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Culturing human intestinal stem cells for regenerative applications in the treatment of inflammatory bowel disease

Culturing human intestinal stem cells for regenerative applications in the treatment of... Review Culturing human intestinal stem cells for regenerative applications in the treatment of inflammatory bowel disease 1 1 2,3,4,5,6 2,3,4,5,6,7 Fredrik EO Holmberg , Jakob B Seidelin , Xiaolei Yin , Benjamin E Mead , 2,3,4,5 1 2,3,4,5,6,7,* 1,** Zhixiang Tong , Yuan Li , Jeffrey M Karp & Ole H Nielsen Abstract Introduction Both the incidence and prevalence of inflammatory bowel disease Inflammatory bowel disease (IBD) of which Crohn’s disease (CD) (IBD) is increasing globally; in the industrialized world up to 0.5% and ulcerative colitis (UC) are the two most prevalent entities, of the population are affected and around 4.2 million individuals constitute a chronic remitting disorder with increasing incidence suffer from IBD in Europe and North America combined. Successful worldwide, reported in the range of up to 50 per 100,000 in the engraftment in experimental colitis models suggests that intestinal Western population (Molodecky et al, 2012). IBD causes lifelong stem cell transplantation could constitute a novel treatment strat- morbidity, including extra-intestinal complications (Larsen et al, egy to re-establish mucosal barrier function in patients with 2010), and can greatly impair quality of life of affected individuals. severe disease. Intestinal stem cells can be grown in vitro in orga- It also constitutes a considerable economic burden for society in noid structures, though only a fraction of the cells contained are terms of direct medical costs (Burisch et al, 2013), and indirect costs stem cells with regenerative capabilities. Hence, techniques to arising from impaired work performance, including sick leave enrich stem cell populations are being pursued through the devel- (Hoivik et al, 2013). opment of multiple two-dimensional and three-dimensional Mucosal healing is associated with a more favorable prognosis culture protocols, as well as co-culture techniques and multiple for patients with IBD, including lower relapse and hospitalization growth medium compositions. Moreover, research in support rates, as well as a diminished risk for surgery (Peyrin-Biroulet et al, matrices allowing for efficient clinical application is in progress. In 2011; Shah et al, 2016). vitro culture is accomplished by modulating the signaling path- Successful transplantation of intestinal stem cells (ISCs), which ways fundamental for the stem cell niche with a suitable culture are responsible for tissue homeostasis and injury response, has been matrix to provide additional contact-dependent stimuli and struc- achieved in murine models of experimental colitis, demonstrating tural support. The aim of this review was to discuss medium that they adhere to and become an integrated part of the epithelium, compositions and support matrices for optimal intestinal stem cell thereby improving mucosal healing (Yui et al, 2012; Fordham et al, culture, as well as potential modifications to advance clinical use 2013; Fukuda et al, 2014). Hence, ISC transplantation might consti- in IBD. tute an appealing therapeutic approach to re-establish the epithelial barrier in IBD. Keywords inflammatory bowel disease; intestinal stem cells; organoids; ISCs are located at the base of the intestinal crypts where they regenerative medicine; support matrix renew the epithelium through differentiation to multiple epithelial DOI 10.15252/emmm.201607260 | Received 26 October 2016 | Revised 16 progenies (Bjerknes & Cheng, 2006), and drive mucosal regenera- January 2017 | Accepted 14 February 2017 | Published online 10 March 2017 tion. Several genes mark the ISC population, including LGR5 (Barker EMBO Mol Med (2017) 9: 558–570 et al, 2007), olfactomedin 4 (OLFM4) (van der Flier et al, 2009a), and ASCL2 (van der Flier et al, 2009b). See the Glossary for abbreviations used in this article. ISCs can be cultured in vitro, giving rise to three-dimensional self-organizing structures called organoids (Sato et al, 2009). 1 Department of Gastroenterology, Herlev Hospital, University of Copenhagen, Herlev, Denmark 2 Division of BioEngineering in Medicine, Department of Medicine, Center for Regenerative Therapeutics, Brigham and Women’s Hospital, Cambridge, MA, USA 3 Harvard Medical School, Boston, MA, USA 4 Harvard Stem Cell Institute, Cambridge, MA, USA 5 Harvard - MIT Division of Health Sciences and Technology, Cambridge, MA, USA 6 David H. Koch Institute for Integrative Cancer Research, MIT, Cambridge, MA, USA 7 Broad 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 Molecular Medicine Vol 9 |No 5 | 2017 ª 2017 The Authors. Published under the terms of the CC BY 4.0 license 558 Fredrik EO Holmberg et al Culturing ISCs for the treatment of IBD EMBO Molecular Medicine Growth medium Glossary Anoikis The basal medium for culturing ISCs often contains Advanced Dissociation-induced apoptosis occurring when anchorage-dependent Dulbecco’s Modified Eagle Medium/F12, supplemented with Gluta- cells, such as epithelial cells, detach from the underlying extracellular max, B-27, N-2, HEPES, acetylcysteine, and penicillin/streptomycin, basement membrane. Cell–cell contact can sometimes prevent anoikis from occurring. though human colonic organoids can be sustained without N-2 supplement (Fujii et al, 2015). It is also possible to replace B-27, Inflammatory bowel disease (IBD) A group of chronic remitting inflammatory conditions localized to the N-2, and acetylcysteine with serum (Van Dussen et al, 2015), but intestine, often debuting in adolescence. The two major subtypes are this approach may pose other challenges for clinical applications, as ulcerative colitis and Crohn’s disease, but it also includes microscopic discussed in the subsequent section. The basal medium prevents colitis and diversion colitis. Crohn’s disease can affect segments of the bacterial contamination and provides buffering capacity, necessary entire gastrointestinal tract, while ulcerative colitis is restricted to the amino acids, vitamins, antioxidants, hormones as well as inorganic colon. Symptoms include abdominal pain, diarrhea, anemia, rectal bleeding, and weight loss. However, the condition is often compounds. complicated by extra-intestinal symptoms, commonly affecting skin, Apart from the basic components, the growth media applied may joints, or eyes. IBD is frequently treated with anti-inflammatory and vary according to the type or composition of growth factors and immunomodulatory drugs, although surgical bowel resection may be small molecules, either in the form of conditioned media, or required in severe disease. high-purity recombinant proteins. Frequently used growth media Intestinal organoid constituents, their working mechanisms and effects, as well as A three-dimensional organlike structure grown in vitro, consisting of applications are summarized in Table 1. 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 WNT/R-spondin signaling structures consisting solely of epithelial components. Intestinal stem cell niche WNT signaling plays a crucial role in tissue development and home- A specific microenvironment which dynamically regulates stem cell ostasis, though over-activity is associated with tumorigenesis renewal and differentiation. It consists of an intricate signaling (Krausova & Korinek, 2014). system of chemical mediators and mechanical cues derived from Two primary branches of WNT signaling exist: canonical and epithelial and mesenchymal sources, as well as from the extracellular matrix. non-canonical. Non-canonical signaling is implicated in the estab- lishment 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 b-catenin dependent, and it is best studied owing to its essential role in preserving the undifferenti- Organoids resemble the intestinal epithelium in vivo, possessing ated stem cell state and promoting proliferation (van de Wetering crypt and villus domains that contain multiple epithelial cell types et al, 2002). The canonical WNT pathway is activated by binding of derived from the ISCs (Sato et al, 2011b). a WNT ligand to the Frizzled receptor and its co-receptor complex Since intestinal stemness is determined by extrinsic signals, low-density lipoprotein receptor-related protein 5/6 (LRP5/6). This multiple culture protocols exist to emulate the in vivo ISC niche, leads to stabilization of b-catenin that translocates to the nucleus and to sustain them in vitro. Protocols for human cell culture are where it interacts with T-cell factor/lymphoid enhancer factor (TCF/ based on a coordinated stimulation of wingless-type mouse LEF), thereby activating downstream target genes such as c-MYC, mammary tumor virus integration site (WNT) signaling, epider- Cyclin D1, and Axin2 (Mah et al, 2016). In the absence of WNT mal growth factor (EGF), as well as inhibition of bone morpho- activation, b-catenin is subject to proteosomal degradation genic protein (BMP), transforming growth factor-b (TGF-b) promoted by the Axin/APC/GSK3b complex-mediated phosphoryla- signaling, and p38 signaling (Jung et al, 2011; Sato et al, 2011a). tion. WNT signaling can in turn be augmented by binding of The primary distinguishing factors between protocols are the R-spondins (RSPOs) to the LGR5 receptor, which suppresses inter- growth medium constituents and the support matrices applied, nalization and degradation of Frizzled by neutralizing transmem- resulting in differences in cellular composition. Nevertheless, most brane ligases RNF43/ZNRF3 (Li et al, 2012). Several other signaling culture protocols for human intestinal organoids are unable to pathways, for example, BMP, Notch, EGF, and prostaglandin E efficiently increase the frequency of ISCs within organoid struc- (PGE ), have been suggested to interact with the canonical WNT tures, as only a few percent of the cells contained are self- pathway as summarized in Fig 1. renewing and multipotent stem cells (Jung et al, 2011). This To culture human intestinal organoids, the growth medium raises the need for devising improved culture techniques to yield needs to be supplemented with a WNT ligand, and conditioned a purer population of ISCs, applicable for clinical transplantation medium is often applied. The use of conditioned media is generally strategies. more cost-effective than recombinant proteins, though conditioned This review provides an updated overview of current growth media contains serum for the purpose of protein stabilization, protocols for human ISCs in vitro, seeking to pinpoint obstacles in and includes the inherent risk for xenogeneic and pathogenic stem cell enrichment and matrix support, which should be contamination, although presumably quite small (Tekkatte et al, addressed to allow for regenerative application of ISCs in IBD. 2011). Serum also contains undefined components and demonstrates ª 2017 The Authors EMBO Molecular Medicine Vol 9 |No 5 | 2017 559 EMBO Molecular Medicine Culturing ISCs for the treatment of IBD Fredrik EO Holmberg et al 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 WNT3a Activates canonical WNT signaling Stimulates crypt cells proliferation and maintains the stem cell state (Clevers & Nusse, 2012) (Clevers & Nusse, 2012; Farin et al, 2012; Krausova & Korinek, 2014) R-spondin 1 Augments WNT/b-catenin signaling Stimulates crypt cell proliferation and maintains stem cell state (de Lau et al, 2014) (Farin et al, 2012; Krausova & Korinek, 2014; de Lau et al, 2014) CHIR99021 Stimulates canonical WNT signaling Stimulates stem cell proliferation and can be used in combination with (Yin et al, 2014) VPA, when growing single mouse ISCs in absence of Paneth cells (Yin et al, 2014) Valproic acid Inhibits histone deacetylase and activates Maintains proliferative crypts and blocks secretory differentiation (Sato Notch signaling (Yin et al, 2014) et al, 2011b). Can be used in combination with CHIR99021 when growing single mouse ISCs in absence of Paneth cells (Yin et al, 2014) Noggin 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) EGF Activates RAS/RAF/MEK/ERK signaling pathway Stimulates stem cell migration, proliferation, and inhibits apoptosis (Suzuki et al, 2010; Date & Sato, 2015) (Frey et al, 2004; Suzuki et al, 2010) PGE Enhances canonical WNT signaling Prevents anoikis as well as promotes stem cell survival and proliferation, (Buchanan & DuBois, 2006) 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 SB431542 Inhibits TGF-b 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) SB202190 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) Mandatory growth medium components for long-term culturing human intestinal stem cells as organoids. batch-to-batch variability that hampers standardization. Nonethe- biologic activity, presumably due to hydrophobic aggregation less, mesenchymal stem cells cultured in serum have already been (Dhamdhere et al, 2014). However, it was recently shown that used in human trials without issues (Panes et al, 2016). Neverthe- the serum glycoprotein afamin stabilizes WNT proteins by form- less, serum substitutes have successfully been applied to circumvent ing water-soluble complexes, thereby preventing aggregation potential issues when culturing human mesenchymal stem cells while at the same time maintaining their biologic activity (Mihara (Kim et al, 2013). et al, 2016). This is reflected in the EC value that is estimated Human recombinant WNT3a is commercially available, but to be 5–10 times lower for afamin/WNT3a versus purified substituting conditioned medium with recombinant WNT3a WNT3a. Hence, afamin/WNT3a complex might be a better means reduces the growth efficiency of intestinal organoids (Fujii et al, to accomplish WNT activation in ISC-derived organoids for clini- 2015). WNT proteins are palmitoylated, which is crucial for inter- cal applications. actions with the Frizzled receptor, though this is difficult to Small molecules such as the GSK3b inhibitor CHIR99021, which express and to purify (Willert et al, 2003). Impurities can activate prevents b-catenin degradation, can further activate the WNT path- mediators of TGF-b and BMP signaling, which is undesirable way (Yin et al, 2014). when culturing ISCs (Carthy et al, 2016). Even though human Augmentation of WNT signaling with RSPO1 is most commonly high-purity recombinant WNT3a has become commercially avail- used, either in the form of conditioned media or as a recombinant able, it is unlikely to be a fitting substitute for WNT3a condi- protein, with similar efficacy in human organoid growth (Fujii et al, tioned medium, since purified WNT proteins rapidly lose their 2015). EMBO Molecular Medicine Vol 9 |No 5 | 2017 ª 2017 The Authors 560 Fredrik EO Holmberg et al Culturing ISCs for the treatment of IBD EMBO Molecular Medicine WNT3a R-spondin 1 CHIR Frizzled LGR5 ISC BMP Gα Axin APC MAPK cAMP GSK3β PGE -R BMP-R PGE Noggin Loss of phosphorylation- PTEN induced degradation PKA SB202190 PI3K / PIP / β-catenin AKT p38 Gβγ SMAD DLL1/4 Adjacent cascade supportive NUMB cells Lysosomal NICD degradation EGF Notch β-catenin Target NICD ERK MEK RAF RAS EGF-R transcription factors and Cytoplasm genes Figure 1. Suggested downstream effects of growth medium components on canonical WNT signaling. Activation of the WNT pathway inhibits phosphorylation-induced degradation of b-catenin mediated by Axin/APC/GSK3b, which precipitates nuclear translocation of b-catenin and activation of target genes. BMP inhibition and EGF activation increase nuclear b-catenin levels, due to phosphorylation and inactivation of GSK3b or phosphorylation of b-catenin itself. Similarly, CHIR99021 can increase WNT signaling by inactivation of GSK3b. PGE can promote b-catenin stability through suppression of GSK3b, but perhaps also through interaction between PGE -R subunits and Axin, activation of cAMP/PKA and PI3K/PIP /AKT activity. SB202190 inhibits p38, thereby decreasing 2 3 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 b-catenin, precipitating its lysosomal degradation, thereby dampening WNT activity. BMP and TGF-b signaling The TGF-b pathway activates the SMAD 2/3 cascade, but it clearly demonstrates context dependency (Hata & Chen, 2016), and BMP signaling gradients promote spatially arranged differentiation is capable of activating several other pathways, including the of ISCs, in part by suppressing WNT signaling, thereby regulating MAPK pathway. The exact mechanism of action in ISCs remains the number of stem cells in vivo (He et al, 2007; Krausova & obscure, but TGF-b appears not to affect ISC proliferation, although Korinek, 2014). it controls clone expansion and extinction, as well as modulates BMP signaling is activated by ligand binding to a multi-compo- the differentiation of secretory lineage precursors (Fischer et al, nent receptor complex and incorporates several complex pathways, 2016). for example, activation of the SMAD cascade (SMAD 1, 5, and 8), TGF-b receptor inhibitors, like A83-01 or SB431542, increase and MAPK, as well as positive regulation of PTEN (He et al, 2007; plating efficiency and are necessary for long-term culture of intesti- Katagiri & Watabe, 2016). In turn, PTEN negatively regulates the nal organoids by maintaining the undifferentiated stem cell state phosphatidylinositol 3-kinase (PI3K)/phosphatidylinositol triphos- (Sato et al, 2011a). phate (PIP )/AKT cascade, which has several downstream substrates, including GSK3b and b-catenin (He et al, 2007). Thus, AKT interacts with the canonical WNT pathway by increasing EGF b-catenin levels in the nucleus due to phosphorylation and inactiva- tion of GSK3b or phosphorylation of b-catenin itself (Fig 1). Hence, EGF is an important regulator of intestinal epithelial cell migration active BMP signaling suppresses the b-catenin/WNT pathway, and proliferation (Suzuki et al, 2010). Binding of EGF to its receptor thereby counteracting the proliferative effects of WNT activation. results in induction of tyrosine kinase activity, with subsequent acti- Noggin is a BMP antagonist, and as such, the addition of recom- vation of the RAS/RAF/MEK/ERK signaling as well as the PI3K/ binant Noggin or conditioned medium, combined with exogenous PIP /AKT cascades, inducing organoid growth (Date & Sato, 2015). WNT activation, leads to preservation and proliferation of ISCs. The PI3K/PIP /AKT pathway overlaps with the EGF and the BMP Without Noggin, intestinal organoids cannot be maintained in pathways, and provides a link to the canonical WNT pathway, as culture (Sato et al, 2009). shown in Fig 1. ª 2017 The Authors EMBO Molecular Medicine Vol 9 |No 5 | 2017 N EMBO Molecular Medicine Culturing ISCs for the treatment of IBD Fredrik EO Holmberg et al EGF in the form of recombinant protein is essential for culturing b-catenin, precipitating its lysosomal degradation (Kwon et al, human intestinal organoids, and lack of EGF or addition of an inhi- 2011). The process appears to be independent of NICD, as bitor of the EGF receptor causes decreased organoid formation and depicted in Fig 1. survival (Matano et al, 2015). Yet, human intestinal organoids have When culturing and mechanically passaging intestinal orga- been cultured without EGF when large amounts of serum were noids, Notch stimulation is supplied by adjacent supportive cells used, in the form of conditioned medium containing WNT, RSPO3, (Sasaki et al, 2016), hence further stimulation is likely redundant. and Noggin (Van Dussen et al, 2015). However, when growing dissociated single ISCs attained through EGF signaling in vivo is partly regulated by a negative feed- enzymatic organoid dissociation, Notch signaling should be stimu- back system, constituted by the p38 MAPK pathway that affects lated. One common approach is to add Jagged-1 peptide to the EGF receptor (Frey et al, 2006). This pathway regulates numerous support matrix for the first couple of days (Sato et al, 2009; Yin cell responses, including inflammation, apoptosis, cell cycle, et al, 2014), although additional studies are required to demon- differentiation, proliferation, and tumorigenesis (Zarubin & Han, strate an increased efficacy. When growing pure murine stem cell 2005). In the intestinal epithelium, p38 determines whether EGF cultures, Notch stimulation can be provided by exogenous supple- stimulation results in migration or in proliferation (Frey et al, mentation of the histone deacetylase inhibitor; valproic acid (VPA) 2004). Pharmacological inhibition of p38 decreases ligand-driven (Yin et al, 2014). In terms of clinical applications, VPA has the degradation of the EGF receptor, without affecting its internaliza- benefit of already being approved by both EMA and FDA for treat- tion (Frey et al, 2006), resulting in increased proliferation. Simi- ment of epilepsy and certain bipolar disorders, which might larly, deletion of p38 in intestinal epithelial cells results in simplify the approval process for its application in clinical stem increased proliferation, but also in a decreased goblet cell dif- cell enrichment. ferentiation (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 Prostaglandin E of human ISCs. IGF-1 can, similarly to EGF, stimulate PI3K/PIP /AKT and RAS/ The physiologically active lipid PGE is produced from arachidonic 3 2 RAF/MEK/ERK signaling, resulting in growth of intestinal orga- acid in cell membranes via the cyclooxygenase pathway and binds noids. However, EGF tends to more efficiently induce budding, to a number of G-coupled cell receptors. PGE promotes ISC expan- corresponding to crypt formation and organoid expansion (Reynolds sion and cell proliferation in vitro (Fan et al, 2014), inducing orga- et al, 2014). noid swelling and spheroid morphology rather than an organoid crypt structure (Fordham et al, 2013). The swelling was recently revealed to be caused by induction of anion and fluid secretion into Notch signaling the organoid lumen (Fujii et al, 2016). PGE upregulates several WNT target genes (Fan et al, 2014), Notch is essential to maintain the ISC pool by controlling stem cell which presumably explains its association with the development of self-renewal, as well as the balance between absorptive and secre- colorectal cancer (Buchanan & DuBois, 2006). It also appears to tory cell lineage specification (Demitrack & Samuelson, 2016). Path- suppress enterocyte differentiation and to promote repair of the way inhibition reduces ISCs proliferation and induces secretory intestinal epithelium (Miyoshi et al, 2017). lineage differentiation, thereby diminishing the ISC population (van Studies of ISCs (Miyoshi et al, 2017) and vertebrate Es et al, 2005; Van Dussen et al, 2012). Conversely, activation of hematopoietic stem cells (Goessling et al, 2009) have revealed the Notch pathway maintains stem cell multipotency and promotes that PGE signaling affects b-catenin stability through suppression stem cell proliferation, while directing progenitors toward an of GSK3b. Several other pathways are suggested to be involved, absorptive, rather than a secretory fate (Stanger et al, 2005; Demi- for example, interaction between PGE receptor-subunits and track & Samuelson, 2016). Axin, activation of cAMP/PKA as well as PI3K/PIP /AKT activity When a Notch ligand binds to the receptor, the Notch intra- (Fig 1) (Evans, 2009). Additionally, PGE upregulates LGR5 cellular domain (NICD) is separated through proteolytic cleavage, protein in human colorectal adenomas through a b-catenin inde- initiating nuclear translocation and activation of target genes pendent pathway, a central mechanism in colorectal tumorigene- (Date & Sato, 2015). However, in some cases, ligand binding is sis (Al-Kharusi et al, 2013). However, data attained from cancer insufficient to cause cleavage and receptor activation. The research cannot be extrapolated directly to normal ISCs, since process requires both ligand stabilization and mechanical force, cancer cells might contain mutations of the WNT or PGE path- inducing conformational changes of the receptor (Varnum-Finney ways. et al, 2000; Musse et al, 2012). Thus, direct activation of Notch PGE has the benefit of already being approved for clinical use pathway using recombinant Notch ligand has shown limited by both EMA and FDA for induction of labor. success. Genetic activation of the Notch pathway in murine ISCs antag- onizes and titrates canonical WNT signaling activity, thereby Other small molecules and cytokines affecting intestinal maintaining the stem cell state and balancing the differentiation stem cell maintenance process (Tian et al, 2015). Similarly, membrane-bound Notch and its adaptor protein NUMB in human embryonic stem cells and To sustain human ISCs, the vitamin nicotinamide is often added to human colon cancer cells associate with unphosphorylated the growth medium (Sato et al, 2011a). Nicotinamide impedes EMBO Molecular Medicine Vol 9 |No 5 | 2017 ª 2017 The Authors 562 Fredrik EO Holmberg et al Culturing ISCs for the treatment of IBD EMBO Molecular Medicine TM sirtuin activity involved in apoptosis, aging, differentiation, and Corning Matrigel Matrix and BD Matrigel Basement transcription regulation (Denu, 2005). However, nicotinamide can Membrane Matrix are the most extensively used three-dimensional be omitted without affecting long-term sustainability of human (3D) support matrices for culturing ISCs. The extensive usage of colonic stem cells (Fujii et al, 2015). Matrigel is attributed to its capacity to support long-term growth of When culturing dissociated single stem cells, the Rho-associated stem cells, while retaining the undifferentiated cell state (Hughes protein kinase (ROCK) inhibitors, Y-27632 (Watanabe et al, 2007) et al, 2010). It is a xenogeneic and proteinaceous matrix derived or thiazovivin (Wang et al, 2013), can be added to the growth from mouse sarcoma cells, mainly composed of laminin, collagen medium for the first few days to prevent anoikis. Research on IV, and entactin (Hughes et al, 2010). Its disadvantages include pluripotent stem cells has suggested that ROCK inhibitors suppress batch-to-batch variability, undefined composition, including varying caspase-dependent cell death (Wu et al, 2015). amounts of sarcoma-derived proteins, cytokines, and growth Amidated gastrin-17 is regularly used when culturing intestinal factors, along with the potential risk for pathogen transmission organoids, though it only marginally improves culture efficiency (Hughes et al, 2010). Such factors make Matrigel an ill-suited and it may therefore be omitted (Sato et al, 2011a). culture platform for clinical application. Therefore, considerable Addition of the cytokine interleukin 22 (IL-22) to the growth efforts have been made to identify well-defined matrices for both medium has shown to increase the proliferation of ISCs and to in vitro ISC expansion and their in vivo transplantation. cause EGF redundancy when culturing human intestinal orga- Collagen is an easily attainable connective tissue constituent, and noids (Lindemans et al, 2015). It activates STAT3, which causes common sources include fibroblasts cultured in vitro, as well as growth of human intestinal organoids independent of Paneth tissue extracts, such as human placenta. Different collagen formula- cells as well as both Notch and WNT signaling (Lindemans tions can be applied to sustain intestinal epithelial cell growth et al, 2015). in vitro (Ootani et al, 2009; Yui et al, 2012). However, reduced Many of the small molecules used to culture ISCs are available in budding has been reported when intestinal organoids are cultured high-purity formulations, though safety data are sparse, which could in support matrices rich in collagen (Pastula et al, 2016), potentially provide translational limitations. However, very low concentrations due to increased mechanical rigidity. Recently, human ISCs isolated of the small molecules are used in culture and can presumably be from small intestine were cultured to confluence on two-dimen- washed off prior to transplantation. sional (2D) monolayers on thin layers of bovine type I collagen and recombinant human laminin isotypes (Scott et al, 2016), although maintenance of the undifferentiated stem cell state was unclear at Culture matrices the protein level. Another possible approach could be to utilize allogenic or xeno- Cell–matrix interactions are implicated in numerous cell functions, geneic tissues as ECM (e.g., small intestinal submucosa or urinary including differentiation, anoikis, proliferation, and gene regulation bladder matrix), which already are being used to culture other cell (Berrier & Yamada, 2007). This is accomplished through a set of types in research and clinical settings. Tissues derived from natural membrane receptors, several of which are integrins (e.g., a2b1), sources are, however, restricted in their amplitude for modification, that anchor the cells to the intestinal basement membrane (Lussier with inconsistencies related to the health and age of the donors et al, 2000). Attachment to the intracellular cytoskeleton and activa- (Fitzpatrick & McDevitt, 2015). tion of signaling pathways are achieved through recruitment of Biologic matrices suffer the disadvantages of batch-to-batch vari- effector and adaptor proteins. This results in modification of anti- ability, relatively high manufacturing costs, limited scalability, and apoptotic pathways, gene expression, cell differentiation, prolifera- risk of pathogen contamination, motivating research on synthetic tion, and motility, as shown in Fig 2 (Lussier et al, 2000; Hofmann supportive matrices to overcome such issues. Synthetic matrices are et al, 2007). In the absence of cell–matrix anchorage or cell–cell chemically defined and malleable in terms of physiochemical and contact, epithelial cells undergo anoikis within hours (Hofmann mechanical properties (Tong et al, 2015). Multiple types of 2D et al, 2007). synthetic substrates have been used to culture hESCs. Further, Substantial efforts have been made to identify and optimize certain isoforms of laminin and vitronectin, fibronectin, as well as suitable matrices for stem cell cultures, particularly for culturing other xeno-free synthetic cell support matrices have successfully human pluripotent stem cells (hPSCs), which include induced been used to support hPSC (Villa-Diaz et al, 2013). Nevertheless, pluripotent stem cells (iPSCs) and human embryonic stem cells the conformation of vitronectin and laminin is sensitive to changes (hESCs). Different culture protocols and support matrices are in temperature and pH, which limits their potential for long-term detailed in Fig 3. usage (Tong et al, 2015). Initial extracellular matrices (ECM) for culturing hPSCs were 3D matrices, as opposed to 2D ones, provide more space for the produced by feeder layers of lethally irradiated fibroblasts in cells to grow, thereby reducing disadvantageous cell clustering (Lei enriched culture medium. Similarly, human colonic stem cells were et al, 2014). Furthermore, they efficiently provide physical and recently successfully cultured on feeder layers of irradiated mouse chemical gradients of importance for numerous cell functions, embryonic fibroblasts over a Matrigel coating (Wang et al, 2015). including differentiation and proliferation (Sant et al, 2010; Tong Variability when using feeder layers, along with the prospect of et al, 2015). Although simple collagen I 3D hydrogel matrices denaturing or degrading peptides and proteins with sterilization support ISCs, they have the disadvantage of low stiffness, limited techniques, as well as the potential risk for pathogen and xeno- long-term stability, and batch-to-batch variability (Caliari & Burdick, geneic transmission has led to the establishment of feeder-free 2016). 3D gels with compositions closer to the supportive matrix culture systems (Villa-Diaz et al, 2013). found in vivo or even mimicking axial gradients in connective tissue ª 2017 The Authors EMBO Molecular Medicine Vol 9 |No 5 | 2017 563 EMBO Molecular Medicine Culturing ISCs for the treatment of IBD Fredrik EO Holmberg et al PROLIFERATION SURVIVAL DIFFERENTIATION Effector and GENE EXPRESSION adaptor proteins e.g. β-actin, non-receptor Cadherins tyrosine kinase MOTILITY e.g. E-cadherin Actin filaments Actin filaments Effector and adaptor proteins e.g. α-actin, talin, non-receptor tyrosine kinase Integrins e.g. α2β1 αβ Basement membrane B Basement membrane asement membrane Pr P Pr roteoglucans oteoglycans oteoglucans F Fibr Fibr ibro onectin onectin nectin Collagen IV Collagen IV Collagen IV Laminin Laminin Laminin Figure 2. Cell–cell and cell–matrix interactions. Physical interactions between the intestinal epithelium, adjacent cells, and the ECM provide pivotal signals for cell survival, proliferation, gene expression, differentiation, and motility. Adhesion molecules, such as integrins (e.g., a2b1) and cadherins (e.g., E-cadherin) that attach to adjacent cells as well as to ECM proteins, mediate this. Adaptor and effector proteins provide linkage to intracellular actin filaments and can activate several signaling pathways, including non-receptor tyrosine kinases. composition might be expected to provide improved viability and However, ISC transplantation constitutes a plausible alternative function of the cultured ISCs. Interestingly, fundamental ECM approach to accelerate mucosal healing. In fact, EGF is an effective factors, such as mechanical properties and biochemical signals that treatment option for certain subtypes of IBD, possibly through its regulate ISC colony and organoid formation, have recently been regenerative capabilities (Sinha et al, 2003). A schematic of the identified (Gjorevski et al, 2016). The efforts resulted in the forma- envisioned process, from harvesting of the ISCs to transplantation, tion of a mechanically dynamic polyethylene glycol (PEG) hydrogel, is depicted in Fig 4. functionalized with RGD (Arg-Gly-Asp) peptides and controlled Autologous transplantation may be performed in order to avoid degradation kinetics, capable of expanding human small intestine the process of finding suitable cell donors, as well as circumventing and colorectal cancer organoids (Gjorevski et al, 2016). Hence, the risk for tissue rejection and the need for further immunomodula- minimal PEG-based hydrogels constitute a well-defined alternative tory therapy due to the procedure itself. ISCs would be harvested that might be applied to overcome the limitations of Matrigel in endoscopically from IBD patients with frequent and severe relapses terms of clinical application of ISCs. during periods with remission, and then expanded and kept frozen until needed. An alternative approach could be to harvest ISCs from non-inflamed areas during flares, or alternatively even from actively Regenerative applications in IBD inflamed areas. However, excessive epithelial cell death can be observed in areas of active disease (Blander, 2016). Also, colonic Introduction of biologics like monoclonal antibodies against tumor organoids derived from patients with flaring UC have in vitro been necrosis alpha (TNF inhibitors) later followed by a4b7 anti-integrins shown to maintain an altered expression of genes associated with has revolutionized the management of IBD. However, despite these antimicrobial defense, absorptive and secretory functions, compared therapeutic advances about one-third of patients with CD and one- to healthy controls (Dotti et al, 2016). Additionally, lasting tran- sixth of patients with UC still require surgical bowel resection within scriptional changes in the affected epithelium have been observed in 5 years after diagnosis (Frolkis et al, 2013). patients with UC despite remission (Planell et al, 2013). Although Much like the majority of other medical therapies for IBD, TNF the consequences of such changes are unknown, for the purpose of inhibitors and anti-integrins act through immunomodulation. transplantation it might be better to harvest ISCs from non-involved EMBO Molecular Medicine Vol 9 |No 5 | 2017 ª 2017 The Authors 564 Fredrik EO Holmberg et al Culturing ISCs for the treatment of IBD EMBO Molecular Medicine Conditioned medium � WNT3a Feeder–layer co-culture Substrates of synthetic polymers � RSPO1 � MEF or ECM-derived substrates � ISEMF 2D � Collagen I � Laminin Gelatinous matrix Co-culture: Gelatinous matrix � Matrigel over a feeder layer 3D � Collagen I � Myofibroblasts Well defined components � Synthetic hydrogels � Noggin � A83-01 � EGF � SB202190 � PGE � Y-27632 � CHIR99021 � IL-22 � VPA � Nicotinamide Figure 3. Culture protocols for ISCs. Culture protocols for ISCs generally consist of two basic components: a support matrix and a growth medium. The support component can be in either 2Dor 3D. 2D matrices are usually derived from feeder cells such as mouse embryonic fibroblasts (MEF), synthetic substrates or from ECM-derived proteins, for example, collagen and laminin. 3D support matrices are usually in the form of gelatinous matrices, for example, Matrigel, collagen I, or synthetic hydrogels. Another approach is to use 3D co-cultures, consisting of a gelatinous matrix over a feeder-layer, for example, myofibroblasts. The growth medium often includes a conditioned medium, such as WNT3a, RSPO1, or intestinal subepithelial myofibroblasts (ISEMF), as well as fully defined growth factors, small molecules, and cytokines, for example, Noggin, EGF, nicotinamide, A83-01,SB202190, PGE , CHIR99021, VPA, and IL-22. epithelium. Another argument for this approach is that patients with could be an alternative route of administration—although the large IBD have ~1.5- to twofold increased risk of developing colorectal volume needed in the latter case would greatly increase the need for cancer (Beaugerie & Itzkowitz, 2015), presumably due to prolonged cell expansion in vitro. Regardless of which method is chosen, a suit- and remitting inflammation. When intestinal epithelial cells are able delivery vehicle will be required to protect and sustain the cells in harvested endoscopically, relatively few clones are afterward transit. Ideally, this should be fully defined and biocompatible, while enriched in vitro. If these cells were to contain genetic mutations allowing for in situ crosslinking and mucosal adhesion. that predispose to malignancy, then transplantation might lead to ISC transplantation may be able to spur the epithelial healing risk of malignant transformation in a greater area of the intestine process, but for a majority of patients, it is unlikely that this would after engraftment. This important issue could, however, be be successful as a monotherapy, as cells presumably will have diffi- addressed by screening for mutations known to be associated with culties engrafting during ongoing inflammation. Hence, concomitant colorectal cancer. immunomodulatory therapy will likely be needed to give the trans- More than 160 susceptibility genes predisposing to IBD so far planted cells optimal conditions to re-establish barrier integrity. have been identified, including inflammatory bowel disease 5 (IBD5) A crucial aspect of all cell-based treatment strategies is to avoid and cadherin 1 (CDH1) that are associated with epithelial barrier inducing chromosomal changes that could lead to malignant trans- function (Miner-Williams & Moughan, 2016). Genetic susceptibility formation or other cell abnormalities. Epithelial stem cells grown does not automatically lead to development of IBD, but transplanta- in vitro can acquire a specific single nucleotide variant (SNV) signa- tion of cells with a genetic susceptibility may potentially have impli- ture differing from the somatic SNV signature seen in vivo in mice cations on epithelial function even after a successful transplantation. (Behjati et al, 2014). Long-term cultivation of human ISCs has Clearly however, more research on this matter is warranted. revealed a low level of genomic instability with a limited copy ISCs should be transplanted as complete organoids or as small cell number and SNV instability for the first 100 days of continuous clusters with intact endogenous Notch stimulation to maintain stem- proliferation (Wang et al, 2015). Yet, a trend toward increasing SNV ness and delay anoikis. Successful engraftment would most likely was observed as a function of passage number, but not involving require integrin activation to accomplish adherence to the ECM of the reported driver genes in human cancer. However, a forthright chro- damaged mucosa, which in turn depends on extracellular divalent mosomal trisomy was noted after 200 days. It is possible that genetic cations (Berrier & Yamada, 2007). In terms of delivery, endoscopic changes acquired in vitro could increase the risk of introducing new transplantation would intuitively be the simplest method, but enema mutations to the recipient of transplanted cells and could potentially ª 2017 The Authors EMBO Molecular Medicine Vol 9 |No 5 | 2017 565 EMBO Molecular Medicine Culturing ISCs for the treatment of IBD Fredrik EO Holmberg et al Intestinal biopsies are harvested endoscopically Intestinal crypts are Culture system isolated is established Organoids are formed Stem cell + LGR5 transplantation stem cells Intestinal stem cells are enriched as organoids LGR5 stem cells Figure 4. The course of human ISC harvesting to transplantation. Human intestinal epithelium can be harvested endoscopically, and ISCs can subsequently be isolated and enriched in vitro as organoids. Organoids enriched in stem cells can then be transplanted back to the patient (or as a suspension of purified stem cells), thereby hopefully promoting mucosal healing. increase the risk of neoplasia. Propagation of a sufficient amount of for use in humans, preferably constituents that are already ISCs for regenerative application would, however, presumably approved by FDA and EMA, or which demonstrate minimal or no require reasonably short culture duration. Accordingly, the risk of toxicity. alterations in SNV signature and copy number could be minimized. Cell–cell and cell–matrix interactions have profound effects on cell phenotype and survival. The continuous development of alter- native synthetic support matrices for ISCs is promising in terms of Future perspectives creating a suitable and indispensable substitute for Matrigel. The number of ISCs attained in vitro is commonly estimated by Alternative growth media compositions and culture protocols to determining LGR5 expression levels. Yet, gene expression does not increase the ISC yield are continuously being explored to allow necessarily correlate to equivalent increases on protein level, and an for successful regenerative applications. This includes growth increase in gene expression may reflect gene upregulation rather factors and small molecules that target the WNT pathway, such than increase in stem cell numbers. It would therefore be imperative as PGE and CHIR99021, along with newly identified pathway to standardize how stem cell amplification is quantified in vitro. targets such as IL-22 and STAT3. Advancing regenerative applica- Modest quantities of LGR5 on the cell surface, along with the lack of tions of ISCs requires additional investigation to identify compo- selective antibodies with high affinity for human LGR5, hinder the nents affecting WNT, Notch, EGF, and BMP signaling that are apt effective quantification of ISC expansion. Still, the use of antibodies EMBO Molecular Medicine Vol 9 |No 5 | 2017 ª 2017 The Authors 566 Fredrik EO Holmberg et al Culturing ISCs for the treatment of IBD EMBO Molecular Medicine Beaugerie L, Itzkowitz SH (2015) Cancers complicating inflammatory bowel Pending issues disease. 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Culturing human intestinal stem cells for regenerative applications in the treatment of inflammatory bowel disease

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Abstract

Review Culturing human intestinal stem cells for regenerative applications in the treatment of inflammatory bowel disease 1 1 2,3,4,5,6 2,3,4,5,6,7 Fredrik EO Holmberg , Jakob B Seidelin , Xiaolei Yin , Benjamin E Mead , 2,3,4,5 1 2,3,4,5,6,7,* 1,** Zhixiang Tong , Yuan Li , Jeffrey M Karp & Ole H Nielsen Abstract Introduction Both the incidence and prevalence of inflammatory bowel disease Inflammatory bowel disease (IBD) of which Crohn’s disease (CD) (IBD) is increasing globally; in the industrialized world up to 0.5% and ulcerative colitis (UC) are the two most prevalent entities, of the population are affected and around 4.2 million individuals constitute a chronic remitting disorder with increasing incidence suffer from IBD in Europe and North America combined. Successful worldwide, reported in the range of up to 50 per 100,000 in the engraftment in experimental colitis models suggests that intestinal Western population (Molodecky et al, 2012). IBD causes lifelong stem cell transplantation could constitute a novel treatment strat- morbidity, including extra-intestinal complications (Larsen et al, egy to re-establish mucosal barrier function in patients with 2010), and can greatly impair quality of life of affected individuals. severe disease. Intestinal stem cells can be grown in vitro in orga- It also constitutes a considerable economic burden for society in noid structures, though only a fraction of the cells contained are terms of direct medical costs (Burisch et al, 2013), and indirect costs stem cells with regenerative capabilities. Hence, techniques to arising from impaired work performance, including sick leave enrich stem cell populations are being pursued through the devel- (Hoivik et al, 2013). opment of multiple two-dimensional and three-dimensional Mucosal healing is associated with a more favorable prognosis culture protocols, as well as co-culture techniques and multiple for patients with IBD, including lower relapse and hospitalization growth medium compositions. Moreover, research in support rates, as well as a diminished risk for surgery (Peyrin-Biroulet et al, matrices allowing for efficient clinical application is in progress. In 2011; Shah et al, 2016). vitro culture is accomplished by modulating the signaling path- Successful transplantation of intestinal stem cells (ISCs), which ways fundamental for the stem cell niche with a suitable culture are responsible for tissue homeostasis and injury response, has been matrix to provide additional contact-dependent stimuli and struc- achieved in murine models of experimental colitis, demonstrating tural support. The aim of this review was to discuss medium that they adhere to and become an integrated part of the epithelium, compositions and support matrices for optimal intestinal stem cell thereby improving mucosal healing (Yui et al, 2012; Fordham et al, culture, as well as potential modifications to advance clinical use 2013; Fukuda et al, 2014). Hence, ISC transplantation might consti- in IBD. tute an appealing therapeutic approach to re-establish the epithelial barrier in IBD. Keywords inflammatory bowel disease; intestinal stem cells; organoids; ISCs are located at the base of the intestinal crypts where they regenerative medicine; support matrix renew the epithelium through differentiation to multiple epithelial DOI 10.15252/emmm.201607260 | Received 26 October 2016 | Revised 16 progenies (Bjerknes & Cheng, 2006), and drive mucosal regenera- January 2017 | Accepted 14 February 2017 | Published online 10 March 2017 tion. Several genes mark the ISC population, including LGR5 (Barker EMBO Mol Med (2017) 9: 558–570 et al, 2007), olfactomedin 4 (OLFM4) (van der Flier et al, 2009a), and ASCL2 (van der Flier et al, 2009b). See the Glossary for abbreviations used in this article. ISCs can be cultured in vitro, giving rise to three-dimensional self-organizing structures called organoids (Sato et al, 2009). 1 Department of Gastroenterology, Herlev Hospital, University of Copenhagen, Herlev, Denmark 2 Division of BioEngineering in Medicine, Department of Medicine, Center for Regenerative Therapeutics, Brigham and Women’s Hospital, Cambridge, MA, USA 3 Harvard Medical School, Boston, MA, USA 4 Harvard Stem Cell Institute, Cambridge, MA, USA 5 Harvard - MIT Division of Health Sciences and Technology, Cambridge, MA, USA 6 David H. Koch Institute for Integrative Cancer Research, MIT, Cambridge, MA, USA 7 Broad 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 Molecular Medicine Vol 9 |No 5 | 2017 ª 2017 The Authors. Published under the terms of the CC BY 4.0 license 558 Fredrik EO Holmberg et al Culturing ISCs for the treatment of IBD EMBO Molecular Medicine Growth medium Glossary Anoikis The basal medium for culturing ISCs often contains Advanced Dissociation-induced apoptosis occurring when anchorage-dependent Dulbecco’s Modified Eagle Medium/F12, supplemented with Gluta- cells, such as epithelial cells, detach from the underlying extracellular max, B-27, N-2, HEPES, acetylcysteine, and penicillin/streptomycin, basement membrane. Cell–cell contact can sometimes prevent anoikis from occurring. though human colonic organoids can be sustained without N-2 supplement (Fujii et al, 2015). It is also possible to replace B-27, Inflammatory bowel disease (IBD) A group of chronic remitting inflammatory conditions localized to the N-2, and acetylcysteine with serum (Van Dussen et al, 2015), but intestine, often debuting in adolescence. The two major subtypes are this approach may pose other challenges for clinical applications, as ulcerative colitis and Crohn’s disease, but it also includes microscopic discussed in the subsequent section. The basal medium prevents colitis and diversion colitis. Crohn’s disease can affect segments of the bacterial contamination and provides buffering capacity, necessary entire gastrointestinal tract, while ulcerative colitis is restricted to the amino acids, vitamins, antioxidants, hormones as well as inorganic colon. Symptoms include abdominal pain, diarrhea, anemia, rectal bleeding, and weight loss. However, the condition is often compounds. complicated by extra-intestinal symptoms, commonly affecting skin, Apart from the basic components, the growth media applied may joints, or eyes. IBD is frequently treated with anti-inflammatory and vary according to the type or composition of growth factors and immunomodulatory drugs, although surgical bowel resection may be small molecules, either in the form of conditioned media, or required in severe disease. high-purity recombinant proteins. Frequently used growth media Intestinal organoid constituents, their working mechanisms and effects, as well as A three-dimensional organlike structure grown in vitro, consisting of applications are summarized in Table 1. 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 WNT/R-spondin signaling structures consisting solely of epithelial components. Intestinal stem cell niche WNT signaling plays a crucial role in tissue development and home- A specific microenvironment which dynamically regulates stem cell ostasis, though over-activity is associated with tumorigenesis renewal and differentiation. It consists of an intricate signaling (Krausova & Korinek, 2014). system of chemical mediators and mechanical cues derived from Two primary branches of WNT signaling exist: canonical and epithelial and mesenchymal sources, as well as from the extracellular matrix. non-canonical. Non-canonical signaling is implicated in the estab- lishment 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 b-catenin dependent, and it is best studied owing to its essential role in preserving the undifferenti- Organoids resemble the intestinal epithelium in vivo, possessing ated stem cell state and promoting proliferation (van de Wetering crypt and villus domains that contain multiple epithelial cell types et al, 2002). The canonical WNT pathway is activated by binding of derived from the ISCs (Sato et al, 2011b). a WNT ligand to the Frizzled receptor and its co-receptor complex Since intestinal stemness is determined by extrinsic signals, low-density lipoprotein receptor-related protein 5/6 (LRP5/6). This multiple culture protocols exist to emulate the in vivo ISC niche, leads to stabilization of b-catenin that translocates to the nucleus and to sustain them in vitro. Protocols for human cell culture are where it interacts with T-cell factor/lymphoid enhancer factor (TCF/ based on a coordinated stimulation of wingless-type mouse LEF), thereby activating downstream target genes such as c-MYC, mammary tumor virus integration site (WNT) signaling, epider- Cyclin D1, and Axin2 (Mah et al, 2016). In the absence of WNT mal growth factor (EGF), as well as inhibition of bone morpho- activation, b-catenin is subject to proteosomal degradation genic protein (BMP), transforming growth factor-b (TGF-b) promoted by the Axin/APC/GSK3b complex-mediated phosphoryla- signaling, and p38 signaling (Jung et al, 2011; Sato et al, 2011a). tion. WNT signaling can in turn be augmented by binding of The primary distinguishing factors between protocols are the R-spondins (RSPOs) to the LGR5 receptor, which suppresses inter- growth medium constituents and the support matrices applied, nalization and degradation of Frizzled by neutralizing transmem- resulting in differences in cellular composition. Nevertheless, most brane ligases RNF43/ZNRF3 (Li et al, 2012). Several other signaling culture protocols for human intestinal organoids are unable to pathways, for example, BMP, Notch, EGF, and prostaglandin E efficiently increase the frequency of ISCs within organoid struc- (PGE ), have been suggested to interact with the canonical WNT tures, as only a few percent of the cells contained are self- pathway as summarized in Fig 1. renewing and multipotent stem cells (Jung et al, 2011). This To culture human intestinal organoids, the growth medium raises the need for devising improved culture techniques to yield needs to be supplemented with a WNT ligand, and conditioned a purer population of ISCs, applicable for clinical transplantation medium is often applied. The use of conditioned media is generally strategies. more cost-effective than recombinant proteins, though conditioned This review provides an updated overview of current growth media contains serum for the purpose of protein stabilization, protocols for human ISCs in vitro, seeking to pinpoint obstacles in and includes the inherent risk for xenogeneic and pathogenic stem cell enrichment and matrix support, which should be contamination, although presumably quite small (Tekkatte et al, addressed to allow for regenerative application of ISCs in IBD. 2011). Serum also contains undefined components and demonstrates ª 2017 The Authors EMBO Molecular Medicine Vol 9 |No 5 | 2017 559 EMBO Molecular Medicine Culturing ISCs for the treatment of IBD Fredrik EO Holmberg et al 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 WNT3a Activates canonical WNT signaling Stimulates crypt cells proliferation and maintains the stem cell state (Clevers & Nusse, 2012) (Clevers & Nusse, 2012; Farin et al, 2012; Krausova & Korinek, 2014) R-spondin 1 Augments WNT/b-catenin signaling Stimulates crypt cell proliferation and maintains stem cell state (de Lau et al, 2014) (Farin et al, 2012; Krausova & Korinek, 2014; de Lau et al, 2014) CHIR99021 Stimulates canonical WNT signaling Stimulates stem cell proliferation and can be used in combination with (Yin et al, 2014) VPA, when growing single mouse ISCs in absence of Paneth cells (Yin et al, 2014) Valproic acid Inhibits histone deacetylase and activates Maintains proliferative crypts and blocks secretory differentiation (Sato Notch signaling (Yin et al, 2014) et al, 2011b). Can be used in combination with CHIR99021 when growing single mouse ISCs in absence of Paneth cells (Yin et al, 2014) Noggin 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) EGF Activates RAS/RAF/MEK/ERK signaling pathway Stimulates stem cell migration, proliferation, and inhibits apoptosis (Suzuki et al, 2010; Date & Sato, 2015) (Frey et al, 2004; Suzuki et al, 2010) PGE Enhances canonical WNT signaling Prevents anoikis as well as promotes stem cell survival and proliferation, (Buchanan & DuBois, 2006) 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 SB431542 Inhibits TGF-b 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) SB202190 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) Mandatory growth medium components for long-term culturing human intestinal stem cells as organoids. batch-to-batch variability that hampers standardization. Nonethe- biologic activity, presumably due to hydrophobic aggregation less, mesenchymal stem cells cultured in serum have already been (Dhamdhere et al, 2014). However, it was recently shown that used in human trials without issues (Panes et al, 2016). Neverthe- the serum glycoprotein afamin stabilizes WNT proteins by form- less, serum substitutes have successfully been applied to circumvent ing water-soluble complexes, thereby preventing aggregation potential issues when culturing human mesenchymal stem cells while at the same time maintaining their biologic activity (Mihara (Kim et al, 2013). et al, 2016). This is reflected in the EC value that is estimated Human recombinant WNT3a is commercially available, but to be 5–10 times lower for afamin/WNT3a versus purified substituting conditioned medium with recombinant WNT3a WNT3a. Hence, afamin/WNT3a complex might be a better means reduces the growth efficiency of intestinal organoids (Fujii et al, to accomplish WNT activation in ISC-derived organoids for clini- 2015). WNT proteins are palmitoylated, which is crucial for inter- cal applications. actions with the Frizzled receptor, though this is difficult to Small molecules such as the GSK3b inhibitor CHIR99021, which express and to purify (Willert et al, 2003). Impurities can activate prevents b-catenin degradation, can further activate the WNT path- mediators of TGF-b and BMP signaling, which is undesirable way (Yin et al, 2014). when culturing ISCs (Carthy et al, 2016). Even though human Augmentation of WNT signaling with RSPO1 is most commonly high-purity recombinant WNT3a has become commercially avail- used, either in the form of conditioned media or as a recombinant able, it is unlikely to be a fitting substitute for WNT3a condi- protein, with similar efficacy in human organoid growth (Fujii et al, tioned medium, since purified WNT proteins rapidly lose their 2015). EMBO Molecular Medicine Vol 9 |No 5 | 2017 ª 2017 The Authors 560 Fredrik EO Holmberg et al Culturing ISCs for the treatment of IBD EMBO Molecular Medicine WNT3a R-spondin 1 CHIR Frizzled LGR5 ISC BMP Gα Axin APC MAPK cAMP GSK3β PGE -R BMP-R PGE Noggin Loss of phosphorylation- PTEN induced degradation PKA SB202190 PI3K / PIP / β-catenin AKT p38 Gβγ SMAD DLL1/4 Adjacent cascade supportive NUMB cells Lysosomal NICD degradation EGF Notch β-catenin Target NICD ERK MEK RAF RAS EGF-R transcription factors and Cytoplasm genes Figure 1. Suggested downstream effects of growth medium components on canonical WNT signaling. Activation of the WNT pathway inhibits phosphorylation-induced degradation of b-catenin mediated by Axin/APC/GSK3b, which precipitates nuclear translocation of b-catenin and activation of target genes. BMP inhibition and EGF activation increase nuclear b-catenin levels, due to phosphorylation and inactivation of GSK3b or phosphorylation of b-catenin itself. Similarly, CHIR99021 can increase WNT signaling by inactivation of GSK3b. PGE can promote b-catenin stability through suppression of GSK3b, but perhaps also through interaction between PGE -R subunits and Axin, activation of cAMP/PKA and PI3K/PIP /AKT activity. SB202190 inhibits p38, thereby decreasing 2 3 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 b-catenin, precipitating its lysosomal degradation, thereby dampening WNT activity. BMP and TGF-b signaling The TGF-b pathway activates the SMAD 2/3 cascade, but it clearly demonstrates context dependency (Hata & Chen, 2016), and BMP signaling gradients promote spatially arranged differentiation is capable of activating several other pathways, including the of ISCs, in part by suppressing WNT signaling, thereby regulating MAPK pathway. The exact mechanism of action in ISCs remains the number of stem cells in vivo (He et al, 2007; Krausova & obscure, but TGF-b appears not to affect ISC proliferation, although Korinek, 2014). it controls clone expansion and extinction, as well as modulates BMP signaling is activated by ligand binding to a multi-compo- the differentiation of secretory lineage precursors (Fischer et al, nent receptor complex and incorporates several complex pathways, 2016). for example, activation of the SMAD cascade (SMAD 1, 5, and 8), TGF-b receptor inhibitors, like A83-01 or SB431542, increase and MAPK, as well as positive regulation of PTEN (He et al, 2007; plating efficiency and are necessary for long-term culture of intesti- Katagiri & Watabe, 2016). In turn, PTEN negatively regulates the nal organoids by maintaining the undifferentiated stem cell state phosphatidylinositol 3-kinase (PI3K)/phosphatidylinositol triphos- (Sato et al, 2011a). phate (PIP )/AKT cascade, which has several downstream substrates, including GSK3b and b-catenin (He et al, 2007). Thus, AKT interacts with the canonical WNT pathway by increasing EGF b-catenin levels in the nucleus due to phosphorylation and inactiva- tion of GSK3b or phosphorylation of b-catenin itself (Fig 1). Hence, EGF is an important regulator of intestinal epithelial cell migration active BMP signaling suppresses the b-catenin/WNT pathway, and proliferation (Suzuki et al, 2010). Binding of EGF to its receptor thereby counteracting the proliferative effects of WNT activation. results in induction of tyrosine kinase activity, with subsequent acti- Noggin is a BMP antagonist, and as such, the addition of recom- vation of the RAS/RAF/MEK/ERK signaling as well as the PI3K/ binant Noggin or conditioned medium, combined with exogenous PIP /AKT cascades, inducing organoid growth (Date & Sato, 2015). WNT activation, leads to preservation and proliferation of ISCs. The PI3K/PIP /AKT pathway overlaps with the EGF and the BMP Without Noggin, intestinal organoids cannot be maintained in pathways, and provides a link to the canonical WNT pathway, as culture (Sato et al, 2009). shown in Fig 1. ª 2017 The Authors EMBO Molecular Medicine Vol 9 |No 5 | 2017 N EMBO Molecular Medicine Culturing ISCs for the treatment of IBD Fredrik EO Holmberg et al EGF in the form of recombinant protein is essential for culturing b-catenin, precipitating its lysosomal degradation (Kwon et al, human intestinal organoids, and lack of EGF or addition of an inhi- 2011). The process appears to be independent of NICD, as bitor of the EGF receptor causes decreased organoid formation and depicted in Fig 1. survival (Matano et al, 2015). Yet, human intestinal organoids have When culturing and mechanically passaging intestinal orga- been cultured without EGF when large amounts of serum were noids, Notch stimulation is supplied by adjacent supportive cells used, in the form of conditioned medium containing WNT, RSPO3, (Sasaki et al, 2016), hence further stimulation is likely redundant. and Noggin (Van Dussen et al, 2015). However, when growing dissociated single ISCs attained through EGF signaling in vivo is partly regulated by a negative feed- enzymatic organoid dissociation, Notch signaling should be stimu- back system, constituted by the p38 MAPK pathway that affects lated. One common approach is to add Jagged-1 peptide to the EGF receptor (Frey et al, 2006). This pathway regulates numerous support matrix for the first couple of days (Sato et al, 2009; Yin cell responses, including inflammation, apoptosis, cell cycle, et al, 2014), although additional studies are required to demon- differentiation, proliferation, and tumorigenesis (Zarubin & Han, strate an increased efficacy. When growing pure murine stem cell 2005). In the intestinal epithelium, p38 determines whether EGF cultures, Notch stimulation can be provided by exogenous supple- stimulation results in migration or in proliferation (Frey et al, mentation of the histone deacetylase inhibitor; valproic acid (VPA) 2004). Pharmacological inhibition of p38 decreases ligand-driven (Yin et al, 2014). In terms of clinical applications, VPA has the degradation of the EGF receptor, without affecting its internaliza- benefit of already being approved by both EMA and FDA for treat- tion (Frey et al, 2006), resulting in increased proliferation. Simi- ment of epilepsy and certain bipolar disorders, which might larly, deletion of p38 in intestinal epithelial cells results in simplify the approval process for its application in clinical stem increased proliferation, but also in a decreased goblet cell dif- cell enrichment. ferentiation (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 Prostaglandin E of human ISCs. IGF-1 can, similarly to EGF, stimulate PI3K/PIP /AKT and RAS/ The physiologically active lipid PGE is produced from arachidonic 3 2 RAF/MEK/ERK signaling, resulting in growth of intestinal orga- acid in cell membranes via the cyclooxygenase pathway and binds noids. However, EGF tends to more efficiently induce budding, to a number of G-coupled cell receptors. PGE promotes ISC expan- corresponding to crypt formation and organoid expansion (Reynolds sion and cell proliferation in vitro (Fan et al, 2014), inducing orga- et al, 2014). noid swelling and spheroid morphology rather than an organoid crypt structure (Fordham et al, 2013). The swelling was recently revealed to be caused by induction of anion and fluid secretion into Notch signaling the organoid lumen (Fujii et al, 2016). PGE upregulates several WNT target genes (Fan et al, 2014), Notch is essential to maintain the ISC pool by controlling stem cell which presumably explains its association with the development of self-renewal, as well as the balance between absorptive and secre- colorectal cancer (Buchanan & DuBois, 2006). It also appears to tory cell lineage specification (Demitrack & Samuelson, 2016). Path- suppress enterocyte differentiation and to promote repair of the way inhibition reduces ISCs proliferation and induces secretory intestinal epithelium (Miyoshi et al, 2017). lineage differentiation, thereby diminishing the ISC population (van Studies of ISCs (Miyoshi et al, 2017) and vertebrate Es et al, 2005; Van Dussen et al, 2012). Conversely, activation of hematopoietic stem cells (Goessling et al, 2009) have revealed the Notch pathway maintains stem cell multipotency and promotes that PGE signaling affects b-catenin stability through suppression stem cell proliferation, while directing progenitors toward an of GSK3b. Several other pathways are suggested to be involved, absorptive, rather than a secretory fate (Stanger et al, 2005; Demi- for example, interaction between PGE receptor-subunits and track & Samuelson, 2016). Axin, activation of cAMP/PKA as well as PI3K/PIP /AKT activity When a Notch ligand binds to the receptor, the Notch intra- (Fig 1) (Evans, 2009). Additionally, PGE upregulates LGR5 cellular domain (NICD) is separated through proteolytic cleavage, protein in human colorectal adenomas through a b-catenin inde- initiating nuclear translocation and activation of target genes pendent pathway, a central mechanism in colorectal tumorigene- (Date & Sato, 2015). However, in some cases, ligand binding is sis (Al-Kharusi et al, 2013). However, data attained from cancer insufficient to cause cleavage and receptor activation. The research cannot be extrapolated directly to normal ISCs, since process requires both ligand stabilization and mechanical force, cancer cells might contain mutations of the WNT or PGE path- inducing conformational changes of the receptor (Varnum-Finney ways. et al, 2000; Musse et al, 2012). Thus, direct activation of Notch PGE has the benefit of already being approved for clinical use pathway using recombinant Notch ligand has shown limited by both EMA and FDA for induction of labor. success. Genetic activation of the Notch pathway in murine ISCs antag- onizes and titrates canonical WNT signaling activity, thereby Other small molecules and cytokines affecting intestinal maintaining the stem cell state and balancing the differentiation stem cell maintenance process (Tian et al, 2015). Similarly, membrane-bound Notch and its adaptor protein NUMB in human embryonic stem cells and To sustain human ISCs, the vitamin nicotinamide is often added to human colon cancer cells associate with unphosphorylated the growth medium (Sato et al, 2011a). Nicotinamide impedes EMBO Molecular Medicine Vol 9 |No 5 | 2017 ª 2017 The Authors 562 Fredrik EO Holmberg et al Culturing ISCs for the treatment of IBD EMBO Molecular Medicine TM sirtuin activity involved in apoptosis, aging, differentiation, and Corning Matrigel Matrix and BD Matrigel Basement transcription regulation (Denu, 2005). However, nicotinamide can Membrane Matrix are the most extensively used three-dimensional be omitted without affecting long-term sustainability of human (3D) support matrices for culturing ISCs. The extensive usage of colonic stem cells (Fujii et al, 2015). Matrigel is attributed to its capacity to support long-term growth of When culturing dissociated single stem cells, the Rho-associated stem cells, while retaining the undifferentiated cell state (Hughes protein kinase (ROCK) inhibitors, Y-27632 (Watanabe et al, 2007) et al, 2010). It is a xenogeneic and proteinaceous matrix derived or thiazovivin (Wang et al, 2013), can be added to the growth from mouse sarcoma cells, mainly composed of laminin, collagen medium for the first few days to prevent anoikis. Research on IV, and entactin (Hughes et al, 2010). Its disadvantages include pluripotent stem cells has suggested that ROCK inhibitors suppress batch-to-batch variability, undefined composition, including varying caspase-dependent cell death (Wu et al, 2015). amounts of sarcoma-derived proteins, cytokines, and growth Amidated gastrin-17 is regularly used when culturing intestinal factors, along with the potential risk for pathogen transmission organoids, though it only marginally improves culture efficiency (Hughes et al, 2010). Such factors make Matrigel an ill-suited and it may therefore be omitted (Sato et al, 2011a). culture platform for clinical application. Therefore, considerable Addition of the cytokine interleukin 22 (IL-22) to the growth efforts have been made to identify well-defined matrices for both medium has shown to increase the proliferation of ISCs and to in vitro ISC expansion and their in vivo transplantation. cause EGF redundancy when culturing human intestinal orga- Collagen is an easily attainable connective tissue constituent, and noids (Lindemans et al, 2015). It activates STAT3, which causes common sources include fibroblasts cultured in vitro, as well as growth of human intestinal organoids independent of Paneth tissue extracts, such as human placenta. Different collagen formula- cells as well as both Notch and WNT signaling (Lindemans tions can be applied to sustain intestinal epithelial cell growth et al, 2015). in vitro (Ootani et al, 2009; Yui et al, 2012). However, reduced Many of the small molecules used to culture ISCs are available in budding has been reported when intestinal organoids are cultured high-purity formulations, though safety data are sparse, which could in support matrices rich in collagen (Pastula et al, 2016), potentially provide translational limitations. However, very low concentrations due to increased mechanical rigidity. Recently, human ISCs isolated of the small molecules are used in culture and can presumably be from small intestine were cultured to confluence on two-dimen- washed off prior to transplantation. sional (2D) monolayers on thin layers of bovine type I collagen and recombinant human laminin isotypes (Scott et al, 2016), although maintenance of the undifferentiated stem cell state was unclear at Culture matrices the protein level. Another possible approach could be to utilize allogenic or xeno- Cell–matrix interactions are implicated in numerous cell functions, geneic tissues as ECM (e.g., small intestinal submucosa or urinary including differentiation, anoikis, proliferation, and gene regulation bladder matrix), which already are being used to culture other cell (Berrier & Yamada, 2007). This is accomplished through a set of types in research and clinical settings. Tissues derived from natural membrane receptors, several of which are integrins (e.g., a2b1), sources are, however, restricted in their amplitude for modification, that anchor the cells to the intestinal basement membrane (Lussier with inconsistencies related to the health and age of the donors et al, 2000). Attachment to the intracellular cytoskeleton and activa- (Fitzpatrick & McDevitt, 2015). tion of signaling pathways are achieved through recruitment of Biologic matrices suffer the disadvantages of batch-to-batch vari- effector and adaptor proteins. This results in modification of anti- ability, relatively high manufacturing costs, limited scalability, and apoptotic pathways, gene expression, cell differentiation, prolifera- risk of pathogen contamination, motivating research on synthetic tion, and motility, as shown in Fig 2 (Lussier et al, 2000; Hofmann supportive matrices to overcome such issues. Synthetic matrices are et al, 2007). In the absence of cell–matrix anchorage or cell–cell chemically defined and malleable in terms of physiochemical and contact, epithelial cells undergo anoikis within hours (Hofmann mechanical properties (Tong et al, 2015). Multiple types of 2D et al, 2007). synthetic substrates have been used to culture hESCs. Further, Substantial efforts have been made to identify and optimize certain isoforms of laminin and vitronectin, fibronectin, as well as suitable matrices for stem cell cultures, particularly for culturing other xeno-free synthetic cell support matrices have successfully human pluripotent stem cells (hPSCs), which include induced been used to support hPSC (Villa-Diaz et al, 2013). Nevertheless, pluripotent stem cells (iPSCs) and human embryonic stem cells the conformation of vitronectin and laminin is sensitive to changes (hESCs). Different culture protocols and support matrices are in temperature and pH, which limits their potential for long-term detailed in Fig 3. usage (Tong et al, 2015). Initial extracellular matrices (ECM) for culturing hPSCs were 3D matrices, as opposed to 2D ones, provide more space for the produced by feeder layers of lethally irradiated fibroblasts in cells to grow, thereby reducing disadvantageous cell clustering (Lei enriched culture medium. Similarly, human colonic stem cells were et al, 2014). Furthermore, they efficiently provide physical and recently successfully cultured on feeder layers of irradiated mouse chemical gradients of importance for numerous cell functions, embryonic fibroblasts over a Matrigel coating (Wang et al, 2015). including differentiation and proliferation (Sant et al, 2010; Tong Variability when using feeder layers, along with the prospect of et al, 2015). Although simple collagen I 3D hydrogel matrices denaturing or degrading peptides and proteins with sterilization support ISCs, they have the disadvantage of low stiffness, limited techniques, as well as the potential risk for pathogen and xeno- long-term stability, and batch-to-batch variability (Caliari & Burdick, geneic transmission has led to the establishment of feeder-free 2016). 3D gels with compositions closer to the supportive matrix culture systems (Villa-Diaz et al, 2013). found in vivo or even mimicking axial gradients in connective tissue ª 2017 The Authors EMBO Molecular Medicine Vol 9 |No 5 | 2017 563 EMBO Molecular Medicine Culturing ISCs for the treatment of IBD Fredrik EO Holmberg et al PROLIFERATION SURVIVAL DIFFERENTIATION Effector and GENE EXPRESSION adaptor proteins e.g. β-actin, non-receptor Cadherins tyrosine kinase MOTILITY e.g. E-cadherin Actin filaments Actin filaments Effector and adaptor proteins e.g. α-actin, talin, non-receptor tyrosine kinase Integrins e.g. α2β1 αβ Basement membrane B Basement membrane asement membrane Pr P Pr roteoglucans oteoglycans oteoglucans F Fibr Fibr ibro onectin onectin nectin Collagen IV Collagen IV Collagen IV Laminin Laminin Laminin Figure 2. Cell–cell and cell–matrix interactions. Physical interactions between the intestinal epithelium, adjacent cells, and the ECM provide pivotal signals for cell survival, proliferation, gene expression, differentiation, and motility. Adhesion molecules, such as integrins (e.g., a2b1) and cadherins (e.g., E-cadherin) that attach to adjacent cells as well as to ECM proteins, mediate this. Adaptor and effector proteins provide linkage to intracellular actin filaments and can activate several signaling pathways, including non-receptor tyrosine kinases. composition might be expected to provide improved viability and However, ISC transplantation constitutes a plausible alternative function of the cultured ISCs. Interestingly, fundamental ECM approach to accelerate mucosal healing. In fact, EGF is an effective factors, such as mechanical properties and biochemical signals that treatment option for certain subtypes of IBD, possibly through its regulate ISC colony and organoid formation, have recently been regenerative capabilities (Sinha et al, 2003). A schematic of the identified (Gjorevski et al, 2016). The efforts resulted in the forma- envisioned process, from harvesting of the ISCs to transplantation, tion of a mechanically dynamic polyethylene glycol (PEG) hydrogel, is depicted in Fig 4. functionalized with RGD (Arg-Gly-Asp) peptides and controlled Autologous transplantation may be performed in order to avoid degradation kinetics, capable of expanding human small intestine the process of finding suitable cell donors, as well as circumventing and colorectal cancer organoids (Gjorevski et al, 2016). Hence, the risk for tissue rejection and the need for further immunomodula- minimal PEG-based hydrogels constitute a well-defined alternative tory therapy due to the procedure itself. ISCs would be harvested that might be applied to overcome the limitations of Matrigel in endoscopically from IBD patients with frequent and severe relapses terms of clinical application of ISCs. during periods with remission, and then expanded and kept frozen until needed. An alternative approach could be to harvest ISCs from non-inflamed areas during flares, or alternatively even from actively Regenerative applications in IBD inflamed areas. However, excessive epithelial cell death can be observed in areas of active disease (Blander, 2016). Also, colonic Introduction of biologics like monoclonal antibodies against tumor organoids derived from patients with flaring UC have in vitro been necrosis alpha (TNF inhibitors) later followed by a4b7 anti-integrins shown to maintain an altered expression of genes associated with has revolutionized the management of IBD. However, despite these antimicrobial defense, absorptive and secretory functions, compared therapeutic advances about one-third of patients with CD and one- to healthy controls (Dotti et al, 2016). Additionally, lasting tran- sixth of patients with UC still require surgical bowel resection within scriptional changes in the affected epithelium have been observed in 5 years after diagnosis (Frolkis et al, 2013). patients with UC despite remission (Planell et al, 2013). Although Much like the majority of other medical therapies for IBD, TNF the consequences of such changes are unknown, for the purpose of inhibitors and anti-integrins act through immunomodulation. transplantation it might be better to harvest ISCs from non-involved EMBO Molecular Medicine Vol 9 |No 5 | 2017 ª 2017 The Authors 564 Fredrik EO Holmberg et al Culturing ISCs for the treatment of IBD EMBO Molecular Medicine Conditioned medium � WNT3a Feeder–layer co-culture Substrates of synthetic polymers � RSPO1 � MEF or ECM-derived substrates � ISEMF 2D � Collagen I � Laminin Gelatinous matrix Co-culture: Gelatinous matrix � Matrigel over a feeder layer 3D � Collagen I � Myofibroblasts Well defined components � Synthetic hydrogels � Noggin � A83-01 � EGF � SB202190 � PGE � Y-27632 � CHIR99021 � IL-22 � VPA � Nicotinamide Figure 3. Culture protocols for ISCs. Culture protocols for ISCs generally consist of two basic components: a support matrix and a growth medium. The support component can be in either 2Dor 3D. 2D matrices are usually derived from feeder cells such as mouse embryonic fibroblasts (MEF), synthetic substrates or from ECM-derived proteins, for example, collagen and laminin. 3D support matrices are usually in the form of gelatinous matrices, for example, Matrigel, collagen I, or synthetic hydrogels. Another approach is to use 3D co-cultures, consisting of a gelatinous matrix over a feeder-layer, for example, myofibroblasts. The growth medium often includes a conditioned medium, such as WNT3a, RSPO1, or intestinal subepithelial myofibroblasts (ISEMF), as well as fully defined growth factors, small molecules, and cytokines, for example, Noggin, EGF, nicotinamide, A83-01,SB202190, PGE , CHIR99021, VPA, and IL-22. epithelium. Another argument for this approach is that patients with could be an alternative route of administration—although the large IBD have ~1.5- to twofold increased risk of developing colorectal volume needed in the latter case would greatly increase the need for cancer (Beaugerie & Itzkowitz, 2015), presumably due to prolonged cell expansion in vitro. Regardless of which method is chosen, a suit- and remitting inflammation. When intestinal epithelial cells are able delivery vehicle will be required to protect and sustain the cells in harvested endoscopically, relatively few clones are afterward transit. Ideally, this should be fully defined and biocompatible, while enriched in vitro. If these cells were to contain genetic mutations allowing for in situ crosslinking and mucosal adhesion. that predispose to malignancy, then transplantation might lead to ISC transplantation may be able to spur the epithelial healing risk of malignant transformation in a greater area of the intestine process, but for a majority of patients, it is unlikely that this would after engraftment. This important issue could, however, be be successful as a monotherapy, as cells presumably will have diffi- addressed by screening for mutations known to be associated with culties engrafting during ongoing inflammation. Hence, concomitant colorectal cancer. immunomodulatory therapy will likely be needed to give the trans- More than 160 susceptibility genes predisposing to IBD so far planted cells optimal conditions to re-establish barrier integrity. have been identified, including inflammatory bowel disease 5 (IBD5) A crucial aspect of all cell-based treatment strategies is to avoid and cadherin 1 (CDH1) that are associated with epithelial barrier inducing chromosomal changes that could lead to malignant trans- function (Miner-Williams & Moughan, 2016). Genetic susceptibility formation or other cell abnormalities. Epithelial stem cells grown does not automatically lead to development of IBD, but transplanta- in vitro can acquire a specific single nucleotide variant (SNV) signa- tion of cells with a genetic susceptibility may potentially have impli- ture differing from the somatic SNV signature seen in vivo in mice cations on epithelial function even after a successful transplantation. (Behjati et al, 2014). Long-term cultivation of human ISCs has Clearly however, more research on this matter is warranted. revealed a low level of genomic instability with a limited copy ISCs should be transplanted as complete organoids or as small cell number and SNV instability for the first 100 days of continuous clusters with intact endogenous Notch stimulation to maintain stem- proliferation (Wang et al, 2015). Yet, a trend toward increasing SNV ness and delay anoikis. Successful engraftment would most likely was observed as a function of passage number, but not involving require integrin activation to accomplish adherence to the ECM of the reported driver genes in human cancer. However, a forthright chro- damaged mucosa, which in turn depends on extracellular divalent mosomal trisomy was noted after 200 days. It is possible that genetic cations (Berrier & Yamada, 2007). In terms of delivery, endoscopic changes acquired in vitro could increase the risk of introducing new transplantation would intuitively be the simplest method, but enema mutations to the recipient of transplanted cells and could potentially ª 2017 The Authors EMBO Molecular Medicine Vol 9 |No 5 | 2017 565 EMBO Molecular Medicine Culturing ISCs for the treatment of IBD Fredrik EO Holmberg et al Intestinal biopsies are harvested endoscopically Intestinal crypts are Culture system isolated is established Organoids are formed Stem cell + LGR5 transplantation stem cells Intestinal stem cells are enriched as organoids LGR5 stem cells Figure 4. The course of human ISC harvesting to transplantation. Human intestinal epithelium can be harvested endoscopically, and ISCs can subsequently be isolated and enriched in vitro as organoids. Organoids enriched in stem cells can then be transplanted back to the patient (or as a suspension of purified stem cells), thereby hopefully promoting mucosal healing. increase the risk of neoplasia. Propagation of a sufficient amount of for use in humans, preferably constituents that are already ISCs for regenerative application would, however, presumably approved by FDA and EMA, or which demonstrate minimal or no require reasonably short culture duration. Accordingly, the risk of toxicity. alterations in SNV signature and copy number could be minimized. Cell–cell and cell–matrix interactions have profound effects on cell phenotype and survival. The continuous development of alter- native synthetic support matrices for ISCs is promising in terms of Future perspectives creating a suitable and indispensable substitute for Matrigel. The number of ISCs attained in vitro is commonly estimated by Alternative growth media compositions and culture protocols to determining LGR5 expression levels. Yet, gene expression does not increase the ISC yield are continuously being explored to allow necessarily correlate to equivalent increases on protein level, and an for successful regenerative applications. This includes growth increase in gene expression may reflect gene upregulation rather factors and small molecules that target the WNT pathway, such than increase in stem cell numbers. It would therefore be imperative as PGE and CHIR99021, along with newly identified pathway to standardize how stem cell amplification is quantified in vitro. targets such as IL-22 and STAT3. Advancing regenerative applica- Modest quantities of LGR5 on the cell surface, along with the lack of tions of ISCs requires additional investigation to identify compo- selective antibodies with high affinity for human LGR5, hinder the nents affecting WNT, Notch, EGF, and BMP signaling that are apt effective quantification of ISC expansion. Still, the use of antibodies EMBO Molecular Medicine Vol 9 |No 5 | 2017 ª 2017 The Authors 566 Fredrik EO Holmberg et al Culturing ISCs for the treatment of IBD EMBO Molecular Medicine Beaugerie L, Itzkowitz SH (2015) Cancers complicating inflammatory bowel Pending issues disease. 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Journal

EMBO Molecular Medicine – Springer Journals

Published: May 1, 2017

Keywords: inflammatory bowel disease; intestinal stem cells; organoids; regenerative medicine; support matrix

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