Clinical efficacy and safety of stem cells in refractory Crohn’s disease: A systematic review

Abstract

Refractory Crohn’s disease, especially when complicated by complex perianal fistula, seriously reduces the patients’ quality of life. Preclinical and clinical studies indicate stem cells may be a promising therapy for refractory Crohn’s disease.

Full text

Accepted Manuscript
Clinical efficacy and safety of stem cells in refractory Crohn’s disease: A systematic
review
Lei Ye, Xiao-Wei Wu, Na Yu, Jing-Xin Pan, Lian-Ming Liao, Fang-Yu Wang, PhD
PII: S2352-1775(16)00002-9
DOI: 10.1016/j.jocit.2016.01.001
Reference: JOCIT 5
To appear in: Journal of Cellular Immunotherapy
Received Date: 15 September 2015
Revised Date: 15 January 2016
Accepted Date: 15 January 2016
Please cite this article as: Ye L, Wu X-W, Yu N, Pan J-X, Liao L-M, Wang F-Y, Clinical efficacy
and safety of stem cells in refractory Crohn’s disease: A systematic review, Journal of Cellular
Immunotherapy (2016), doi: 10.1016/j.jocit.2016.01.001.
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Clinical efficacy and safety of stem cells in refractory Crohn’s disease :
A systematic review
Lei Ye
1
, Xiao-Wei Wu
1
, Na Yu
1
, Jing-Xin Pan
2
, Lian-Ming Liao
3
Fang-Yu
Wang
1
1. Department of Gastroenterology and Hepatology, Jinling Hospital,
Medicine School of Nanjing University, Nanjing 210002, China
2. Medicine School of Nanjing University, Nanjing 210009, China
3. Central Laboratory, Union Hospital of Fujian Medical University,
Fuzhou, 350000, China
Author contributions: Ye L and Wang FY designed the study; Ye L ,Pan JX
and Yu N searched all articles; Ye Land Wu XW developed the methodology;
Ye L, Wu XW, Yu N, Liao LM and Wang FY wrote the manuscript.
Correspondence to: Fang-Yu Wang, PhD, Department of Gastroenterology
and Hepatology, Jinling Hospital, Medicine School of Nanjing University,
305 Zhongshan East Road, Nanjing 210002, Jiangsu Province, China.
E-mail:wangfangyul@126.com

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Abstract
Background Refractory Crohn’s disease, especially when complicated by complex
perianal fistula, seriously reduces the patients’ quality of life. Preclinical and clinical
studies indicate stem cells may be a promising therapy for refractory Crohn’s disease.
Objective To systematically review evidence on clinical efficacy and safety of stem cells
in refractory Crohn’s disease. Methods A detailed search was performed to identify
randomized controlled trials (RCT), systematic reviews of RCTs or high-quality
non-RCTs published before September 2015 in the Cochrane Library, PubMed, Medline,
EMBASE, and the ISI Web of Knowledge databases. Search terms included: MSC
(mesenchymal stem cell), stem cells, HSC(hematopoietic stem cell),IBD (inflammatory
bowel disease), CD (Crohn’s disease), UC (ulcerative colitis). Two authors independently
extracted data for analysis using predefined selection criteria and quality indicators.
Trials were also retrieved from the website of www.Clinicaltrials.gov. Results 18 of the
225 articles identified met the inclusion criteria. However, there were only two studies
that had control groups. Therefore, a brief qualitative analysis of the evidence was
considered to be more adequate. It seems stem cells can reduce Crohn’s Disease Activity
Index (CDAI) and help alleviate CD symptoms. Moreover, the incidence of serious
adverse events caused by stem cell transplantation was very low (1.75%, 7/400) .
Conclusions Stem cell therapy for CD is very promising, but large, multicenter and
randomized clinical trials are needed to further support their therapeutic benefits.
Key Words: stem cells, Crohn’s disease, clinical efficacy, mesenchymal stem cell,
hematopoietic stem cell

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Introduction
The etiology of Crohn’s disease (CD) is still not well understood. Interaction among the
gut microbiota, the intestinal mucosa and the host immune system has been implicated in
the pathogenesis of CD[1]. Perianal fistula is the most notorious complication of CD,
which is formidable for both patients and clinicians, as there are around 25-30% patients
having no other alternative choices but accepting surgery[2]. The past two decades have
seen an explosion of scientific and clinical interest in stem cell transplantation for a
variety of diseases including CD. There are several kinds of candidate stem cells with
potential application for CD, such as hematopoietic stem cells (HSCs), amniotic fluid
stem cells (AFSCs), induced pluripotent stem cells (iPSCs) and mesenchymal stem cells
(MSCs)[3]. Among them, HSCs and MSCs have been evaluated in clinical trials [4].
Thus, the present review will focus on published clinical trials using HSCs and MSCs.
Accumulating literature has showed MSCs and HSCs may be beneficial for CD
patients. We searched all studies available to evaluate clinical efficacy and safety of stem
cells in severe refractory CD. Located in bone marrow, HSCs can give rise to all blood
cells and transplanted HSCs can reconstitute the hematopoietic system of the hosts [5].
HSCs transplantation is widely considered to be a potential therapy for severe
autoimmune diseases[6].With the synergistic effect of adhesion molecules and platelets,
HSCs are able to home to the injured tissues [7].
Originally isolated from bone marrow, MSCs can also be isolated from a variety of
other sources such as placenta, umbilical cord, adipose tissue, teeth and menstrual
fluid[8]. Bone marrow-derived MSCs are best characterized for their biological functions

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and most widely used for immunoregulation and regenerative cell therapy[9-11]. Like
HSCs, they are also capable of migrating to injured tissues. In contrast to HSCs, MSCs
transplantation do not induce graft-versus-host-disease (GVHD) and have even been used
to treat GVHD[12].
Methods
Research question and objectives:
This review aimed to answer the following question: Is autologous/allogeneic stem cell
transplantation a safe and effective treatment for refractory CD? The primary objectives
were to assess whether patients receiving stem cells transplantation achieved:
1. any response to the stem cell-based therapies;
2. any alleviation in both clinical symptoms and endoscopic findings of CD;
The secondary objectives were to assess whether the frequency and nature of adverse
events was acceptable.
Data Sources
The search strategy was developed in accordance with the Center for Reviews and
Dissemination’s Guidance for Systematic Reviews in Health Care[13]and the Preferred
Reporting Items for Systematic Reviews and Meta-Analyses(PRISMA)guidelines[14].
The Cochrane Library, PubMed, Medline, EMBASE, and the ISI Web of Knowledge
databases were searched for papers published from the data of the database set-up to
September 2015 using the following key words: MSC (mesenchymal stem cell), stem
cells, IBD (inflammatory bowel disease), CD (Crohn’s disease), UC (ulcerative colitis),
and combined words using Boolean logic (AND, OR). We also searched

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ClinicalTrials.gov for relevant clinical studies. To increase sensitivity, no restrictions
were placed on study/trial/review type.
Eligibility criteria for the review (PICOS elements)
Detailed information on the participants, interventions, comparisons, and outcomes to be
included was provided in Table 1, as advocated by the 2009 Centre for Reviews and
Dissemination handbook.
Study Selection
Two investigators (LY and NY) undertook the initial screen of titles and abstracts. Any
disagreement about selection decisions was then discussed until concordance was
reached. Full manuscripts of papers identified as potentially relevant were reviewed
independently by the same two authors according to the pre-specified eligibility criteria
showed in Table 1. Duplicate articles and those published in a language other than
English were excluded. The selection process was showed in Figure 1.
Critical evaluation and quality assessment
Researches may vary considerably in methodology in the design or conduct of a study,
obscuring the benefit/harm of an intervention. Each study included in a review must
therefore undergo a quality assessment process. In accordance with the Cochrane
Reviewers’ Handbook and the Newcastle-Ottawa[15], each literature was assessed by the
same investigators. Disagreement was resolved according to the pre-defined criteria.
Results and data synthesis
Data extraction sheets were completed for each study. Quantitative data analyses were
performed for eligible studies with adequate data; due to study heterogeneity and the

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small number of incoporated studies, meta-analysis was considered inappropriate for this
review. Therefore, a brief qualitative analysis of the evidence was presented in narrative
form shown in Tables 2, 3, and 4.
Review of objectives
Do patients response to the stem cell-based therapies?
As shown in Tables 2, 3, and 4, a total of 18 articles were eligible for the qualitative
analyses. To be more specific, there were 6 clinical trials using HSCs and 12 using MSCs
respectively. Clinical response was defined as a decrease in Crohn’s Disease Activity
Index (CDAI) >100. Endpscopic improvement referred to a decrease of Crohn’s disease
endoscopic index of severity greater than 5, or a Crohn’s disease endoscopic index of
severity of less than 3. Considering some patients have developed perianal fistulas, the
combined measurement of clinical response was defined as fistula closure. Totally there
were 483 patients (Tables 2, 3, and 4) with 400 evaluable CD patients.
The data concerning luminal CD was limited. Five available studies had 45 CD
patients, with improvement in clinical signs in 42. The results indicated a promising role
of MSCs in CD therapy. As regarding the local treatment of perianal CD, similar
satisfactory results have been published. Tow recently published studies [31,32],one
study showed that a complete closure was achieved in 27 out of 33 patients after the final
injection of ASCs [32]. However, a study conducted by Herreros et al[33] including 200
participants stated that ASCs alone or in combination with fibrin glue was equivalent to
fibrin glue alone.
Is there any remission in signs or symptoms of Crohn’s disease?

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Of the six studies examining the efficacy of HSCs, the results were encouraging.
Among 66 patients, 57 CD patients achieved complete remission as indicated by physical
examination, CDAI level(CDAI<150), bowel ultrasound sonography and/or radiological
exam [16-21]. HSCs transplantation seemed to be superior to MSCs transplantation in
terms of remission rate (86.36% (57/66) vs 44.90% (154/343)). However, due to the
heterogeneity of the studies, caution should be took when HSCs and MSCs are compared
with. What’s more, there were few patients receiving HSCs transplantation. On the
contract, there were 2 multicenter, randomized and double-blinded trials that evaluated
the efficacy of MSCs [28,33].
Preparation of MSCs is more easy and cheaper than that of HSCs, which needs
leukapheresis. In addition, no donor-recipient matching is required for on-the-shelf
MSCs from donors (such as Prochymal
TM
) [22], which is in contract to HSCs, for it may
cause GVHD after transplantation.
Whether adverse events are acceptable?
Each study evaluated the safety of the treatment and found that the incidence of serious
adverse events (SAEs) was low. Only 3 studies reported several cases of SAEs (Tables 2,
3, and 4). Hasselblatt et al[19]reported 4 individuals who developed SAEs during stem
cell mobilization. Of which, one patient suffered prolonged neutropenic fever and
reversible acute renal failure as well as vaginal bleeding. Lopez-cubero et al[20] recorded
1 case of SAEs. During the long-term follow-up period (4.5-15.3 yr), 1 patient developed
septicemia which was considered to be not related to stem cell therapy. In another study
reported by De la Portilla et al[30], there were 2 cases of SAEs, which were “pyrexia”

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and “perianal abscess”respectively. Overall, the incidence of SAEs was low.
Discussion
Up to now, mechanisms for stem cells in treating CD are still not well understood.
Stem cells may suppress the immune reaction in injured tissues and differentiate into
epithelia cells to promote tissue healing. However, what happens during the
differentiation process remains mysterious. In the present review, we included 6 and 12
trials using HSCs and MSCs respectively. Despite variations in dosage, timing, and
frequency of administration, stem cells seemed to significantly benefit the patients with
refractory CD in terms of both primary and secondary endpoints. These results were very
encouraging.
Inflammatory bowel diseases include UC and CD. Though they share similar
pathologic and epidemiological features, the clinical manifestations differ from each
other[34]. Unlike UC, CD causes intestinal transmural inflammation (ulceration) and
fistulas may occur in up to 50% of the patients [2]. Moreover, the global incidence and
prevalence of CD are increasing [35]. To date, much progress has been made in the
pharmacological therapy for CD [36].CD therapy is based on a so-called step-up
approach. 5-Aminosalicylates remain a mainstay of treating first-episode CD. If it failed,
patients would be given by glucocrticoids to achieve rapid symptom relief. For further
therapy, glucocrticoids would be followed by methotrexate or acetazolamide or 6-MP or
cyclosporine follow to gain long-term disease control[37]. For patients with severe CD
who fail corticosteroids and immunosuppressor such as methotrexate, then biological
agents are a viable alternative.They target specific molecules.Infliximab, one of anti-TNF

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agents, is a chimeric monoclonal antibody directed against the proinflammatory cytokine
tumor necrosis factor- α, and has been approved by the US Food and Drug
Administration for use in moderate to severe CD. Anti-adhesion molecules represent a
new promising immunmodulatory therapy in CD. Vedolizumab is a monoclonal antibody,
blocking the pathway of mucosal addressin cell adhesion molecule-1, was approved in
May 2014 for treating CD[38]. The ultimate goal for CD therapy is to gain sustained
clinical and endoscopic remission[39]. Nevertheless, there are still about half of CD
patients who do not response to pharmacological therapy and have to receive intestinal
resection in about 10 years after CD onset[40].
Stem cells can home to the damaged tissues and differentiate into specific cells to
help restore tissue function or regulate host immune function through an army of
cytokine and signaling molecules[41-43]. Before HSC transplantation, patients need to
undergo a high-dose immune ablation regimen to eliminate T-lymphocytes and memory
T cells. After retransfusion of HSCs, they generate naïve cells and restore the immune
system[44]. In addition, HSCs may integrate into the existing tissues and differentiate
into other types of cells[42]. MSCs may exert its action by immunoregulation [43]. To be
more exciting, Sanchez et al [45]have shown for the first time that specific inhibition of
SMAD-2/3 signaling pathway in hESCs facilitates the conversion of hESCs into
multipotent MSCs with fully immunosuppressive and anti-inflammatory properties in
vivo, opening up new avenues for future clinical applications of MSCs. Information on
homing and differentiation of transplanted stem cells in the injured intestines are rapidly
accumulating. In indomethacin(IDM)-treated rats, Qu et al[46] found that transplanted

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BM-MSCs labelled with PKH26 resided in the injured intestinal areas, mainly in the
crypts and villi. Surprisingly, cotransplanted stem cells expressed Lgr-5, a marker of
intestinal stem cells(ISCs) two weeks after cell transfusion[46-47], which indicated that
transplanted stem cells are capable of migrating to the damaged tissue and further
transdifferentiate into ISCs to restore epithelium function.
While a large number of clinical trials have been completed or underway, there is a
lack of strong evidence of their effectiveness and safety. Some experts pointed out that
autologous transplantation may put patients at a risk of receiving the genetically
defective immune cells, while allogeneic transplantation has a potential to cause
myeloablation[35].Moreover, recent studies stated that cultured MSC may undergo
transdifferentiation into proinflammatory cells in the chronic inflammatory environment.
Yasuhiro et al [48]first demonstrated that MSCs secreted IL-7 and play a pathological
role in IBD by forming the niche for colitogenic CD4 memory T cells in bone marrow.
More researches are needed to further understand the mechanisms of stem cells before
they enter clinical application. We still do not know what kind of stem cells is best for
CD. In addition to HSCs and MSCs, amniotic fluid stem cells have been used and the
results were encouraging [49]. IFN-γ-treated human amniotic fluid stem cells could
inhibit T-cell proliferation and increase CD4(+) CD25(+) FOXP3(+) regulatory T cells.
In vivo, IFN- γ-treated human amniotic fluid stem cells were capable of
immunoregulatory function, promoting allograft survival in a mouse model of allogeneic
skin transplantation. Thus, amniotic fluid stem cells may be a new source of stem cells
for immunotherapy.

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There exit several limitations of the review. First, the number of included studies was
small and none of these trials were RCT. Second, most of the trials enrolled a few
patients. In one trial with 200 patients enrolled, there was no greater improvement in
patients receiving ASCs and fibrin glue compared to those receiving fibrin glue alone.
Third, quantitative data analyses could not be performed due to heterogeneity in
designing, classifications of fistulas, treatment and the duration of follow-up.
Conclusion
Stem cell therapy is a field that has developed considerably in the past decade.
Currently available clinical data indicates that stem cell therapy for CD is very promising.
In spite of the huge accomplishments, stem cell-based therapy is still in its infancy. There
are only 2 ongoing Phase III trials using stem cells for the treatment of CD (Table 5).
However, there is a long way to go before they are officially approved to be used for CD.
Further large randomized trials are warranted. Enormous advances are needed to bridge
the translational gap between the benchtop researches and clinical applications of stem
cells for CD.
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Table 1
Pre-specified eligibility criteria
Participants Interventions Comparison Outcomes Study types
CD patients,age>=18yr
Refractory to or
unsuitable for current
available therapies
Autologous/Allogeneic
MSCs
Autologous/Allogeneic
HSCs
Self-control or placebo
controls using fibrin glue
or routine therapies
Clinical/Endoscopic
remission/response
(defined by CADI)
perianal fistulas
healing/closure
SAEs should be included
RCTs/Systematic reviews
of RCTs/ high-quality
Non-RCTs
CD: Crohn’s disease; yr: year; MSCs: mesenchymal stem cells; HSCs: hematopoietic stem cells;CDAI: Crohn’s Disease Activity Index;SAEs:
serious adverse events;RCT: randomized controlled trials
Table 2
Hemapoietic stem cells transplantation studies in Crohn’s disease
Study ID Participants Procedure Remission(number of
patients, time of
evaluation)
SAEs Recurrence(number
of patients, time of
evaluation)
Oyama et al
[16]
12 Autologous HSCs
Clinical:11 (12m) None 1 (15m)
Burt et al
[17]
24 Autologous HSCs
Clinical:24 (6-12m) 1SAEs not related
9% (12m)
37% (24m)
43% (36m)
61% (48m)
81% (60m)
Cassionotti et al
[18]
10 Autologous HSCs
Clinical:10 (3m)
Endoscopic:5 (3m) None 20% (12m)
50% (24m)

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60% (36m)
70% (48m)
70% (60m)
Hasselblatt et al
[19]
12 Autologous HSCs
Clinical:5 (6m)
Endoscopic:5 (6m) 4SAES related
(fever,renal failure) 7 (37.2m)
Lopez-cubero et al
[20]
6 (5 evaluable) Allogeneic HSCs
Clinical:4 (4.5-15.3yr) 1SAEs related
septicemia 1 (1.5yr)
Hommes et al
[21]
3 Autologous HSCs
Clinical:3 (5-6yr) None 1 (2yr)
HSCs: hematopoietic stem cells; m: month; yr: year; SAEs: serious adverse events
Table 3
Mesenchymal stem cell transplantation studies in luminal Crohn’s diseases
Study ID Participants Procedure Response/Remission(num-
ber of patients, time of
evaluation)
SAEs Recurrence(number
of patients, time of
evaluation)
Onken et al
[22]
10 (9 evaluable) Prochymal
TM
Clinical:9/3 (14d) 1SAES unrelated NA
Duijvestein et al
[23]
10 (6 evaluable) Autologous BMMSCs
Clinical:3/0 (6w)
Endoscopic:2/0 (6w) None NA
Liang et al
[24]
7 (4CD/3UC) Allogeneic
BMMSCs/UC-MSCs Clinical:7/3 (3m)
Endoscopic:3/0 (3-5m) None 2 (6-7m)
Forbes et al
[25]
16 (15 evaluable) Allogeneic BMMSCs Clinical:12/8 (42d)
Endoscopic:7/0 (42d) 1 SAES unrelated
NA
Lazebnik et al
[26]
50 (11CD/39UC) Allogeneic BMMSCs Clinical:11/0 (4-8m,CD)
39/0 (4-8m,UC) NA NA

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CD: Crohn’s disease; UC: ulcerative colitis; BMMSCs: Bone marrow-derived MSCs; UC-MSCs:umbilical cord-derived MSCs; ProchymalTM:
Ex vivo cultured mesenchymal stem cells from marrow of screened, healthy volunteers; SAES: serious adverse events; NA: Not available;
d: day; w: week; m: month
Table 4
Mesenchymal stem cell transplantation studies in perianal Crohn’s diseases
Study ID Participants Procedure Response/Closure(number of
patients, time of evaluation) SAEs Recurrence(number
of patients, time of
of evaluation)
Garcia-Olmoet al
[27]
5 (4 evaluable)
Autologous ASCs
8Fistulas in 4 :2/6 (8w) None NA
Garcia-Olmoet al
[28]
49 (14CD)
Autologous
Group A:25 (7CD)
ASCs+Fg
Group B:24 (7CD)
Fg
Group A:NA/1 (7CD,8w)
Group B:2/5 (7CD,8w) None Group B:3 (12m)
Data for CD:NA
Ciccocioppo et al
[29]
12 (10 evaluable)
Autologous BMMSCs
3/7 (12m) None None (12m)
De laPortilla et al
[30]
24 (22 evaluable)
Allogeneic ASCs
23Fistulas in 22:5/18 (6m) 2SAES
Pyrexia
Perianal abscess
NA
Cho et al
[31]
10 Autologous ASCs
1/3 (2m) None 3 (8m)
Lee et al
[32]
33
Autologous ASCs
5/27 (2m) None 3 (12m)
Herreros et al
[33]
200 Autologous Group A:NA/25 (12w) None NA

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Group A: ASCs
GroupB:ASCs+Fg
Group C: Fg
Group B: NA/26 (12w)
Group C: NA/22 (12w)
CD: Crohn’s disease; ASCs: Adipose-derived MSCs; Fg: Fibrin glue; SAES: serious adverse events; NA: Not available; w: week; m: month
Table 5
Large ongoing Phase III trials using stem cells for perianal Crohn’s diseases
Trial Code Condition Sponsor Status Intervention Enrollment
NCT0154157 Perianal fistula Tigenix Active, not recruiting Two groups:
ASCs, placebo 208
NCT00482092 Perianal fistula Osiris Therapeutics Active, not recruiting Three groups:
Placebo High-dose ASCs
Low-dose ASCs
270
ASCs: Adipose-derived MSCs; Source:Clinicaltrials.gov

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