Mesenchymal Stem Cells to Treat Crohn's Disease with Fistula

Abstract

Crohn's disease, which mainly affects the gastrointestinal tract, is a refractory inflammatory disease that has clinical manifestations of abdominal pain, fever, bowel obstruction, and diarrhea with blood or mucus. Together, these symptoms can severely impair a patient's quality of life. Besides the common complication of intestinal obstruction, fistulas, particularly anorectal fistulas, are common in Crohn's disease patients. Since radical surgical cures can be difficult to achieve and relapse is common, Crohn's disease patients often seek other effective treatments in addition to surgery. Stem-cell therapies have recently been proposed as a method to address the challenges and prospective medical needs of Crohn's disease patients in general and those with fistulas. Several studies suggest that mesenchymal stem cells (MSCs) could improve Crohn's disease and Crohn's fistula. Moreover, studies concerning MSC transplantation or local rejection of stem cells derived from bone marrow or adipose tissue–derived stem cells have assessed stem cell–based treatments for refractory Crohn's disease. Many patients in these studies are now in remission. A number of clinical trials for refractory Crohn's disease have also evaluated transplantation of autologous or allogenic MSCs and showed that MSCs can be safely administered to Crohn's disease patients, with some achieving positive clinical responses.

Introduction

Stem cells are progenitor cells that have the capacity to self-renew and differentiate into multiple cell types. In recent years, scientists have taken advantage of the self-replication and multi-lineage differentiation potential of various types of stem cells to investigate the mechanisms by which these cells can mediate tissue repair in related diseases, with promising results. Stem-cell therapy involves transplantation of autologous or allogenic healthy stem cells to promote repair of pathological cell damage and to rebuild normal function of cells and tissues.¹ To date, stem-cell therapy has been used to treat leukemia, Parkinsonism, liver cirrhosis, kidney failure, systemic lupus erythematosus, and myocardial infarction, as well as diabetes and its complications.^1,2 In the United States, Japan, Korea, and the United Kingdom, significant efforts have been made to translate basic research on stem-cell therapies to clinical applications, and several national research projects have been established to develop core technologies for stem-cell therapy, which has facilitated clinical trials, evaluation of the safety and effectiveness of stem-cell therapy, research training, and expansion of infrastructure.

Crohn's disease, ulcerative colitis and indeterminate colitis comprise inflammatory bowel diseases (IBD).^3,4 Crohn's disease was first described and named by Crohn, Ginzburg, and Oppenheimer in 1932 and was referred to as “regional enteritis,” “segmental enteritis,” or “chronic intestinal wall full-thickness inflammation.” In 1973, The Council for International Organizations of Medical Sciences (CIOMS) named the disease Crohn's disease. Although Crohn's disease can affect the intestinal wall layers of all parts of the gastrointestinal tract, the terminal ileum and right colon are mostly commonly affected, and can result in a significant reduction in a patient's quality of life. Clinical manifestations of Crohn's disease vary according to lesion type and the range and degree of symptoms such as abdominal pain, fever, bowel obstruction, and diarrhea with blood or mucus.⁵

Genetic and environmental factors can affect the pathophysiology of Crohn's disease.⁵ Current drugs used to treat Crohn's disease include steroids, anti-tumor necrosis factor (TNF) therapy, thiopurines, methotrexate, and infliximab, which can all afford rapid control of disease but often do not provide complete healing, and thus Crohn's disease has a high recurrence rate.^6,7 Moreover, some patients have no response to these drugs. The antitumor drug infliximab was recommended for use in combination with surgical treatment to achieve better therapeutic effects, although surgical treatment may also have a high recurrence rate and short-term efficacy.^8
–10 Therefore, novel therapeutic strategies such as stem cell–based therapy are under investigation.

Stem-cell therapy involving infusion of mesenchymal stem cells (MSCs) or hematopoietic stem cells (HSCs) is a promising approach for the treatment of Crohn's disease.¹¹ Many recent studies of stem-cell transplantation or local rejection reported successful treatment of refractory Crohn's disease and frequent disease remission.³ A search of the clinical trial registry (www.clinicaltrials.gov) using the key phrase “stem cell” showed that since 2016, 5,509 clinical trials were registered. Of these, 4,977 involved MSCs. Meanwhile, among 7,788 clinical trials for Crohn's disease, 896 involved stem cells, and of these 35 used MSCs.¹²

Besides the common complication of intestinal obstruction, Crohn's disease patients often have fistulas, with anorectal fistula being the most common type. Anorectal fistulas can be treated with surgical approaches such as fistulotomy, ligation therapy, and anal fistulectomy. The recurrence rate for fistula therapy is high, and some fistulas are lifelong, even after surgical excision. Crohn's disease patients with fistulas frequently suffer from pain and negative side effects associated with sustained use of antibacterial agents and corticosteroids. Although autologous HSC transplantation via intravenous infusion by some hospitals has shown some promise, Crohn's disease patients with severe illness may have many complications and do not respond well to stem cell–based treatments. Given the refractory and complex nature of Crohn's disease, MSC may provide new therapeutic spaces for reconstruction of healthy crissum tissue, decrease of pain, and improvement in the quality of life for Crohn's disease patients.

In 2009, the U.S. Food and Drug Administration (FDA) approved the therapy Prochymal^® (Osiris Therapeutics, Inc., Columbia, MD) to treat graft-versus-host disease (GvHD) and Crohn's disease.¹³ The main component of Prochymal^® is bone marrow–derived MSCs. In 2012, an autologous adipose-derived stem cell–based product, Cupistem^® (Anterogen Co. Ltd., Seoul, South Korea) was approved by the Ministry of Food and Drug Safety of Korea. For this therapy, administration of 3 × 10⁷ adipose-derived stem cells/mL promoted gastrointestinal tissue repair and effectively reduced the recurrence of fistula. Other clinical trials are ongoing to gain a better understanding of the complexity of Crohn's disease and what therapeutic approaches will yield maximal benefits.

This review summarizes the latest clinical studies of MSC transplantation for treatment of Crohn's disease and fistula compared to HSC transplantation to provide a theoretical basis for the cure of Crohn's disease and improvement of patients' quality of life.

Mesenchymal Stem/Stromal Cell Transplantation

MSCs have many properties that would be useful for therapeutic approaches to treat Crohn's disease. For example, MSCs are multipotent, mesoderm-derived cell progenitors that can generate osteogenic, adipogenic, and chondrogenic cells.¹⁴ Through these cells, MSCs have immunomodulatory properties and are associated with the release of cytokines, chemokines, and growth factors, whereas proinflammatory lymphocytes cause cell-cycle arrest and induce T-cell apoptosis.¹⁵ MSCs can also induce healing of damaged tissue by their immunomodulatory actions. Yet, MSCs themselves are non-immunogenic and therefore do not require immune ablation in allogeneic transplantation.¹⁶

MSCs can be obtained from bone marrow (BMSCs) and used for autologous or allogeneic transplantation in Crohn's disease. A study by Duijvestein et al. ¹⁷ involved delivery of twice-daily intravenous infusions of 1–2 × 10⁶ BMSCs/kg body weight for 7 days to nine adult patients with refractory Crohn's disease. These patients were then clinically assessed for 14 weeks. For six patients, the Crohn's disease activity index (CDAI) scores decreased, and three of these patients had a 70% reduction in CDAI scores. The remaining three patients required surgical intervention to treat worsening disease. No serious side effects were seen during or after the BMSC infusion.

For Crohn's disease patients with fistulas, in a study by Ciccocioppo et al.,¹⁸ 10/12 consecutive outpatients (two refused) received monthly intrafistular injections of 2 × 10⁷ MSCs isolated from bone marrow and expanded ex vivo. The patients were monitored by magnetic resonance imaging (MRI), and underwent surgical and endoscopic evaluation during the 12-month follow-up period. MSC expansion was successful in all cases, wherein 70% of the patients had sustained complete fistula closure and 30% had incomplete closure of fistula tracks, with a parallel reduction of Crohn's disease and perianal disease activity indexes (p < 0.01). In addition, rectal mucosal healing was induced after treatment, and no serious adverse effects were seen.

An early Phase II study by Molendijk et al.¹⁹ involved 21 Crohn's disease patients with refractory perianal fistulizing who had been treated with anti-TNF agents and other traditional drugs but saw no improvement in CDAI. The patients were randomly assigned into four groups that received local injections with placebo (solution with no cells; n = 6) or different amount of BMSCs (1 × 10⁷, 3 × 10⁷, or 9 × 10⁷ cells) isolated from healthy donors. Six weeks after transplantation, 85.7% of patients in the group that received 3 × 10⁷ cells saw an improvement, and these effects were stable through week 24. The treated groups also had a statistically significant improvement in fistula healing compared to the placebo group. A study by Liang et al. obtained similar results.²⁰ In these studies, lower BMSC doses appeared to produce higher fistula healing rates, although larger Phase III trials are needed to confirm this result (Table 1).

Table 1.

Clinical trials for treatment of Crohn's disease with bone marrow–derived mesenchymal stromal-cell transplantation

Ref.	Patients (n)	Pre-CDAI	Follow-up (months)	Remission (type/n/time in months)	Recurrence (n/time in months)	Side effect/n	Post-CDAI	Administration method	Clinical Phase
17	10	326 (224–378)	1.5	None	NA	Allergic reaction/1	252 (160–473)	Intravenously	Phase I
						Headache/3
18	10	294 (237–384)	12	Clinical/7/1.5	None	NA	99 (50–150)	Intrafistularly	Phase I
				MRI/7/1.5
20	4	308 (223–378)	19 (6–32)	Clinical/4/1.5	1/6, 1/7	Face hot/1	184 (126–284)	Intrafistularly	Phase I
[19]	15	114	5.6	Clinical/8/1.5	NA	Abdominal pain/5	NA	Intrafistularly	Early Phase II
						Fever/1

NA, not available; CDAI, Crohn's disease activity index; MRI, magnetic resonance imaging.

The above studies used intrafistular or intravenous administration of MSCs isolated from bone marrow of the patients or healthy donors, and these cells were expanded in vitro. However, this expansion requires several weeks,¹⁹ which could limit the use of BMSCs, particularly for patients with severe Crohn's disease. Meanwhile, adipose tissue–derived MSCs (AMSCs) have immune suppressive and immunophenotypic properties that are similar to BMSCs and can be readily isolated from liposuction aspirates. Given their availability, several studies have examined the use of AMSCs in autologous or allogeneic transplantation to treat refractory Crohn's disease.²¹

The first report using autologous stem cells obtained from a lipoaspirate in a Phase I clinical trial of the treatment of Crohn's fistula showed encouraging results.²² In this study, adipose tissue was obtained by liposuction, and the stem cells were purified, expanded, and characterized in vitro. Five adult patients who had been diagnosed with Crohn's disease at least 5 years before the trial and who had been unresponsive to medical treatment and unsuccessfully treated with classic surgery at least twice were enrolled. Six of the nine fistulas in four patients (one patient was excluded because of bacterial contamination of cultured cells) saw at least a 75% improvement after transplantation with autologous AMSCs from passage 3 or earlier. The other two fistulas had incomplete closure of the external opening (not healed; 25%). In a Phase II clinical trial,²³ Crohn's disease patients received one or two (as necessary) injections of either fibrin glue alone or fibrin glue with AMSCs. The patients who received fibrin glue with AMSCs saw on average 71% fistula healing compared to only 4% seen for patients who received the glue alone.²⁴ In the 1-year follow-up period, the recurrence rate in patients treated with AMSCs was 17.6%.²⁵ Recent long-term and Phase II clinical trials^8,26 also showed encouraging effects of AMSCs for treatment of fistulas in Crohn's disease. In a Phase III randomized clinical trial reported by Herreros et al.,²⁷ 200 adult patients with complex fistula-in-ano were randomly assigned to receive adipose-derived stem cells (2 × 10⁷), stem cells plus fibrin glue, or fibrin glue alone; an additional 4 × 10⁷ stem cells could be administered if the fistula had not healed at 12 weeks. At 1 year, the healing rates for the three groups were 57.1%, 52.4% and 37.3%, respectively (p = 0.13). Although the healing rates were >50% at 1-year follow-up, there were no statistically significant differences among the three groups. No severe adverse events associated with the transplantation were reported. Similar results were obtained in studies conducted in 2013^6,28,29 and 2015.⁸

A Phase III randomized, double-blind, parallel-group, placebo-controlled study by Panés et al.³⁰ used allogeneic, expanded AMSCs (Cx601) to treat adult patients with Crohn's disease and treatment-refractory, draining complex perianal fistulas. The primary endpoint was combined remission at week 24 (clinical assessment of closure of external openings combined with MRI). Results showed that 24 weeks after treatment, 50% of patients treated with Cx601 cells achieved combined remission compared to 34% who received placebo treatment. No adverse events were seen after Cx601 treatment. This clinical trial showed effective and well-tolerated new treatment options for patients with Crohn's disease and complex perianal fistulas. Tigenix received FDA approval for a new clinical trial that could accelerate the availability of Cx601 for treatment of Crohn's disease (Table 2).

Table 2.

Clinical trials for treatment of Crohn's disease with adipose mesenchymal stem-cell transplantation

Ref.	Patients (n)	Fistula (n)	Follow-up (months)	Fistula healed rate%/time (months)	Recurrence (n/time in months)	Adverse event (n)	Administration methods	Clinical Phase
22	4	9	22 (12–30)	75%/2	None	No SAEs	Intravenously	Phase I
25	4	NA	(12–48)	73%/2	None	No SAEs	Intravenously	Phase I
27	200	NA	12	40%/6	NA	Proctalgia (80 [43.7%]), abscess drainage (41 [22.4%]), pain (25 [13.7%]), perianal abscess (24 [13.1%]), pyrexia (17 [9.3%]), swelling (12 [6.6%]), and pruritus (12 [6.6%])	Intravenously	Phase III
				49%/12
26	24	NA	38–42.6	40%/38	5/12	2 adverse events unrelated to the treatment	Intravenously	Phase II
30	212	NA	6	50%/6	None	Anal abscess (6 [6%])	Intralesionally	Phase III
						Proctalgia (5 [5%])
28	24	NA	6	56.3%/6	5/6	Pyrexia 1, perianal abscess 1	Intralesionally	Phase I/IIa
29	43	NA	12	81%/2	NA	Pain 60%, anal bleeding 7%	Intravenously	Phase I
				88%/12
6	9	NA	8	78%/1.5	None	Pain (3)	Intravenously	Phase I
						Diarrhea (2)
8	26 (mPP)	NA	24	80.8%/24	None	No SAEs	Intravenously	Phase II
	36 (ITT)			75%/24

mPP, modified per protocol; SAE, severe adverse event; ITT, intention-to-treat.

Another source of stem cells is human umbilical cord MSCs (hUCMSCs), which are derived from umbilical cords and have unique characteristics in clinical studies. hUCMSCs are readily available, have differentiation potential, proliferation ability, low immunogenicity, and few ethical limitations. Moreover, hUCMSCs retain the potential for multidirectional differentiation, even after multiple passages and cryopreservation. Based on these properties, hUCMSCs could be used as seed cells for generation of pluripotent stem cells for use in clinical applications.

HSC Transplantation

HSCs have self-renewal activity and can differentiate into diverse types of mature blood cells, which to date have primarily been used to treat hematological malignant tumors. HSCs also have immune function, including control of inflammation, regulation of immune response, and immune-cell regeneration.³¹ During tissue recovery and restoration in the normal immune system, HSCs can home to damaged tissue and migrate to the injury site directly or differentiate into epithelial or immunomodulatory cells.^11,32 In theory, allogeneic HSC transplantation (HSCT) should yield similar therapeutic effects to autologous HSCT, which requires complete ablation of the patient's existing immune system. Due to the high rate of death caused by allogeneic HSCT, the number of clinical trials concerning use of autologous HSCT to treat refractory Crohn's disease is limited.

Richard et al.³³ first reported two patients with severe Crohn's disease, as defined by CDAI >250, who were treated with autologous HSCT obtained after isolation, expansion, and enrichment. The CDAI for these patients remained <100 for 1 year, suggesting that autologous HSCT may be a useful tool to treat severe Crohn's disease. A Phase I clinical study performed by Oyama et al.³⁴ involving 12 patients with refractory Crohn's disease showed that during the follow-up period that ranged between 7 and 37 months, 11 patients achieved sustained remission, and only one patient had recurrence of active Crohn's disease within 15 months of HSCT. Meanwhile, Cassinotti et al.³⁵ treated four moderate-severe Crohn's disease patients with unselected stem cells isolated from peripheral blood (PBSCs) during autologous HSCT and found that three patients maintained remission after a median follow-up of 16.5 months. There were no deaths or life-threatening infections in this study. A Phase I/II trial with long-term follow-up of autologous HSCT for severe refractory Crohn's disease performed by Burt et al.³⁶ showed a clinical relapse-free survival percentage of 91% at 1 year after transplantation and 19% at 5 years. Hommes et al.³⁷ reported a similar long-term follow-up outcome. However, in a randomized clinical trial to examine outcomes for refractory Crohn's disease treated with autologous HSCT or conventional therapy, the HSCT-treated group showed no statistically significant improvement in sustained disease remission at 1 year, and there were some indications that significant toxicity may be associated with HSCT.³⁸ Overall, these studies highlight the need for larger randomized clinical trials to characterize the effects and outcomes for HSCT (Table 3).

Table 3.

Clinical trials for treatment of Crohn's disease with autologous hemapoietic stem-cell transplantation

Ref.	Patients (n)	Pre-CDAI	Follow-up (months)	Remission (type/n/time in months)	Recurrence (n/time in months)	Adverse event (n)	Post-CDAI	Clinical Phase
33	2	305, 271	15, 12	2	None	Culture-negative fever (2)	58, 60	Phase I
34	12	291 (250–358)	18.5 (7–37)	11	1/15	SAEs (76),	75 (18–185)	Phase I
						No SAEs (265)
35	4	319 (272–345)	16.5 (11–20)	Clinical/4/3	1/4	Culture-negative fever (4), chest pain (1), BK virus-related macrohematuria	91 (56–102)	Phase I–II
				Endoscopic/4/3
36	24	235 (91–307)	64 (6–108)	24/12	9% at 1 year	No SAEs	<150	Phase I/II
37	3	NA	5 (48–72)	2	1/2, 1/60, 1/18	Patient 2 had a quick and transient deterioration of kidney function	NA	Phase I
38	23	326 (251–414)	12	2 (8.7%)	NA	1 died at 20 days	<150	Phase I

Designing Tomorrow's Therapies for Crohn's Disease Based on Current Results

Current preclinical and clinical data suggest that among stem cell–based therapies for patients with refractory Crohn's disease, MSCs may be the optimal cell type for transplantation. Indeed, MSC therapy for Crohn's disease has unique advantages in that MSCs are readily obtained and produce a smaller wound at the harvesting site, as well as having a lower probability of virus infection. Moreover, autologous MSCs may have low immunogenicity, which can reduce the risk of GvHD. MSCs also have immunoregulatory activity that promotes matrix support as well as paracrine and homing migration.¹ MSCs used in the above-mentioned trials were predominantly harvested from autologous tissue and were reported to be a safe and feasible option for stem cell–based therapies. However, isolation and expansion of autologous MSCs to yield sufficient numbers of cells for transplantation requires several weeks, which can be an issue for patients with severe Crohn's disease. More research is also needed to determine the optimal administration route and stem-cell dose for transplantation. One safety concern for stem cell–based therapy is the potential for malignant transformation of the administered cells, although the design of personalized treatments can reduce these side effects while also decreasing preparation and treatment costs.

Although there is no definitive study concerning the mechanisms associated with the beneficial effects provided by MSC treatment for Crohn's disease, some basic studies^39,40 demonstrated that MSCs exert an immunomodulatory action in the presence of inflammatory mediators (e.g., interferon-γ, TNF-r, interleukin-10). MSCs also inhibit Th17 cell differentiation and can exert anti-inflammatory effects by inducing the incidence of a regulatory T-cell phenotype in these cells.⁴¹

Although the CDAI of Crohns he CDAIe patients decreases after infusion of autologous HSCs, the clinical outcome was not satisfactory, and negative effects such as systemic lupus erythematosus, BK virus infection, fever, chest pain, renal function impairment, or even death have occurred in some patients. In patients treated with BMSCs and AMSCs, the CDAI and perianal disease activity index (PDAI) decreased significantly. Furthermore, most of the wound area healed, whereas MRI and computed tomography showed gradual shrinkage of internal fistulas. Although these patients still experienced some side effects, including headache, abdominal pain, and fever, no severe complications such as systemic lupus erythematosus were seen, and the patients' quality of life improved gradually, which together suggests that BMSC and AMSC transplantation could be a safe and efficacious treatment for Crohn. tdisease. Such approaches are urgently needed, since of the 17 currently approved stem-cell therapy products, only two aim to improve Crohnket internationallyl safety and effic.¹³

Despite the low success rate for biological agents such as anti-TNF-α monoclonal antibodies, biologics are usually used as a first-line therapy for Crohn's disease fistula. In general, current treatments for Crohn's disease with fistula are unsatisfactory due to a failure to achieve complete fistula closure, lower recurrence rates, and limit adverse effects. Traditional Crohn's disease treatments involving steroid, anti-TNF, thiopurines, methotrexate, and infliximab may create an inflammatory imbalance that could be avoided by wider use of MSC-based therapies.

Conclusions

MSC therapy has unique advantages and significant potential clinical applications as a novel therapeutic option for Crohn's fistula. MSC-based therapies show durable efficacy with low recurrence, even in cases for which healing was not achieved with biologics or for which conventional surgical procedures cannot be performed.

Footnotes

Acknowledgments

This work was supported by the Hi-tech Research and Development Program (863 Program) of China (2012AA021004), Key Programs for Science and Technology Development of Sichuan Province (2014SZ0122), and the National Natural Science Foundation of China (81472486).

Author Disclosure

No competing financial interests exist.

References

Trounson

, McDonald

. Stem cell therapies in clinical trials: progress and challenges. Cell Stem Cell, 2015; 17:11–22.

Locke

, Windsor

, Dunbar

. Human adipose-derived stem cells: isolation, characterization and applications in surgery. ANZ J Surg, 2009; 79:235–244.

Flores

, Gómez-Gómez

, Masedo-González

, et al. Stem cell therapy in inflammatory bowel disease: a promising therapeutic strategy?. World J Stem Cells, 2015; 7:343.

Chen

Q-Q

, Yan

, Wang

C-Z

, et al. Mesenchymal stem cells alleviate TNBS-induced colitis by modulating inflammatory and autoimmune responses. World J Gastroenterol, 2013; 19:4702–4717.

Baumgart

, Sandborn

. Crohn's disease. Lancet, 2012; 380:1590–1605.

Cho

, Lee

, Park

, et al. Autologous adipose tissue-derived stem cells for the treatment of Crohn's fistula: a Phase I clinical study. Cell Transplant, 2013; 22:279–285.

Crescenzo Di Stazio

, Limongelli

, Guadagni

, et al. Treatment of complex perianal fistulas in Crohn disease: infliximab, surgery or combined approach. Can J Surg, 2010; 53:299.

Cho

, Park

, Yoon

, et al. Long-term results of adipose-derived stem cell therapy for the treatment of Crohn's fistula. Stem Cells Transl Med, 2015; 4:532–537.

Van Koperen

, Safiruddin

, Bemelman

, et al. Outcome of surgical treatment for fistula in ano in Crohn's disease. Br J Surg, 2009; 96:675–679.

10.

Ritchie

, Sackier

, Hodde

. Incontinence rates after cutting seton treatment for anal fistula. Colorectal Dis, 2009; 11:564–571.

11.

Gazouli

, Roubelakis

, Theodoropoulos

. Stem cells as potential targeted therapy for inflammatory bowel disease. Inflamm Bowel Dis, 2014; 20:952–955.

12.

Chinnadurai

, Ng

, Velu

, et al. Challenges in animal modelling of mesenchymal stromal cell therapy for inflammatory bowel disease. World J Gastroenterol, 2015; 21:4779.

13.

Syed

, Evans

. Stem cell therapy market. Nat Rev Drug Discov, 2013; 12:185–186.

14.

English

, French

, Wood

. Mesenchymal stromal cells: facilitators of successful transplantation?. Cell Stem Cell, 2010; 7:431–442.

15.

Keating

. Mesenchymal stromal cells. Curr Opin Hematol, 2006; 13:419–425.

16.

O'Donoghue

, Chan

, de la Fuente

, et al. Microchimerism in female bone marrow and bone decades after fetal mesenchymal stem-cell trafficking in pregnancy. Lancet, 2004; 364:179–182.

17.

Duijvestein

, Vos

ACW

, Roelofs

, et al. Autologous bone marrow-derived mesenchymal stromal cell treatment for refractory luminal Crohn's disease: results of a Phase I study. Gut, 2010; 59:1662–1669.

18.

Ciccocioppo

, Bernardo

, Sgarella

, et al. Autologous bone marrow-derived mesenchymal stromal cells in the treatment of fistulising Crohn's disease. Gut, 2011; 60:788–798.

19.

Molendijk

, Bonsing

, Roelofs

, et al. Allogeneic bone marrow–derived mesenchymal stromal cells promote healing of refractory perianal fistulas in patients with Crohn's disease. Gastroenterology, 2015; 149:918–927.

20.

Liang

, Zhang

, Wang

, et al. Allogeneic mesenchymal stem cell transplantation in seven patients with refractory inflammatory bowel disease. Gut, 2012; 61:468–469.

21.

Bassi

, Pacelli

, Carusone

, et al. Adipose-derived stromal cells (ASCs). Transfus Apher Sci, 2012; 47:193–198.

22.

García-Olmo

, García-Arranz

, Herreros

, et al. A Phase I clinical trial of the treatment of Crohn's fistula by adipose mesenchymal stem cell transplantation. Dis Colon Rectum, 2005; 48:1416–1423.

23.

Garcia-Olmo

, Garcia-Arranz

, Herreros

. Expanded adipose-derived stem cells for the treatment of complex perianal fistula including Crohn's disease. Expert Opin Biol Ther, 2008; 8:1417–1423.

24.

Garcia-Olmo

, Herreros

, Pascual

, et al. Expanded adipose-derived stem cells for the treatment of complex perianal fistula: a Phase II clinical trial. Dis Colon Rectum, 2009; 52:79–86.

25.

Garcia-Olmo

, Herreros

, Pascual

, et al. Treatment of enterocutaneous fistula in Crohn's disease with adipose-derived stem cells: a comparison of protocols with and without cell expansion. Int J Colorectal Dis, 2009; 24:27–30.

26.

Guadalajara

, Herreros

, De-La-Quintana

, et al. Long-term follow-up of patients undergoing adipose-derived adult stem cell administration to treat complex perianal fistulas. Int J Colorectal Dis, 2012; 27:595–600.

27.

Herreros

, Garcia-Arranz

, Guadalajara

, et al. Autologous expanded adipose-derived stem cells for the treatment of complex cryptoglandular perianal fistulas: a Phase III randomized clinical trial (FATT 1: fistula Advanced Therapy Trial 1) and long-term evaluation. Dis Colon Rectum, 2012; 55:762–772.

28.

Portilla

FDL

, Alba

, García-Olmo

, et al. Expanded allogeneic adipose-derived stem cells (eASCs) for the treatment of complex perianal fistula in Crohn's disease: results from a multicenter Phase I/IIa clinical trial. Int J Colorectal Dis, 2013; 28:313–323.

29.

Lee

, Park

, Cho

, et al. Autologous adipose tissue-derived stem cells treatment demonstrated favorable and sustainable therapeutic effect for Crohn's fistula. Stem Cells, 2013; 31:2575–2581.

30.

Julián Panés

DG-O

, Gert Van

Assche

, et al. Expanded allogeneic adipose-derived mesenchymal stem cells (Cx601) for complex perianal fistulas in Crohn's disease: a Phase 3 randomised, double-blind controlled trial. Lancet, 2016; 388:1281–1290.

31.

Copelan

. Hematopoietic stem-cell transplantation. New Engl J Med, 2006; 354:1813–1826.

32.

Kavanagh

DPJ

, Kalia

. Hematopoietic stem cell homing to injured tissues. Stem Cell Rev Rep, 2011; 7:672–682.

33.

Burt

, Traynor

, Oyama

, et al. High-dose immune suppression and autologous hematopoietic stem cell transplantation in refractory Crohn disease. Blood, 2003; 101:2064–2066.

34.

Oyama

, Craig

, Traynor

, et al. Autologous hematopoietic stem cell transplantation in patients with refractory Crohn's disease. Gastroenterology, 2005; 128:552–563.

35.

Cassinotti

, Annaloro

, Ardizzone

, et al. Autologous haematopoietic stem cell transplantation without CD34+ cell selection in refractory Crohn's disease. Gut, 2008; 57:211–217.

36.

Burt

, Craig

, Milanetti

, et al. Autologous nonmyeloablative hematopoietic stem cell transplantation in patients with severe anti-TNF refractory Crohn's disease: long-term follow-up. Blood, 2010; 116:6123–6132.

37.

Hommes

, Duijvestein

, Zelinkova

, et al. Long-term follow-up of autologous hematopoietic stem cell transplantation for severe refractory Crohn's disease. J Crohns Colitis, 2011; 5:543–549.

38.

Hawkey

, Allez

, Clark

, et al. Autologous hematopoetic stem cell transplantation for refractory Crohn disease: a randomized clinical trial. JAMA, 2015; 314:2524–2534.

39.

Lechanteur

, Briquet

, Giet

, et al. Clinical-scale expansion of mesenchymal stromal cells: a large banking experience. J Transl Med, 2016; 14:1–15.

40.

Ciccocioppo

, Cangemi

, Kruzliak

, et al. Concise review: cellular therapies: the potential to regenerate and restore tolerance in immune-mediated intestinal diseases. Stem Cells, 2016; 34:1474–1486.

41.

Ghannam

, Pène

, Torcymoquet

, et al. Mesenchymal stem cells inhibit human Th17 cell differentiation and function and induce a T regulatory cell phenotype. J Immunol, 2010; 185:302–312.