Tissue-Engineering the Intestine: The Trials before the Trials
2019; Elsevier BV; Volume: 24; Issue: 6 Linguagem: Inglês
10.1016/j.stem.2019.04.018
ISSN1934-5909
AutoresHans Clevers, Ryan Conder, Vivian Li, Matthias P. Lütolf, Ludovic Vallier, Sarah Chan, Tracy C. Grikscheit, Kim B. Jensen, Paolo De Coppi,
Tópico(s)3D Printing in Biomedical Research
ResumoBuilding complex tissues requires the development of innovative interdisciplinary engineering solutions. In this Forum, the INTENS Consortium discuss experimental considerations and challenges for generating a tissue-engineered intestine for the treatment of short bowel syndrome, taking into account cell source, scaffold choice, and design strategy for achieving proper assembly and function. Building complex tissues requires the development of innovative interdisciplinary engineering solutions. In this Forum, the INTENS Consortium discuss experimental considerations and challenges for generating a tissue-engineered intestine for the treatment of short bowel syndrome, taking into account cell source, scaffold choice, and design strategy for achieving proper assembly and function. Current cell and gene therapies with market authorization (Cuende et al., 2018Cuende N. Rasko J.E.J. Koh M.B.C. Dominici M. Ikonomou L. Cell, tissue and gene products with marketing authorization in 2018 worldwide.Cytotherapy. 2018; 20: 1401-1413Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar) include multiple approaches for skin, cornea, bone, or cartilage and CAR-T cell therapies. In addition, pluripotent stem cells (PSCs) have been delivered into the immune-privileged space of the retina. This first-in-human progress occurred with single cell types that do not require spatial or mechanical obligations. However, moving to more complex tissues, such as the intestine, presents increased obstacles such as motility, numerous differentiated cell types, three-dimensional convolution, conductance of luminal flow, and microbial interaction. Even for partial organ replacement, which might be clinically useful in certain cases, these requirements must be fulfilled by any therapeutically viable tissue-engineered candidate. Typically, cells must be delivered in vivo on a biocompatible product or on a construct that biodegrades to limit ongoing foreign body reactions and inflammation while initially offering support and possibly patterning clues. More complex tissues, such as the intestine, will require pioneering scientific regulatory pathways to ensure safe, efficient, and reproducible delivery of combined therapies that include stem cells, devices, biomaterials, and surgical techniques in preregistered trials. Here we discuss these parameters through the lens of replacing the small intestine for children with short bowel syndrome (SBS). SBS results when patients have inadequate functional intestinal epithelium, whether from congenital absence or subsequent loss, often resulting from massive surgical resection after infectious or vascular catastrophe. Currently, intestinal transplants may save some patients, but at high medical and human cost; roughly 50% of grafts survive after 5 years. Moreover, as intestinal transplants are highly immunogenic, patients require continuous and life-long immunosuppression, resulting in the majority (97%) developing infectious complications, with high rates of chronic kidney disease (25% at 5 years) and additional problems including rejection and graft versus host disease (Kesseli and Sudan, 2019Kesseli S. Sudan D. Small Bowel Transplantation.Surg. Clin. North Am. 2019; 99: 103-116Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). There is remarkable recent progress in both organ-specific stem cell cultures and differentiation of PSCs, leading to real possibilities for in vivo replacement of intestinal function from both sources. Unlike solid organs, the hollow viscus of the intestine requires substantially less tissue generation: 2πrh compared to πr2h for a solid organ. Therefore, the intestine is an attractive initial candidate for the translation of organ replacement with stem and progenitor cells as the gateway to understanding cell delivery regulatory and immunologic considerations for other abdominal organs. The structural complexity of the intestine, including elevated villi and invaginated crypts, supports differentiation and proliferation, respectively (Figure 1). Moreover, the complex interactions between multiple cell types constitute a major engineering task for SBS therapies. The small intestine is composed of concentric rings of muscle fibers (longitudinal and circular), as well as a submucosa that provides a mesenchymal framework for the epithelium, which forms the absorptive surface and protective barrier that manages nutrient absorption (Figures 1A and 1B). Within these layers, the enteric nervous system (ENS) regulates peristalsis and the controlled secretion of hormones and enzymes. Blood vessels ramify into capillaries accompanied by lymphatics that distribute nutrition and immune components (Gehart and Clevers, 2019Gehart H. Clevers H. Tales from the crypt: new insights into intestinal stem cells.Nat. Rev. Gastroenterol. Hepatol. 2019; 16: 19-34Crossref PubMed Scopus (363) Google Scholar). Tissue-engineered small intestine (TESI) should not be conflated with constructs that do not function. Although the physiological parameters (e.g., enzyme activity or villus height) may not exactly replicate those of the native intestine, TESI must support the complex relationships between resident cell types and does not include examples in which epithelium or mesenchyme survive alone or contain substantially different or non-functional cell populations (Figure 1C). Moreover, macroscopically, an engineered intestine must contain the native intestinal epithelial mucosa, submucosa, smooth muscle, ENS, and serosa in the correct orientation. Peristalsis, segmentation, mixing, digestion, absorption, secretion, and excretion also require reconstitution of an ENS (Schlieve et al., 2017Schlieve C.R. Fowler K.L. Thornton M. Huang S. Hajjali I. Hou X. Grubbs B. Spence J.R. Grikscheit T.C. Neural Crest Cell Implantation Restores Enteric Nervous System Function and Alters the Gastrointestinal Transcriptome in Human Tissue-Engineered Small Intestine.Stem Cell Reports. 2017; 9: 883-896Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). On a subcellular level, appropriate barrier function, digestion, and absorption depends on the correct localization of enzymes and membrane proteins. Intimate juxtaposition with blood flow for nutrient delivery and waste removal is required for the growth of engineered tissues, and to date there are no reliable engineered vascular beds ramified to capillaries that are proven to remain functional for long-term blood flow without thrombosis. Therefore, most current in vivo engineered organ approaches rely on vascularization of the construct after transplantation. A common approach to tissue engineering the small intestine includes delivering cells onto a scaffold, which is typically either derived from a decellularized organ (Urbani et al., 2018Urbani L. Camilli C. Phylactopoulos D.E. Crowley C. Natarajan D. Scottoni F. Maghsoudlou P. McCann C.J. Pellegata A.F. Urciuolo A. et al.Multi-stage bioengineering of a layered oesophagus with in vitro expanded muscle and epithelial adult progenitors.Nat. Commun. 2018; 9: 4286Crossref PubMed Scopus (56) Google Scholar) or a synthetic biodegradable matrix (Grant et al., 2015Grant C.N. Mojica S.G. Sala F.G. Hill J.R. Levin D.E. Speer A.L. Barthel E.R. Shimada H. Zachos N.C. Grikscheit T.C. Human and mouse tissue-engineered small intestine both demonstrate digestive and absorptive function.Am. J. Physiol. Gastrointest. Liver Physiol. 2015; 308: G664-G677Crossref PubMed Scopus (76) Google Scholar). Importantly, the scaffold must maintain its shape, provide an appropriate surface for tissue growth and organization, and be porous to allow for angiogenesis and vasculogenesis. Scaffold materials already approved for human use include polyglycolic acid/poly-L-lactic acid (PGA/PLLA), on which engineered tissues have been derived from small intestine, colon, esophagus, stomach, and liver; but many candidate biodegradable biomaterials or, in some cases, decellularized structures may suffice. There are scaling problems with these constructs as rates of hydrolysis are altered with larger and thicker materials, such as those that might be required for human neonate intestine. As cells proliferate, secrete extracellular matrix, and establish cell-to-cell interactions within the developing engineered tissue, the supportive scaffold is gradually degraded (Finkbeiner et al., 2015Finkbeiner S.R. Freeman J.J. Wieck M.M. El-Nachef W. Altheim C.H. Tsai Y.H. Huang S. Dyal R. White E.S. Grikscheit T.C. et al.Generation of tissue-engineered small intestine using embryonic stem cell-derived human intestinal organoids.Biol. Open. 2015; 4: 1462-1472Crossref PubMed Scopus (122) Google Scholar). Decellularized regions of the gastrointestinal tract may also serve as scaffolds (Urbani et al., 2018Urbani L. Camilli C. Phylactopoulos D.E. Crowley C. Natarajan D. Scottoni F. Maghsoudlou P. McCann C.J. Pellegata A.F. Urciuolo A. et al.Multi-stage bioengineering of a layered oesophagus with in vitro expanded muscle and epithelial adult progenitors.Nat. Commun. 2018; 9: 4286Crossref PubMed Scopus (56) Google Scholar) but similarly will rely on in vivo vascularization. For the latter, batch to batch variation might constitute a regulatory bottleneck. Future engineered intestine will contain cells that are either extracted from the intestine or from PSCs following in vitro differentiation (see below). Intestinal epithelial cells can be isolated and expanded as stem cells or as crypts with associated mesenchyme, ENS, endothelial cells, and muscle cells. For example, Lgr5 marks intestinal epithelial stem cells that can self-renew in vitro under defined cell culture conditions and differentiate into all intestinal epithelial cell types. Beyond single-cell strategies, clusters of various intestinal cell types can be isolated and maintained in vitro, vitrified for storage, or grown in vivo with varying amounts and types of small molecules. Typically, when mesenchymal cells remain with the epithelium, fewer to no extrinsic molecules are required for generating functional TESI (Grant et al., 2015Grant C.N. Mojica S.G. Sala F.G. Hill J.R. Levin D.E. Speer A.L. Barthel E.R. Shimada H. Zachos N.C. Grikscheit T.C. Human and mouse tissue-engineered small intestine both demonstrate digestive and absorptive function.Am. J. Physiol. Gastrointest. Liver Physiol. 2015; 308: G664-G677Crossref PubMed Scopus (76) Google Scholar). As an alternative to adult stem cells, the directed differentiation of PSCs into essentially all specialized cell types of the intestine enables off-the-shelf approaches and opportunities for patients, who no longer have residual tissue to provide starting material. Moreover, PSC-derived enteric neurons, mesenchymal cells, muscle cells, and epithelial cells can generate TESI in vivo that demonstrates appropriate cellular identities and ENS-driven contractility (Schlieve et al., 2017Schlieve C.R. Fowler K.L. Thornton M. Huang S. Hajjali I. Hou X. Grubbs B. Spence J.R. Grikscheit T.C. Neural Crest Cell Implantation Restores Enteric Nervous System Function and Alters the Gastrointestinal Transcriptome in Human Tissue-Engineered Small Intestine.Stem Cell Reports. 2017; 9: 883-896Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). As with other PSC-derived tissues, in vivo transplantation markedly improves the maturity of the epithelium. PSCs have been extensively reviewed for ethical considerations as a donor source and could still impose obvious safety concerns if PSC-derived cells after differentiation still contain insufficiently differentiated cell populations, resulting in the formation of undesired tissue types or malignancies. Extensive testing for specific release criteria, including the absence of pluripotency markers, will be required to proceed forward with this cell source. While autologous adult stem cells might not require immunosuppression, it remains unclear whether autologous PSCs would avoid immune surveillance. Initial therapies based on PSC derivatives are unlikely to be autologous due to safety, complexity, and costs associated with establishing personalized GMP-compliant manufacturing procedures. Thus, transplantation of an off-the-shelf PSC therapy for SBS would require immunosuppression. Engineered intestines could however be modified to contain fewer passenger lymphocytes, or cell derivatives could be generated from universal donor PSC lines with altered anti-HLA or anti-donor profiles. An alternative strategy is the development, as in Japan, of multiple lines to cover the MHC classes of a population; however, many more lines may still be required in countries with a wider immunological variation. Under all circumstances, donor cells and the differentiated cell products will still need to meet stringent GMP standards for implantation, including release criteria that identify standards for biological activity, viability, sterility, freedom from pathogen contamination such as mycoplasma, and stable karyotypes. Multiple companies and academic groups, including consortia at Tokyo Medical and Dental University, Yokohama City University, Cincinnati Children's Hospital Medical Center, and the INTENS framework supported by the European Union, are developing GMP conditions for growth of transplantable donor cells (Takebe et al., 2018Takebe T. Wells J.M. Helmrath M.A. Zorn A.M. Organoid Center Strategies for Accelerating Clinical Translation.Cell Stem Cell. 2018; 22: 806-809Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Cellular therapies that employ various donor cell types (Table 1) will most likely follow a clinical progression from less to more expensive, complex, and personalized. For inflammatory conditions or mucosal defects of the intestine, intestinal stem cells (ISCs) harvested from an alternative intestinal region could constitute an autologous clinical solution for epithelial resurfacing. However, in order to salvage SBS patients, full thickness intestine capable of absorption, secretion, and movement of the food bolus should be engineered. In order to achieve this, TESI can be generated from multicellular clusters extracted from tissues, thereby including differentiated epithelial cell types, mesenchyme, muscle, and components of the ENS. These grow well beyond the size of the implanted construct but can display disorganized morphology despite having appropriate intestinal functions, including nutrient absorption (Grant et al., 2015Grant C.N. Mojica S.G. Sala F.G. Hill J.R. Levin D.E. Speer A.L. Barthel E.R. Shimada H. Zachos N.C. Grikscheit T.C. Human and mouse tissue-engineered small intestine both demonstrate digestive and absorptive function.Am. J. Physiol. Gastrointest. Liver Physiol. 2015; 308: G664-G677Crossref PubMed Scopus (76) Google Scholar). Importantly, transplantation of ISCs alone or generating full thickness intestine can both serve as autologous therapies and will not require immunosuppression. For the latter, this might also be regulated similarly to other surgical procedures, particularly if storage steps such as vitrification or in vitro expansion are not required.Table 1Possible Strategies for Replacement of Human Intestine and the Enteric Nervous System Vary by Cost, Indication, and Donor MaterialCell TypeSourceAutologousImmunosuppressionCostMesenchymeLymphatic/ VascularENSPossible IndicationReferenceISCISC cultures/intestinal tissueyesno$$nohostnoepithelial resurfacingSugimoto et al., 2018Sugimoto S. Ohta Y. Fujii M. Matano M. Shimokawa M. Nanki K. Date S. Nishikori S. Nakazato Y. Nakamura T. et al.Reconstruction of the Human Colon Epithelium In Vivo.Cell Stem Cell. 2018; 22: 171-176 e175Abstract Full Text Full Text PDF PubMed Scopus (115) Google ScholarMulticellular clustersintestinal tissueyesno$yeshost and donoryesSBS, requires adequate remnant intestine, but cells can be expanded in vitroGrant et al., 2015Grant C.N. Mojica S.G. Sala F.G. Hill J.R. Levin D.E. Speer A.L. Barthel E.R. Shimada H. Zachos N.C. Grikscheit T.C. Human and mouse tissue-engineered small intestine both demonstrate digestive and absorptive function.Am. J. Physiol. Gastrointest. Liver Physiol. 2015; 308: G664-G677Crossref PubMed Scopus (76) Google ScholarHIO, non-autologousPSCnoyes$$$yeshostnoabsorptive surface without ENSFinkbeiner et al., 2015Finkbeiner S.R. Freeman J.J. Wieck M.M. El-Nachef W. Altheim C.H. Tsai Y.H. Huang S. Dyal R. White E.S. Grikscheit T.C. et al.Generation of tissue-engineered small intestine using embryonic stem cell-derived human intestinal organoids.Biol. Open. 2015; 4: 1462-1472Crossref PubMed Scopus (122) Google Scholar, Schlieve et al., 2017Schlieve C.R. Fowler K.L. Thornton M. Huang S. Hajjali I. Hou X. Grubbs B. Spence J.R. Grikscheit T.C. Neural Crest Cell Implantation Restores Enteric Nervous System Function and Alters the Gastrointestinal Transcriptome in Human Tissue-Engineered Small Intestine.Stem Cell Reports. 2017; 9: 883-896Abstract Full Text Full Text PDF PubMed Scopus (79) Google ScholarHIO, autologousPSCyesunknown$$$$yeshostnoabsorptive surface without ENSN/AMultiple separately derived cell typesaMultiple cells types could include mesenchyme, enteric neural crest derived cells, and intestinal stem cell and muscle cells derived either from PSCs or from intestinal tissues.intestinal tissue/PSCunknownunknown$$$$$yesunknownyesSBS, regulatory complexity increases with additional cell typesFinkbeiner et al., 2015Finkbeiner S.R. Freeman J.J. Wieck M.M. El-Nachef W. Altheim C.H. Tsai Y.H. Huang S. Dyal R. White E.S. Grikscheit T.C. et al.Generation of tissue-engineered small intestine using embryonic stem cell-derived human intestinal organoids.Biol. Open. 2015; 4: 1462-1472Crossref PubMed Scopus (122) Google Scholar, Schlieve et al., 2017Schlieve C.R. Fowler K.L. Thornton M. Huang S. Hajjali I. Hou X. Grubbs B. Spence J.R. Grikscheit T.C. Neural Crest Cell Implantation Restores Enteric Nervous System Function and Alters the Gastrointestinal Transcriptome in Human Tissue-Engineered Small Intestine.Stem Cell Reports. 2017; 9: 883-896Abstract Full Text Full Text PDF PubMed Scopus (79) Google ScholarIntestinal epithelial stem cells (ISCs) can be expanded in vitro and resurface damaged epithelium in animal models (Sugimoto et al., 2018Sugimoto S. Ohta Y. Fujii M. Matano M. Shimokawa M. Nanki K. Date S. Nishikori S. Nakazato Y. Nakamura T. et al.Reconstruction of the Human Colon Epithelium In Vivo.Cell Stem Cell. 2018; 22: 171-176 e175Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). ISCs are unlikely to increase intestinal length by themselves, as additional cell types are required for support. Clusters of epithelial, mesenchymal, and neural crest cells isolated from intestinal tissues can be expanded in vitro and used to generate TESI upon transplantation (Grant et al., 2015Grant C.N. Mojica S.G. Sala F.G. Hill J.R. Levin D.E. Speer A.L. Barthel E.R. Shimada H. Zachos N.C. Grikscheit T.C. Human and mouse tissue-engineered small intestine both demonstrate digestive and absorptive function.Am. J. Physiol. Gastrointest. Liver Physiol. 2015; 308: G664-G677Crossref PubMed Scopus (76) Google Scholar). Human intestinal organoids (HIOs) derived from PSCs as a mixture of mesenchyme and epithelium will generate a functional TESI upon transplantation. However, this TESI will not feature enteric neurons unless they are additionally supplemented, either isolated from the tissue or derived from PSCs (Finkbeiner et al., 2015Finkbeiner S.R. Freeman J.J. Wieck M.M. El-Nachef W. Altheim C.H. Tsai Y.H. Huang S. Dyal R. White E.S. Grikscheit T.C. et al.Generation of tissue-engineered small intestine using embryonic stem cell-derived human intestinal organoids.Biol. Open. 2015; 4: 1462-1472Crossref PubMed Scopus (122) Google Scholar, Schlieve et al., 2017Schlieve C.R. Fowler K.L. Thornton M. Huang S. Hajjali I. Hou X. Grubbs B. Spence J.R. Grikscheit T.C. Neural Crest Cell Implantation Restores Enteric Nervous System Function and Alters the Gastrointestinal Transcriptome in Human Tissue-Engineered Small Intestine.Stem Cell Reports. 2017; 9: 883-896Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Additional multicellular approaches will likely be developed and may include different cell types derived from PSCs or combinations with cells derived from adult intestinal tissues.a Multiple cells types could include mesenchyme, enteric neural crest derived cells, and intestinal stem cell and muscle cells derived either from PSCs or from intestinal tissues. Open table in a new tab Intestinal epithelial stem cells (ISCs) can be expanded in vitro and resurface damaged epithelium in animal models (Sugimoto et al., 2018Sugimoto S. Ohta Y. Fujii M. Matano M. Shimokawa M. Nanki K. Date S. Nishikori S. Nakazato Y. Nakamura T. et al.Reconstruction of the Human Colon Epithelium In Vivo.Cell Stem Cell. 2018; 22: 171-176 e175Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). ISCs are unlikely to increase intestinal length by themselves, as additional cell types are required for support. Clusters of epithelial, mesenchymal, and neural crest cells isolated from intestinal tissues can be expanded in vitro and used to generate TESI upon transplantation (Grant et al., 2015Grant C.N. Mojica S.G. Sala F.G. Hill J.R. Levin D.E. Speer A.L. Barthel E.R. Shimada H. Zachos N.C. Grikscheit T.C. Human and mouse tissue-engineered small intestine both demonstrate digestive and absorptive function.Am. J. Physiol. Gastrointest. Liver Physiol. 2015; 308: G664-G677Crossref PubMed Scopus (76) Google Scholar). Human intestinal organoids (HIOs) derived from PSCs as a mixture of mesenchyme and epithelium will generate a functional TESI upon transplantation. However, this TESI will not feature enteric neurons unless they are additionally supplemented, either isolated from the tissue or derived from PSCs (Finkbeiner et al., 2015Finkbeiner S.R. Freeman J.J. Wieck M.M. El-Nachef W. Altheim C.H. Tsai Y.H. Huang S. Dyal R. White E.S. Grikscheit T.C. et al.Generation of tissue-engineered small intestine using embryonic stem cell-derived human intestinal organoids.Biol. Open. 2015; 4: 1462-1472Crossref PubMed Scopus (122) Google Scholar, Schlieve et al., 2017Schlieve C.R. Fowler K.L. Thornton M. Huang S. Hajjali I. Hou X. Grubbs B. Spence J.R. Grikscheit T.C. Neural Crest Cell Implantation Restores Enteric Nervous System Function and Alters the Gastrointestinal Transcriptome in Human Tissue-Engineered Small Intestine.Stem Cell Reports. 2017; 9: 883-896Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Additional multicellular approaches will likely be developed and may include different cell types derived from PSCs or combinations with cells derived from adult intestinal tissues. However, most patients with SBS will not have their initial intestinal resection at a tertiary care hospital with a biorepository team or ongoing clinical trial. For these patients, an off-the-shelf solution is required. Currently, PSCs can be differentiated into human intestinal organoids (HIOs) consisting of either a mixture of epithelial and mesenchymal cells or only epithelial cells. Importantly, TESI formed by HIO consisting of both mesenchymal and epithelial cells generates all relevant differentiated epithelial cell types, but ENS has to be supplied (Finkbeiner et al., 2015Finkbeiner S.R. Freeman J.J. Wieck M.M. El-Nachef W. Altheim C.H. Tsai Y.H. Huang S. Dyal R. White E.S. Grikscheit T.C. et al.Generation of tissue-engineered small intestine using embryonic stem cell-derived human intestinal organoids.Biol. Open. 2015; 4: 1462-1472Crossref PubMed Scopus (122) Google Scholar, Schlieve et al., 2017Schlieve C.R. Fowler K.L. Thornton M. Huang S. Hajjali I. Hou X. Grubbs B. Spence J.R. Grikscheit T.C. Neural Crest Cell Implantation Restores Enteric Nervous System Function and Alters the Gastrointestinal Transcriptome in Human Tissue-Engineered Small Intestine.Stem Cell Reports. 2017; 9: 883-896Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). It can be speculated whether ENS function is required if the TESI is constructed as a stoma or a pouch given that it could still have physiologically relevant absorptive capacity. For all of these patients, foreign PSC-derived TESI should be considered in the context of immunosuppression similar to patients, who currently receive intestinal transplants. Although it is possible to derive autologous PSC tissues, the most likely scenario will comprise non-autologous cell sources as the costs otherwise will be prohibitively expensive due to GMP requirements. Despite all of these obstacles, the speed by which PSC discoveries are on track for clinical trials in other organ systems such as the retina, heart, and brain indicates the great possibilities. It is critical to establish criteria to report the degree and grade of organ development while designing a path to translation. The goal in tissue-engineering organ replacements is to provide adequate function and to understand and reliably deliver those parameters while minimizing patient risk (Cossu et al., 2018Cossu G. Birchall M. Brown T. De Coppi P. Culme-Seymour E. Gibbon S. Hitchcock J. Mason C. Montgomery J. Morris S. et al.Lancet Commission: Stem cells and regenerative medicine.Lancet. 2018; 391: 883-910Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). Some physiologic rescue might be accomplished without entirely recapitulating the target organ—for example, by providing a small segment of intestine that adds just enough absorptive surface area to wean the patient off of intravenous parenteral nutrition. Additionally, if one implanted construct is inadequate, multiple constructs could be combined in reconstructive surgery. Although both adult stem cell and PSC-based approaches generate TESI that has key parameters of function, the resulting tissue architecture in large animal models remains to be adequately determined. Additionally, blood flow must be provided, and in approaches that generate large volumes of tissue without contemporaneous angiogenesis or vasculogenesis, either provision of a pedicle flap or concurrent engineered vasculature will be required. It is likely that constructs that grow in vivo will therefore be the initial therapies. Finally, there is an opportunity for the field to progress with transparent and ethically designed clinical trials while overcoming these hurdles. The first instance of organ engineering outside of an immune privileged space must be appropriately studied and reported through preregistered trials. We apologize to the researchers whose work could not be cited due to article restrictions. The INTENS Consortium is supported by the European Union's Horizon 2020 research and innovation program (668294). Ryan Conder is an employee of STEMCELL Technologies Inc.
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