Background: Inflammatory bowel disease (IBD) is characterized by idiopathic and chronic inflammation arising elsewhere within the gastrointestinal tract. Consequently, the mucosal tissue is destroyed during the active phase of the disease, and therefore, spontaneous repair of damaged tissue is required to restore the function and long-term homeostasis of the intestinal mucosa. Also, in patients with refractory Crohn’s disease, loss of massive intestinal function can lead to short bowel syndrome or may lead to fatal intestinal failure. Summary: The concept of mucosal healing shares the idea that both regulation of mucosal inflammation and repair of the damaged mucosa are critical to achieve the ideal clinical outcome in patients with IBD. However, current treatments lack the option of those targeted to mucosal repair, and therefore, patients must achieve mucosal healing depending on their intrinsic system. To counteract inflammation-induced mucosal damage, various biologics or cell-based treatments are currently being developed. In the early developmental phase, various growth factors have been tested for their ability to promote mucosal repair. However, most of these factors did not show clinical benefit, except the recombinant glucagon-like peptide-2 (GLP-2). On the contrary, cell-based treatments are rapidly emerging, using both somatic stem cells and pluripotent stem cells. Key Messages: In this review, we focus on the current state of factor-based or cell-based regenerative medicine in the treatment of IBD. Additionally, we would like to introduce current examples of tissue engineering technologies and provide future prospects for the application of regenerative medicine in IBD.
Inflammatory bowel disease (IBD) consists of two main diseases, ulcerative colitis (UC) and Crohn’s disease (CD) and is characterized by chronic and idiopathic inflammation of the gastrointestinal tract. It may develop from the coexistence of various risk factors, including genetic susceptibility, dysregulation of the immune system, environmental factors, and dysbiosis . Upon the onset or recurrence of mucosal inflammation, the inflammatory environment usually causes destruction of the mucosal architecture and thus disrupts a wide range of mucosal-specific functions. Based on the concept that our intestinal mucosa functions as one of the key systems to maintain immunological homeostasis , functional and structural repair of the intestinal mucosa is a critical issue for the treatment of patients with IBD. In fact, the clinical concept of mucosal healing shares the idea that regulation of inflammation and proper regeneration of the damaged intestinal mucosa are two indispensable factors for long-term remission and improvement of clinical outcomes of patients with IBD. However, almost all the treatments currently available for patients with IBD target the immune cells or the inflammatory cytokine network and therefore do not have a robust effect in promoting post-inflammation tissue repair.
To provide a new option for achieving mucosal healing using an alternative concept, various growth factors or cell-based treatments are being developed. It should be noted that the development of tissue engineering technologies is rapidly emerging and may provide next-generation treatment for patients with IBD accompanied by refractory ulcers, refractory strictures, critical anastomoses, and short bowel syndrome (SBS).
In the current review, we would like to overview the landscape of factor-based or cell-based regenerative medicine in the treatment of IBD. Furthermore, future prospects are presented based on emerging tissue engineering technologies that may lead to novel treatments for patients with refractory IBD.
Requirement of Tissue Repair/Regeneration in the Treatment of IBD
The concept of mucosal healing has been proposed under the great clinical success of anti-TNF-a therapy, infliximab, in the treatment of UC and CD. Since the initial study by Froslie et al.  many aspects of the prognosis of IBD have been shown to improve with the achievement of mucosal healing. Also, in the treatment of CD, an extended concept of transmural remission has been proposed, indicating remission and repair of all intestinal layers, including the intestinal mucosa . These concepts are quite reasonable and easily accepted when we think back to our basic knowledge regarding the functional role of the intestinal mucosa and the pathogenesis of IBD.
When we start to think about the fundamental role of the intestinal mucosa, we must recall that the intestinal mucosa is located between the internal and external borders of our whole-body system. Thus, the intestinal mucosa plays its role as the structural primary barrier, or the frontline, in the protection of the border of our body. For example, intestinal epithelial cells (IECs) can form the mucosal layer to protect unregulated invasion of pathogens that may usually exist in the gut lumen. Also, IECs may interact with and regulate the gut microbiota by secreting antimicrobial peptides or responding to bacterial components through TLR-mediated pathways. At the same time, IECs can interact with and regulate the internal immune system. For example, a specific lineage of IECs can function as antigen-presenting cells to regulate the immune response to luminal pathogens . Additionally, IECs can produce various cytokines, including IL-7, to regulate the formation and cell population of the mucosal immune system . Therefore, IECs function as a key player in adjusting or optimizing the activity of the mucosal immune system in response to dynamic changes in the external luminal environment. However, in patients with IBD, these structural and functional functions of the intestinal mucosa are lost by immune-mediated tissue damage. Mucosal tissue damage can be recognized as multiple ulcers arising in the gastrointestinal tract during the endoscopic examination. Additionally, inflammation-associated carcinogenesis can be experienced in the long-term treatment of patients with IBD. Such tissue damage or the pathogenic transformation of the intestinal mucosa is not always treated or can be prevented only by regulating the immune system. Some proportion of patients with IBD may not achieve endoscopic mucosal healing due to the persistent existence of refractory ulcers. Furthermore, colitic cancers may occur in UC patients who are in long-term clinical remission. To counteract these problems, an alternative treatment choice must be provided to promote proper regeneration of the intestinal mucosa and restore the intestinal mucosa-mediated homeostatic environment in patients with IBD.
Development of Factor-Based Regenerative Medicine in IBD
In the early years of development, various growth factors have been tested to promote tissue regeneration in patients with IBD. In the first clinical trials, the tissue regeneration potential of keratinocyte growth factor (KGF) or epidermal growth factor (EGF) has been tested in patients with IBD. KGF has shown its tissue regenerative potential in several preclinical studies, and early phase clinical studies have been executed . However, it did not show a clear beneficial effect in patients with IBD, and the development was canceled. EGF was another factor which showed beneficial effects in various preclinical studies. In contrast to KGF, treatment with EGF enema successfully showed a beneficial effect in a small number of UC patients . However, it could not go further, possibly because of its limited effect. This consequence is quite acceptable after the declaration of key factors of the intestinal stem cell (ISC) niche. The innovative study by Sato et al. has shown that ISCs can survive and exert their function only under the coordinated growth factor signals provided in the stem cell niche . Indispensable factors for the ISC niche include Wnt3a, R-Spondin, Noggin, and EGF. Therefore, providing EGF alone may have a limited effect on stem cell-driven regenerative response, without support for activation of the Wnt pathway. Consequently, R-Spondin-1 has shown its mucosal growth effect in a preclinical model . The effect may represent the dominant role of Wnt signaling in mucosal growth. However, recombinant human R-Spondin-1 did not achieve approval for its clinical use. One of the concerns may arise from the carcinogenic potential of these growth factors since cancer cells usually share the main growth factor pathways with benign ISCs.
An example of successful development in factor-based regenerative medicine (Fig. 1) is the glucagon-like peptide-2 (GLP-2). GLP-2 is one of the factors secreted by L cells of the intestinal mucosa. It can regulate a wide range of our metabolic system and, in addition, might have a positive effect on ISC function . A GLP-2 analog teduglutide has gone through a series of clinical trials for its use in the treatment of SBS. In the trial, it showed a clear effect in the reduction of parenteral support in patients with SBS, who are under continuous treatment for intestinal failure. In the study by Jeppesen et al. , up to 63% of SBS patients responded to treatment with daily subcutaneous treatment of teduglutide, resulting in a 1 day or more reduction in parenteral support in 54% of SBS patients. The mechanism of its effect is suspected to increase the mucosal mass of the remnant intestinal tissue, as shown by the increase in plasma citrulline concentration. Now, teduglutide is approved and provided for clinical use in the USA, EU, and Asian countries. The GLP-2-mediated treatment is under further development [13, 14], using several long-acting versions of the GLP-2 analog (glepaglutide, apraglutide). Improvement in a once-a-week treatment would greatly improve the supportive care of patients with severe SBS.
Development of Stem Cell-Based Regenerative Medicine in IBD
Efforts to establish stem cell-based treatment for IBD have been made in many areas (Fig. 2). Common technical barriers were an efficient method to culture and expand the number of functional stem cells ex vivo and also to clear the immunogenicity of the transplanted stem cells. The first trial for this stem cell-based therapy was aimed at refractory CD, using hematopoietic stem cells. The therapy aimed to replace or renew the patient’s immune system through autologous hematopoietic stem cell transplantation (HSCT). It was a partly successful result in small-scale pilot studies and also showed clinical and endoscopic benefit in the multicenter randomized controlled ASTIC trial . However, it was stated that the application of HSCT should be strictly limited to a carefully selected proportion of CD patients as serious adverse events were experienced in the trial.
The application of HSCT to CD was aimed at regulating the immune system, and therefore, any direct beneficial effect on tissue repair could be expected. Furthermore, HSCT for CD must be limited to autologous transplantation as allogenic transplantation would require strict control of the graft versus host disease. On the contrary, mesenchymal stem cells (MSCs) have low immunogenicity and have also been shown to have tissue repair function in various preclinical studies. In fact, MSCs have been used to treat luminal and fistulizing CDs in small-scale pilot studies. The most successful result so far was obtained from a developmental trial using darvadstrocel, adipose tissue-derived MSCs, to fistulize CD . In the randomized controlled trial phase 3 of ADMIRE-CD, darvadstrocel-treated complex perianal fistulas in 46 patients with CD. In the trial, up to 67.4% of darvadstrocel-treated patients achieved clinical remission (compared to 52.2% clinical remission in the control group), and this effect appeared to last up to 3 years (156 weeks) [17, 18]. Based on these trials, darvadstrocel is now approved and available for use in the USA, EU, and Japan.
Another stem cell that is being developed for its use in patients with IBD is ISC. ISCs represent the stem cells of the epithelial tissue, which is located in the lower part of the intestinal crypt . For the clinical use of ISCs, the technical development of ex vivo ISC culture was achieved by innovative organoid technology. The initial report by Sato et al.  showed continuous growth of ISCs in a 3D-culture environment, with the support of non-ISC cells and various growth factors. The system also required the existence of a proper extracellular matrix, reflecting the indispensable components of the stem cell niche in vivo. Yui et al.  also showed that organoid culture that includes functional ISCs could be done by a fully defined factor culture system in combination with type I collagen as a supportive extracellular matrix. For the manufacture of organoids for clinical use, it is quite important to approve the safety of all materials used in the culture system. Therefore, we planned to develop a clinical-grade organoid manufacturing method based on the culture system using fully defined factors and type I collagen . In addition, a new endoscopic method for administering organoids to the target ulcer region was developed for the clinical use of patient-derived organoids. Based on these culture methods and the endoscopic delivery system, a first in human study (FIH study) was designed and approved in Japan (jRCT b032190207). The study aimed to discover the safety of autologous organoid transplantation in UC patients who have sustained refractory ulcers even under the control of advanced immunoregulatory treatments. The first autologous organoid transplantation was completed at our institute in July 2022. The results of the trial will be released after 1 year of follow-up of all enrolled patients.
Future Prospects for the Development and Application of Regenerative Medicine in IBD
In the coming years, the development and application of cell-based regenerative medicine would expand to much wider areas and patients. In this regard, we should discuss the development of treatments using somatic stem cells (MSCs and ISCs) and also those using pluripotent stem cells.
For MSCs, it can be applied to a wider range of patients with CD to take advantage of their immunosuppressive effect . Some trials are carried out for luminal CD, where optimization of the cell source or cell transplantation conditions remains to be determined.
The use of autologous ISCs, based on organoid culture technology, could be expanded to diseases other than UC. One of the good candidates is CD. For the application of organoid transplantation for CD, a clinical-grade culture for small intestinal organoids should be newly established. Also, endoscopic techniques for enteroscopic devices should be developed or modified. Deep remission of the small intestinal regions is a key factor in the treatment of CD. However, sustained ulcers are found in patients under the control of various biologics , and additional treatment with ISC transplantation may benefit those patients by achieving endoscopic remission. An alternative way to use autologous intestinal organoids is to restore region-specific intestinal functions by ectopic transplantation of intestinal organoids. The concept was proposed by the study by Sugimoto et al. , showing the rescue of an intestinal failure model by replacing the epithelial layer of the proximal colon with the small intestinal epithelium. Previous studies have shown that transplanted organoids preserve their regional identity upon engraftment in vivo , and therefore, transplantation of autologous small intestinal organoids to the colon, after efficient de-epithelialization as a pretreatment, would result in the establishment of an ectopic small intestinal mucosa. Surprisingly, their results showed remodeling of the submuscular architecture similar to that of the original small intestinal submucosa after the engraftment of small intestinal organoids. Although several critical issues must be resolved, the study provided a novel concept for treating patients with SBS accompanied by severe intestinal failure.
Another alternative way to generate intestinal grafts in size is to combine intestinal organoid culture technology with patient-derived scaffolds. Meran et al.  showed successful engineering of an intestinal graft for infants with severe intestinal failure. They prepared allogenic intestinal scaffolds and transplanted donor-derived organoids into the decellularized scaffolds. The organoids were successfully engrafted onto the scaffolds and formed a re-epithelialized graft. Those grafts maintained their luminal structures for up to 2 weeks after in vivo transplantation to mice recipients.
Another promising cell source for the future development of stem cell-based treatment is pluripotent stem cells. Both induced pluripotent stem cells and embryonic stem cells can be guided to differentiate into multilayer intestinal organoid tissue . Using the most recent updated protocol, intestinal organoids can be combined with neural cells to gain intrinsic motility function . Pluripotent cell-derived intestinal organoids can be transplanted into the mesentery, and promotion of full-layer tissue maturation can be achieved in the in vivo environment. These technologies could be extended to rescue CD patients suffering from SBS or intestinal failure. Furthermore, if full-layer intestinal tissue could be engineered, those ex vivo generated intestinal grafts can be applied to treat patients with CD with severe strictures or possibly to patients with UC who required total colectomy. Several technical barriers remain for this type of clinical application, such as induced intestinal lumen formation or organoids generating on a large scale, to finally engineer the graft tissue in a proper size for each patient. The immunogenicity of allogenic pluripotent stem cells may be another technical barrier. However, pluripotent cells of low immunogenicity, or universal pluripotent cells, are under development , and therefore, using those cells may allow wide use of allogenic intestinal grafts engineered from pluripotent cells.
Finally, stem cell-based therapy can be further improved by combining organoid technology or tissue engineering methods with gene editing. By extending the experience of chimeric antigen receptor T cell therapy, it may be possible to generate patient-specific organoids or pluripotent cell-derived grafts that have low immunogenicity and low disease susceptibility. For example, patients with monogenic IBD may benefit from a stem cell-based replacement of the gene-modified intestinal tissue.
Factor-based or cell-based regenerative medicine is in the early stage of development for the treatment of IBD. However, organoid-based tissue engineering technologies are rapidly emerging and will presumably promote the development of next-generation regenerative medicine. Those newly developed treatments may be applied to patients in combination with the immune regulatory treatments and contribute to improve achieve deep mucosal remission in an increased number of patients with IBD.
The authors thank Ms. Akiko Chiyoda for helping with manuscript preparation.
Conflict of Interest Statement
Ryuichi Okamoto declares financial support from Takeda Pharmaceutical Company Limited and Mochida Pharmaceutical Company Limited.
The study was supported by JSPS KAKENHI (Grant No. 22H00465, 22K19519, 21H02895, 21K15992), Japan Agency for Medical Research and Development (AMED) (Grant No. 22bm0304001h0010), and Naoki Tsuchida Memorial Foundation.
Ryuichi Okamoto, Tomohiro Mizutani, and Hiromichi Shimizu contributed to the conceptual design and preparation of the current manuscript.