In Vitro Expansion and Transplantation of Intestinal Crypt Stem Cells

Abstract

Adult tissue stem cells hold great promise for regenerative medicine strategies. Major hurdles in their clinical application persist and include the identification of such stem cells, their isolation and in vitro expansion, and—finally—their transplantation. These hurdles have recently been overcome for stem cells of the intestinal tract. Thus, exciting avenues are now opening up to apply these stem cells for a variety of therapeutic strategies. Herein, we highlight recent developments in intestinal stem cell biology and discuss this progress from the perspective of clinical application. Adult tissue maintenance and repair involves cell production by somatic stem cells and their transit-amplifying (TA) progenitor cells, followed by lineage commitment and terminal differentiation. The intestinal epithelium is the most vigorously regenerating tissue and is renewed every 3–5 days. This rapid cell renewal, in combination with its modular architecture of crypts and villi, makes it an ideal model to study stem cell biology. The intestinal stem cells reside at the bottoms of crypts in the small intestine and colon. Their TA daughters occupy the remainder of the crypts. Differentiated cells reside on the villi in the small intestine and in crypt tops and in the flat surface epithelium of the colon. The differentiated cell types of the small intestine include the absorptive enterocytes and multiple secretory lineages (Paneth cells, goblet cells, enteroendocrine cells, and tuft cells), as well as the M cells of the Peyer's patches.1van der Flier L.G. Clevers H. Stem cells, self-renewal, and differentiation in the intestinal epithelium.Annu Rev Physiol. 2009; 71: 241-260Crossref PubMed Scopus (1229) Google Scholar Intestinal homeostasis is tightly controlled by well-characterized signaling pathways. In particular, the Wnt-, Bmp-, epidermal growth factor (EGF), and Notch pathways have been shown to be major players in this regulation. Wnt signaling constitutes the key pathway to maintain the stem cells and drive proliferation at crypt bottoms. Mice lacking Tcf7l2/Tcf4, a key transcription factor in this pathway, lose all proliferation in crypts.2Korinek V. Barker N. Moerer P. et al.Depletion of epithelial stem-cell compartments in the small intestine of mice lacking Tcf-4.Nat Genet. 1998; 19: 379-383Crossref PubMed Scopus (1325) Google Scholar Conversely, colon cancer is caused by loss of the Apc gene, which encodes a key negative regulator of the Wnt pathway. In addition to Wnt signals, Notch signaling is required to maintain crypt cells in the undifferentiated state.3Sancho E. Batlle E. Clevers H. Signaling pathways in intestinal development and cancer.Annu Rev Cell Dev Biol. 2004; 20: 695-723Crossref PubMed Scopus (437) Google Scholar EGF signals maintain the proliferative state of the crypt cells. Bmp and Tgf-β signaling are activated in villus cells and are believed to regulate differentiation. Bmp ligands are expressed in villi, whereas Bmp antagonists such as noggin and gremlins are expressed at crypt mesenchyme to block Bmp signals from the crypts.4Kosinski C. Li V.S. Chan A.S. et al.Gene expression patterns of human colon tops and basal crypts and BMP antagonists as intestinal stem cell niche factors.Proc Natl Acad Sci U S A. 2007; 104: 15418-15423Crossref PubMed Scopus (420) Google Scholar All these pathways cross-talk with each other, forming complex molecular networks to govern tissue homeostasis. Regeneration of intestinal epithelial cells is fueled by stem cells at crypt bottoms. Stem cells, Paneth cells and TA cells together form the crypts of small intestine. Although the stem cells have long remained elusive, they have been known to produce rapidly cycling daughter cells, the TA cells. These TA cells undergo a few, unusually short, rounds of cell division followed by terminal differentiation into specific lineages. Two schools of thought have dominated the discussion over the identity of the intestinal stem cells for several decades. First, Potten et al5Potten C.S. Kovacs L. Hamilton E. Continuous labelling studies on mouse skin and intestine.Cell Tissue Kinet. 1974; 7: 271-283PubMed Google Scholar have proposed that the so-called +4 cells, located directly above the Paneth cells are cells that cycle every 24 hours, yet retain DNA labels owing to asymmetric (“immortal” strand) DNA segregation. Although current papers on those cells erroneously describe these +4 cells as quiescent (based on the observed label retention by Potten et al), several markers have been proposed for this stem cell. The most notable example, Bmi1, was used in a lineage tracing experiment that demonstrated that Bmi1 cells can give rise to long-lived clones containing all cell types of the epithelium.6Sangiorgi E. Capecchi M.R. Bmi1 is expressed in vivo in intestinal stem cells.Nat Genet. 2008; 40: 915-920Crossref PubMed Scopus (967) Google Scholar Thus, Bmi1 marks intestinal stem cells. Second, an alternative candidate stem cell type was first described 4 decades ago, but received little attention until recently. Leblond et al7Cheng H. Leblond C.P. Origin, differentiation and renewal of the four main epithelial cell types in the mouse small intestine V. Unitarian theory of the origin of the four epithelial cell types.Am J Anat. 1974; 141: 537-561Crossref PubMed Scopus (1118) Google Scholar, 8Cheng H. Leblond C.P. Origin, differentiation and renewal of the four main epithelial cell types in the mouse small intestine I. Columnar cell.Am J Anat. 1974; 141: 461-479Crossref PubMed Scopus (540) Google Scholar were the first to describe these slender crypt base columnar cells (CBC) located at the crypt base, intercalated with the much larger, postmitotic Paneth cells. These CBC cells go through their cycle once every 24 hours, and—based on their location at crypt bottoms—seemed to be excellent stem cell candidates. We have recently identified Lgr5 as a specific marker for these CBC cells. Lgr5 belongs to a small subfamily of 7-transmembrane receptors. Its expression is induced by Wnt signals. Using a lineage tracing strategy in mice, we could show that these CBC cells produce progeny every day, yet are long lived. Moreover, among the progeny of these cells, all differentiated cell lineages were identified.9Barker N. van Es J.H. Kuipers J. et al.Identification of stem cells in small intestine and colon by marker gene Lgr5.Nature. 2007; 449: 1003-1007Crossref PubMed Scopus (3988) Google Scholar Thus, the CBC cells—marked by Lgr5—represent long-lived and multipotent stem cells. Every day, each CBC cell is responsible for the production of 16–32 differentiated epithelial cells. Remarkably, the CBC cells keep this up for the lifetime of a mouse. Several other markers, OlfM4, Musashi, and CD133,10Zhu L. Gibson P. Currle D.S. et al.Prominin 1 marks intestinal stem cells that are susceptible to neoplastic transformation.Nature. 2009; 457: 603-607Crossref PubMed Scopus (553) Google Scholar have confirmed these observations. Every crypt contains approximately 15 Lgr5 stem cells. Detailed analysis of the behavior of these stem cells has revealed unusual and expected characteristics: As stated, the Lgr5 stem cells are not quiescent. Moreover, they do not divide asymmetrically. Rather, each of these cells divides every day in a symmetrical fashion. Subsequently, the daughter cells stochastically adopt stem cell or TA cell fates.11Snippert H.J. van der Flier L.G. Sato T. et al.Intestinal crypt homeostasis results from neutral competition between symmetrically dividing Lgr5 stem cells.Cell. 2010; 143: 134-144Abstract Full Text Full Text PDF PubMed Scopus (1334) Google Scholar Recent studies have compared the +4 stem cells and the Lgr5 stem cells. From these studies, it seems that the Lgr5 stem cells constitute the “workhorses” of the crypts. Bmi1-marked cells can give rise to Lgr5-expressing cells when the latter cells are selectively killed. Thus, Bmi1-expressing cells may represent a “reserve” stem cell population to replenish Lgr5-cells upon damage.12Tian H. Biehs B. Warming S. et al.A reserve stem cell population in small intestine renders Lgr5-positive cells dispensable.Nature. 2011; 478: 255-259Crossref PubMed Scopus (854) Google Scholar Similar plasticity was observed with another marker for the +4 cells, HopX.13Takeda N. Jain R. LeBoeuf M.R. et al.Interconversion between intestinal stem cell populations in distinct niches.Science. 2011; 334: 1420-1424Crossref PubMed Scopus (545) Google Scholar Although these studies support the notion that 2 distinct stem cell populations can be characterized, this distinction may be less absolute then currently believed: A recent expression study has employed a novel highly sensitive in situ hybridization technology to define the expression of most stem cell markers at the single cell level. The study shows that the expression of many reported stem cell candidates including Bmi1 coincides in CBC cells.14Itzkovitz S. Lyubimova A. Blat I.C. et al.Single-molecule transcript counting of stem-cell markers in the mouse intestine.Nat Cell Biol. 2011; 14: 106-114Crossref PubMed Scopus (261) Google Scholar Current in vitro studies of the intestinal epithelium are typically performed using epithelial cell lines derived from human colon cancer. Until recently, it has been believed that it would be inherently impossible to establish long-term cultures from primary adult tissues without inducing genetic transformation. A recent study has described an air–liquid interface model to culture small intestinal fragments from newborn mice in vitro.15Ootani A. Li X. Sangiorgi E. et al.Sustained in vitro intestinal epithelial culture within a Wnt-dependent stem cell niche.Nat Med. 2009; 15: 701-706Crossref PubMed Scopus (620) Google Scholar Rather than isolating epithelium, the entire intestinal wall was minced and cultured directly in a 3-dimensional collagen-based gel. The study demonstrated prolonged culture of epithelial–mesenchymal, sphere-like intestinal organoids in which expansion and multilineage differentiation was maintained. The cultures could be propagated without specific growth factors, presumably owing to the presence of the mesenchymal elements, yet were shown to be sensitive to Wnt- as well as Notch signals. The mixed culture of epithelial cells together with surrounding mesenchyme and stroma allows the organoid to be self-sustainable, yet the complex nature of the tissues restricts detailed analysis of stem cell homeostasis and the interaction with the stem cell niche. Recent genetics studies in mice have provided insights into growth factor–dependency of Lgr5 stem cells. Using this knowledge in conjunction with the Lgr5-GFP knockin mice has allowed researchers researchers to establish a long-term in vitro culture model in which 3-dimensional intestinal organoids can be grown from a single Lgr5 stem cell for periods of up to a year,16Sato T. Vries R.G. Snippert H.J. et al.Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche.Nature. 2009; 459: 262-265Crossref PubMed Scopus (4177) Google Scholar defying the Hayflick limit, which states that somatic cells have a limited proliferative potential.17Hayflick L. The limited in vitro lifetime of human diploid cell strains.Exp Cell Res. 1965; 37: 614-636Crossref PubMed Scopus (4287) Google Scholar As a source of Wnt, we utilized a small secreted protein that was known to potentiate low Wnt signals, Rspondin-1. As a Bmp antagonist, we used another small secreted protein termed Noggin. Additionally, we included EGF. As a 3-dimensional matrix, we utilized laminin- and collagen-rich Matrigel, which is liquid at 4°C but forms a gel at 37°C. Single, entire crypts can easily be isolated from mouse or human small intestine. Such crypts invariably grow into 3-dimensional organoids under the culture conditions described. The organoids present as cysts with a central lumen flanked by villus-like epithelium, whereas multiple crypt-like structures with stem cells and Paneth cells protruded in all directions into the Matrigel. The basal side of the epithelial cells is oriented toward the outside of the organoid. The organoids can be passaged at a 1:5 ratio weekly for many months, while chromosome structure and numbers of the stem cells remain normal. Mechanically disrupted organoids rapidly reseal in the passaging process. Self-renewal kinetics and cell-type composition of organoids closely resemble the in vivo situation. Interestingly, careful analysis of these organoids reveals that there are no nonepithelial/mesenchymal cells surrounding the organoid epithelium, posing the question about the identity of the stem cell niche. The stem cell niche is defined as the microenvironment that is in close proximity to the stem cells and controls stem cell activity and maintenance by providing short-range molecular signals (ie, surface receptors and secreted factors). It has long been believed that the intestinal mesenchyme surrounding crypt bottoms constitutes the stem cell niche. This mesenchyme consists of smooth muscle cells, fibroblasts cells and the intestinal subepithelial myofibroblasts. The observation that crypts can be grown continuously for long periods of time and that even single stem cells can be grown into organoids—albeit inefficiently—argued that these nonepithelial cells were dispensable for the creation of stem cell niches in vitro. We then realized that Lgr5/CBC stem cells are in intimate connection with Paneth cells, which made these Paneth cells interesting candidates to constitute the niche. The number of Paneth cells is constant, and equivalent to the number of stem cells in small intestinal crypts. Paneth cells were known to secrete a variety of bactericidal products, such as cryptidins/defensins and lysozyme. In addition, we found that they secret EGF, the related molecule Tgf-α and high-level Wnt3. Moreover, they express high levels of the Notch ligand Dll4. All of these signals are essential in our 3D culture system. We then showed that, although while culture of single stem cells is highly inefficient, stem cell/Paneth cells doublets efficiently formed organoids in vitro.18Sato T. van Es J.H. Snippert H.J. et al.Paneth cells constitute the niche for Lgr5 stem cells in intestinal crypts.Nature. 2011; 469: 415-418Crossref PubMed Scopus (1738) Google Scholar Also, Paneth cells depletion in vivo results in concomitant loss of Lgr5-expressing stem cells. Together, the data imply that Paneth cells constitute the niche for Lgr5 stem cells in intestinal crypts. This indicates that intestinal stem cells do not depend on a preexisting niche structure, but can generate their own niche. Thus far, only 1 type of adult stem cell therapy has found wide applications: The transplantation of bone marrow stem cells for hematologic malignancies, but also applied in solid cancer treatment, treatment of rare hereditary deficiency diseases, and autoimmune syndromes. Despite considerable efforts, it has remained impossible to expand bone marrow stem cells in vitro. Mesenchymal stem cells have more recently been identified in bone marrow, as well as in adipose tissues, for example. These stem cells are believed to generate bone, cartilage, and mesenchymal tissues. Mesenchymal stem cells can be cultured for brief periods but have limited ex vivo expansion potential. The only examples of stem/progenitor cells that can be expanded in vitro and retransplanted involve the epidermis19Gallico 3rd, G.G. O'Connor N.E. Compton C.C. et al.Permanent coverage of large burn wounds with autologous cultured human epithelium.N Engl J Med. 1984; 311: 448-451Crossref PubMed Scopus (1115) Google Scholar and the cornea.20Rama P. Matuska S. Paganoni G. et al.Limbal stem-cell therapy and long-term corneal regeneration.N Engl J Med. 2010; 363: 147-155Crossref PubMed Scopus (827) Google Scholar In both cases, only a single differentiated cell type is produced. Thus, the recently developed technology of intestinal organoid culture from single sorted intestinal stem cell provides innovative angles to take adult stem cells into the clinic. As stated, intestinal organoid cultures can be established from single Lgr5-sorted stem cells. The resulting organoids can be expanded dramatically and over long periods of time, without losing their tissue identity or genomic integrity. Since the initial publication, robust protocols have been developed for stomach organoids and for human small intestinal and colon organoids.21Barker N. Huch M. Kujala P. et al.Lgr5(+ve) stem cells drive self-renewal in the stomach and build long-lived gastric units in vitro.Cell Stem Cell. 2010; 6: 25-36Abstract Full Text Full Text PDF PubMed Scopus (1123) Google Scholar, 22Sato T. Stange D.E. Ferrante M. et al.Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett's epithelium.Gastroenterology. 2011; 141: 1762-1772Abstract Full Text Full Text PDF PubMed Scopus (2133) Google Scholar, 23Jung P. Sato T. Merlos-Suarez A. et al.Isolation and in vitro expansion of human colonic stem cells.Nat Med. 2011; 17: 1225-1227Crossref PubMed Scopus (500) Google Scholar Significantly, the growth factors that are present in the growth medium are the natural growth factors to which the stem cells are exposed in vivo. This makes the organoid cultures physiologically compatible with normal tissues for the purpose of transplantation. Indeed, no genetic manipulation, for example, by transfecting transcription factor genes, is necessary in the process. These aspects of the technology contribute to the expected safety of the procedure. As a first step toward the development of stem cell transplantation, it has been shown that significant amounts of tissue can be grown in vitro from a single adult colon stem cell (Figure 1).23Jung P. Sato T. Merlos-Suarez A. et al.Isolation and in vitro expansion of human colonic stem cells.Nat Med. 2011; 17: 1225-1227Crossref PubMed Scopus (500) Google Scholar, 24Yui S. Nakamura T. Sato T. et al.Functional engraftment of colon epithelium expanded in vitro from a single adult Lgr5(+) stem cell.Nat Med. 2012; 18: 618-623Crossref PubMed Scopus (576) Google Scholar In vitro expanded colon organoids were reintroduced per anum into the colons of multiple mice, pretreated with dextran sulphate sodium, which causes superficial mucosal lesions (Figure 2A–D). The engrafted organoids, grown from a single stem cell taken from a red fluorescent protein (RFP)+ mouse, were able to regenerate epithelial patches that were integrated perfectly into the existing epithelium (RFP−) and generated histologically and functionally normal crypts containing all differentiated cell types (Figure 2E, F). Of note, these transplanted organoids persisted for ≥6 months without changing their histologic appearance. Together, these data demonstrate the feasibility of adult intestinal stem cell therapy by transplanting organoids expanded from single sorted adult stem cells in vitro. Graft rejection can be managed by standard approaches, that is, by human leukocyte antigen matching of donor and acceptor and by immunosuppressive therapy as currently used for bone marrow/organ transplantation. This crypt/stem cell isolation and expansion system thus provides a simple and safe strategy for regenerative medicine.Figure 2Colon organoid transplantation. (A) Colon organoid cartoon with crypt and differentiated-domain. (B, C) Expanded organoids are introduced into the colon damaged by dextran sulphate sodium. (D) Transplanted organoids eventually fuse to cover the lesion. (E, F) Confocal image of long-term engrafted (6 months) clonal organoids derived from an RFP+ donor mouse into RFP− recipient mouse. Of note, transplanted organoids form complete colonic crypts with all cell lineages.24Yui S. Nakamura T. Sato T. et al.Functional engraftment of colon epithelium expanded in vitro from a single adult Lgr5(+) stem cell.Nat Med. 2012; 18: 618-623Crossref PubMed Scopus (576) Google Scholar Images are shown with (F) or without (E) 4′,6-diamidino-2-phenylindole staining.(Courtesy of Mamoru Watanabe and Tetsuya Nakamura).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Protocols have been developed to expand human small intestine and colon organoids from small biopsies22Sato T. Stange D.E. Ferrante M. et al.Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett's epithelium.Gastroenterology. 2011; 141: 1762-1772Abstract Full Text Full Text PDF PubMed Scopus (2133) Google Scholar and from single sorted stem cells using the intestinal stem cell surface marker EphB2.23Jung P. Sato T. Merlos-Suarez A. et al.Isolation and in vitro expansion of human colonic stem cells.Nat Med. 2011; 17: 1225-1227Crossref PubMed Scopus (500) Google Scholar It thus seems that the basic technology is in place. Clinical application awaits translation of the protocols to good medical practice standards, a scale-up to generate amounts of tissue required to treat human subjects, and the development of efficient transplantation approaches. In the transplantation setting, it appears essential to quantitatively remove preexisting crypts. This can be done, for instance, by low-dose radiation, 5-fluorouracil treatment, or modest hyperthermia. As a first application, we are considering the replacement by transplantation of the small intestinal epithelium in microvillus inclusion disease, a hereditary pediatric syndrome in which the intestinal epithelium looks superficially normal but fails to form brush borders.25Muller T. Hess M.W. Schiefermeier N. et al.MYO5B mutations cause microvillus inclusion disease and disrupt epithelial cell polarity.Nat Genet. 2008; 40: 1163-1165Crossref PubMed Scopus (278) Google Scholar As a consequence, infants with microvillus inclusion disease are dependent on intravenous alimentation and ultimately can only survive after a gut transplantation. Because intestinal organoids can be expanded dramatically from single stem cells, this technology may also allow a novel venue into gene therapy approaches. Gene therapy involves the introduction of DNA sequences that are typically integrated into the genomes of cells of the pertinent patient. The safe transfer of DNA represents the major hurdle, which has largely prohibited the introduction of gene therapy into the clinic despite 3 decades of intensive efforts. One of the few success stories of gene therapy involves the treatment of severe combined immunodeficiency (SCID) by ex vivo gene transfer to hematopoietic stem cells. Gene therapy of IL2RG deficiency (SCID-X1) patients involves the transfer of the gamma chain gene in vitro into autologous bone marrow cells through retroviral integration, after which the progenitor cells are reinfused back into patients.26Staal F.J. Pike-Overzet K. Ng Y.Y. et al.Sola dosis facit venenum Leukemia in gene therapy trials: a question of vectors, inserts and dosage?.Leukemia. 2008; 22: 1849-1852Crossref PubMed Scopus (23) Google Scholar The success of SCID-X1 gene therapy was unfortunately accompanied by the development of leukemia in several patients, which has aroused concerns about the safety of gene therapy. The current protocol results in multiple random integration events of the recombinant retrovirus in the genomes of the transplanted bone marrow stem cells. The random integration carries significant risk for activation of neighboring host oncogenes by viral sequences. Because organoids can be grown from single sorted stem cells, one could envisage an approach in which individual stem cells are analyzed after integration of the recombinant DNA sequences. Only stem cells with safe integrations could then be expanded clonally for subsequent transplantation. Virally mediated gene transfer has already been proven feasible in these organoids systems both using retrovirus and lentivirus.27Koo B.K. Stange D.E. Sato T. et al.Controlled gene expression in primary Lgr5 organoid cultures.Nat Methods. 2011; 9: 81-83Crossref PubMed Scopus (225) Google Scholar Treatment of human disease by organ transplantation is inherently limited by the availability of donor organs. Stem cell therapy promises the treatment of multiple patients by tissues “harvested” from a single, potentially live donor. Rapid progress in the field of intestinal stem cell biology now lends promise to the application of adult stem cell therapy in gastroenterology. The apparently unlimited scale at which these stem cells can be expanded in vitro offers particularly exciting therapeutic possibilities. It is expected that similar protocols (now available for stomach, small intestine, and colon), will also become available for other organ types, such as the liver and pancreas. Crucial elements in the organoid technology are the Lgr5 stem cell marker and the growth factor Rspondin1. It may not be surprising that several recent studies have identified Lgr5 as the receptor for the Rspondin growth factor.28Carmon K.S. Gong X. Lin Q. et al.R-spondins function as ligands of the orphan receptors LGR4 and LGR5 to regulate Wnt/beta-catenin signaling.Proc Natl Acad Sci U S A. 2011; 108: 11452-11457Crossref PubMed Scopus (637) Google Scholar, 29de Lau W. Barker N. Low T.Y. et al.Lgr5 homologues associate with Wnt receptors and mediate R-spondin signalling.Nature. 2011; 476: 293-297Crossref PubMed Scopus (936) Google Scholar It is this combination that allows the limitless growth of adult Lgr5 stem cells, allowing them to defy the Hayflick limit.