notch ligand has shown limited success. Genetic activation of the Notch pathway in murine ISCs antagonizes and titrates canonical WNT signaling activity, thereby maintaining the stem cell state and balancing the differentiation process (Tian et al, 2015). Similarly, membrane-bound Notch and its adaptor protein NUMB in human embryonic stem cells and human colon cancer cells associate with unphosphorylated β-catenin, precipitating its lysosomal degradation (Kwon et al, 2011). The process appears to be independent of NICD, as depicted in Fig 1. When culturing and mechanically passaging intestinal organoids, Notch stimulation is supplied by adjacent supportive cells (Sasaki et al, 2016), hence further stimulation is likely redundant. However, when growing dissociated single ISCs attained through enzymatic organoid dissociation, Notch signaling should be stimulated. One common approach is to add Jagged-1 peptide to the support matrix for the first couple of days (Sato et al, 2009; Yin et al, 2014), although additional studies are required to demonstrate an increased efficacy. When growing pure murine stem cell cultures, Notch stimulation can be provided by exogenous supplementation of the histone deacetylase inhibitor; valproic acid (VPA) (Yin et al, 2014). In terms of clinical applications, VPA has the benefit of already being approved by both EMA and FDA for treatment of epilepsy and certain bipolar disorders, which might simplify the approval process for its application in clinical stem cell enrichment. Prostaglandin E2 The physiologically active lipid PGE2 is produced from arachidonic acid in cell membranes via the cyclooxygenase pathway and binds to a number of G-coupled cell receptors. PGE2 promotes ISC expansion and cell proliferation in vitr
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