Therapeutic potential of mesenchymal stromal cells in experimental

Bronchopulmonary dysplasia: protocol for a systematic review.

1Sajit Augustine, 2Marc T Avey, 2,3David Moher, 4,5Bernard Thébaud.

* Corresponding Author: Bernard Thébaud. Email: bthebaud@toh.on.ca, Ottawa

Hospital Research Institute, Sprott Centre for Stem Cell Research, 501 Smyth Rd.,

Ottawa, ON K1H 8L6, Canada, Phone: 613-737-8899, Fax: 613-739-6294.

1. Department of Neonatology, Children’s Hospital of Eastern Ontario, Ottawa,

ON, Canada.

2. Clinical Epidemiology Program, The Ottawa Hospital Research Institute,

Ottawa, ON, Canada

3. School of Epidemiology, Public Health and Preventive Medicine, Faculty of

Medicine, University of Ottawa, Ottawa, ON, Canada

4. Ottawa Hospital Research Institute, Regenerative Medicine Program, Sprott

Centre for Stem Cell Research, Ottawa, Ontario, Canada

5. Department of Pediatrics, Children’s Hospital of Eastern Ontario Research

Institute, University of Ottawa, Ottawa, Ontario, Canada

Abstract

Background: Bronchopulmonary dysplasia (BPD), the chronic lung disease

of prematurity, complicates the medical course of approximately 40% of

infants born <28 weeks preterm, resulting in long term pulmonary and

neurodevelopmental sequelae with a high economic burden. Current

therapies are supportive. Preclinical evidence suggests that mesenchymal

stromal cells (MSC) improve neonatal lung inflammation, function and

structure in experimental models of BPD. This systematic review of

preclinical studies in experimental BPD will assess the current evidence on

the therapeutic potential of MSC in experimental BPD and thus help guide

the design of human clinical trials.

Methods/Design: We will include preclinical, in vivo, controlled comparative

studies of neonatal animal models of BPD who would have received MSCs

or cell free MSC-derived conditioned media compared to healthy controls or

animal models of BPD that haven’t received MSCs. The primary outcome

measure is improved lung histology and secondary outcomes will include

survival, lung function, exercise capacity, lung fibrosis, lung inflammation,

oxidative stress, pulmonary hypertension and right ventricular hypertrophy,

pulmonary vascular growth, safety, weight gain, and long term outcome.

Electronic searches of MEDLINE, Embase, Pubmed and Web of Science will

be constructed and results will be screened independently and in duplicate.

Study selection would be according to PRISMA guidelines. Data from

eligible studies will be analyzed using random effects models. Risk of bias

will be assessed by two reviewers using SYRCLE’s Risk of Bias tool for

animal studies.

Discussion: The results of this systematic review would put into perspective

all the preclinical evidence on the therapeutic potential and safety of MSC in

BPD and also highlight methodological flaws and bring more rigour in the

design of future studies, both experimental and clinical.

Keywords: Mesenchymal stromal cells, Mesenchymal stem cells,

Bronchopulmonary dysplasia, Preclinical, Systematic review protocol.

Background

Bronchopulmonary dysplasia (BPD), the chronic lung disease of

prematurity, is the most common and serious sequelae in extreme

premature infants. BPD, defined as oxygen dependence at 36 weeks post-

menstrual age, complicates the course of approximately 40% of infants

born <28 weeks gestation (1). Over the last decade, the incidence of BPD

increased slightly (2, 3). BPD is a multifactorial disease. While the

strongest association is with preterm birth, other factors such as prenatal

infection and inflammation, mechanical ventilation, oxygen toxicity,

decreased host antioxidant defenses, patent ductus arteriosus, and

postnatal infection all contribute to the pathogenesis of BPD (4).

The “old form” of BPD, originally described by Northway and colleagues (5)

was characterized by severe lung injury, including inflammation, protein-

rich lung edema, extensive airway epithelial metaplasia, peribronchial

fibrosis, and marked airway and pulmonary vascular smooth muscle

hypertrophy (6–8). In contrast, in the era of antenatal steroids, surfactant

replacement, lung protective ventilation and improved nutrition, survival of

more immature infants born during the canalicular and saccular stages of

lung development appears to disrupt the normal program of alveolar and

vascular development, resulting in the “new BPD,” characterized by

alveolar simplification, dysmorphic capillaries, and remodeling of the

vascular and airway smooth muscle layer (9-11).

Although of great clinical relevance, elucidating the pathophysiology of

neonatal chronic lung disease in the postsurfactant era has become

increasingly challenging. With the reduction in BPD-associated mortality

(12), the availability of pathologic specimens has decreased. Furthermore,

there is a paucity of in vitro systems that effectively model the complex 3-

dimensional processes of alveolar formation and vascularization. Thus,

defining the pathophysiology of BPD has relied, to a large extent, on the

detailed observations made in animal models that mimic many features of

this condition. Knowledge gained from these animal models has provided

great insight into the pathophysiology of the old and new forms of BPD and

has led to changes in clinical management (13).

BPD results in long term pulmonary and neurodevelopmental sequelae that

extend beyond childhood with a high economic burden (14) (15)(16). Many

pharmacological and non-pharmacological approaches have been tested for

the prevention and treatment of BPD. Antenatal steroids, protective

ventilation strategies, targeted oxygen saturation goals, caffeine, vitamin

A, and optimization of nutrition have contributed modestly in decreasing

the incidence/severity of BPD. Most current therapies are supportive.

Recently the therapeutic potential of stem cells has attracted attention.

Stem cells have the potential of self-renewal and can differentiate into

specialized cell types thereby promoting organogenesis, tissue

regeneration, maintenance and repair (17). Mesenchymal stromal cells

(MSCs) attracted particular interest because of their ease of isolation,

characterization, apparent multipotency and pleiotropic effects (18). In

experimental models of BPD, MSC reverse alveolar injury and vascular

remodelling, promote alveolarization, attenuate lung fibrosis and

inflammation, decrease pulmonary hypertension, improve exercise

tolerance and survival (19). MSCs exert their therapeutic benefit mainly

through a paracrine activity and this may explain their pleiotropic effects

(20). These data suggest MSCs as a promising therapy to reduce the

incidence/severity of BPD in extreme premature infants.

This systematic review of preclinical studies in animal models mimicking

BPD will assess the current evidence on the therapeutic potential of MSC in

experimental BPD and thus help guide the design of human clinical trials.

Research question

The aim of this systematic review is to address the following research

question: In controlled preclinical studies of BPD, do MSCs reduce the

severity of lung injury?

Methods & Design

Protocol & Registration

The systematic review protocol was developed after extensive discussion

with the scientific research team consisting of clinical (SA, BT) and

preclinical research scientists (BT) and experts in knowledge synthesis and

translation (DM, MA). The PRISMA-P checklist was used to draft the

protocol (21). The PRISMA-P checklist contains 17 items considered to be

essential and minimum components of a systematic review or meta-

analysis protocol. The systematic review protocol is registered on the

Collaborative Approach to Meta-Analysis and Review of Animal Data from

Experimental Studies website (Date XXXX;

http://www.dcn.ed.ac.uk/camarades/research.html#protocols). In the

event of protocol amendments, the date of each amendment will be

accompanied by description of the change and the rationale behind it.

Study eligibility criteria for Experimental Model

Study Design

We will include preclinical, in vivo, controlled comparative studies of

neonatal animal models of BPD. The studies may be randomised, quasi-

randomized, or non-randomised and evaluate the therapeutic potential and

safety of MSCs. Non-interventional studies, studies without controls will be

excluded.

Population

The state of development of the lung at birth greatly varies among species.

At term, the human lung is at the alveolar stage of lung development.

Sheep possess already alveolarized lungs at birth. Conversely, marsupials

and rodents are born with lungs in the late canalicular/early saccular stage,

equivalent to the lung developmental stage of extreme preterm infants,

and are thus ideally suited for structural, ultrastructural and quantitative

investigations. Therefore studies in neonatal rodents have provided

excellent insights into postnatal developmental events (22-24). We will

include all neonatal preclinical in vivo animal models of experimentally

induced BPD. The model will replicate a pattern that is similar to human

BPD. The similarities will include impaired alveolarization, resulting in fewer

and enlarged alveolar air spaces, pulmonary hypertension, disrupted

vascular growth, vascular leakage, accumulation of plasma proteins,

extravascular fibrin deposition, increased lung collagen content, increased

inflammatory cell influx, and disorganized elastin deposition (25).

Experimental BPD may be induced by hyperoxia, mechanical ventilation,

chemical induction (for example Bleomycin, thalidomide, Su-5416 and

fumagillin), or inflammatory mediators (for example LPS, TGF-, TNF-).

We will exclude non-neonatal animal models of lung injury because many of

the pulmonary responses to injury are developmentally regulated. For

example, chronic O2 exposure induces opposite apoptotic and proliferative

responses in neonatal and adult lung cells in rats (26). In mice, hyperoxia

reduces endothelial progenitor cells in the developing lung but not in adult

animals (27).

Intervention

The intervention groups will receive MSCs or cell free MSC-derived

conditioned media (MSC-CM). MSCs can be syngenic or xenogenic and be

derived from various sources (bone marrow, umbilical cord blood, Wharton

jelly, placenta and adipose tissue). MSCs will be defined using the minimal

criteria set out in the International Society for Cellular Therapy (ISCT)(28)

consensus statement. MSCs will be administered intravenously,

intratracheally, intraperitoneally or any other route. MSCs can be

administered prior, during or following the induction of experimental BPD.

Comparison

The comparison group will consist of experimental BPD animal models that

haven’t received MSCs to evaluate the therapeutic potential and safety of

MSC. The comparison group may have received a control cell (for example

pulmonary artery smooth muscle cell, PASMC, lung fibroblasts, dermal

fibroblasts), a control vehicle (phosphate buffered saline) or media (for

example Dulbecco’s modified Eagle’s medium, DMEM). To examine the

severity of BPD we will use other comparison groups with healthy or sham-

surgery animals.

Outcome

Primary outcome: Improved lung histology

The primary outcome measure is improved lung histology as determined by

lung morphometry on histopathology using the following parameters: mean

linear intercept (MLI), radial alveolar count (RAC), or the number of

secondary crests. This will be determined in the experimental BPD animal

model group, which has received MSC or MSC-CM as well as in

experimental BPD animal controls.

Secondary outcome:

Our secondary outcome will include

1. Survival

2. Lung function (for example reversal of bronchial hypereactivity

following methacholine)

3. Exercise capacity (for example by tread mill test)

4. lung fibrosis ( for example by collagen content etc)

5. Lung inflammation (lung macrophage influx, lung neutrophil influx,

TNF-, TGF-, interleukins, myeloperoxidase activity)

6. Oxidative stress

7. Pulmonary hypertension as assessed by echodoppler (pulmonary

arterial acceleration time, PAAT), right ventricular hypertrophy

(Fulton index), and remodelling of the smooth muscle layer of

pulmonary arteries (medial wall thickness)

8. Pulmonary vascular density (von Willebrand positive lung blood

vessels using immunohistochemistry, barium lung angiogram).

9. Safety as determined by tumor formation at predetermined time

points

10. Weight gain

11. Long term outcome (histology, lung function, exercise capacity)

Information sources

Search Strategy: No study design, date or language limits will be imposed

on the search. MEDLINE, EMBASE and Web of Science will be searched

from database inception, PubMed will be searched for specific categories of

records not yet added to MEDLINE. The search strategies will be created by

a Health Sciences Librarian with expertise in systematic review searching,

and may incorporate published strategies to increase the yield of animal

studies (29). The MEDLINE strategy will be developed with input from the

project team, then peer reviewed by a second librarian, not otherwise

associated with the project, using the PRESS standard (30). An exploratory

MEDLINE search strategy is included in Appendix 1. After the MEDLINE

strategy is finalized, it will be adapted to the syntax and subject headings

of the other database.

We will perform a grey literature search of selected conference websites not

covered in the aforementioned databases, and will search the websites of

animal research organizations. The research team will contact authors of

included studies to invite further contribution of any unpublished data

relevant to this review. The bibliographies of included studies and pertinent

reviews will also be hand searched for further preclinical studies.

Unpublished studies will be described in the results section and data from

these studies will be included in any quantitative analyses.

The search will be updated toward the end of the review, after being

validated to ensure that the MEDLINE strategy retrieves a high proportion

of eligible studies found through any means but indexed in MEDLINE.

Information Source Data Management

The literature search results will be used to build the Reference Manager

database and to de-duplicate references.

Study selection

The titles and abstracts of search results will be screened independently by

two investigators. The full text of all potentially eligible studies will be

retrieved and reviewed for eligibility, independently, by two members of the

team using the a priori inclusion criteria described above. Disagreements

between reviewers will be resolved by consensus or by a third member of

the systematic review team. Reasons for exclusion of potentially eligible

studies will be recorded to enable a transparent selection process and to be

in accordance with the Preferred Reporting Items for Systematic Reviews

and Meta-Analyses (PRISMA)(31) guidelines developed for proper reporting

of clinical systematic reviews.

Data Collection and Data Items

Using standardized forms, two reviewers would independently extract data

from each study. Data abstracted will include study design, animal model,

methodology, intervention details, and all reported outcomes. The research

team will contact (maximum of three email attempts) authors of included

studies to invite further contribution of any unpublished data relevant to

this review. Data collected would be deposited in a public repository.

Assessment of Risk of Bias

Risk of bias will be assessed by two reviewers, for each included preclinical

study, using SYRCLE’s Risk of Bias tool for animal studies (32). This tool

contains 10 entries. These entries are related to selection bias,

performance bias, detection bias, attrition bias, reporting bias and other

biases.

Assessment of Construct and External Validity

We will extract study characteristics that are related to the construct and

external validity (33). For construct validity these will include: age, sex,

strain, and animal species, co-morbidities, type of BDP model, timing, dose

and mode of MSC administration, and the use of any co-interventions. For

external validity these will include: if treatment effects have been

demonstrated in more than one model and if they have been replicated by

an independent research group.

Data Synthesis

We will conduct meta-analyses using random-effects model if the data

extracted from eligible studies are homogenous. Dichotomous data

(survival, lung histology, exercise capacity, lung inflammation, lung

fibrosis) will be determined by using risk ratio (RR) with 95% confidence

interval (CI). Continuous outcomes will be analyzed using mean differences

(with 95% CI) or standardized mean differences (95% CI) if different

measurement scales are used.

Sensitivity analyses to examine heterogeneity on the primary outcome of

improvement in lung histology will be carried out according to risk of bias

assessments. Several subgroup analyses to examine preclinical

heterogeneity will be conducted on the primary outcome and will include:

the study design, animal age, sex, species and strain; presence of co-

morbidities; type of experimental induction of BPD; severity of BPD; MSC

preparation; timing of administration of MSCs from induction of

experimental BPD; dose of MSCs; route of MSC administration; type of

controls; use of co-interventions, antibiotics, and mechanical ventilation;

single versus multi-centre study; and presence of an a priori sample size

calculation.

Knowledge Translation

This systematic review will be reported using PRISMA (30). The results of

this systematic review will be distributed by our principal knowledge user

(BT) in Canada through the Stem Cell Network and CellCAN and

internationally through the International Society for Cell Therapies (ISCT).

Several others like Canadian Council on Animal Care in science (CCAC),

Canadian Premature Babies foundation, would be identified and invited to

act as a knowledge user.

BT will also facilitate organization of an end of study knowledge translation

workshop at the Annual Academy of Pediatrics meetings and at the

Canadian Pediatric Society (CPS) meetings with the Canadian Neonatal

Network. Abstracts will be presented at the Academy of Pediatrics meeting,

at CPS and at the Till&McCulloch meeting (Canadian Stem cell meeting).

Discussion

The results of this systematic review will put all the preclinical evidence on

the therapeutic potential of MSC in perspective. The timing cannot be more

appropriate considering the fact that MSCs have attracted most attention

and numerous clinical trials are underway to test their therapeutic potential

for regenerative purposes (34). This systematic review should help

highlight methodological flaws and bring more rigour in the design of new

studies, both experimental and clinical.

Authors’ contributions

BT is the guarantor and principal knowledge user. SA and BT were

responsible for initial drafting and manuscript revisions. SA will be

responsible for the data collection. MA and DM provided critical revisions.

All authors reviewed several drafts of the manuscript and approved the

final version.

Acknowledgements

BT holds a University of Ottawa Partnership Research Chair in Regenerative

Medicine, and is supported by the Canadian Institutes of Health Research

(CIHR), the Canadian Stem Cell Foundation, the Ontario Lung Association,

the Children’s Hospital of Eastern Ontario Research Institute and the

Ottawa Hospital Research Institute. DM holds a University Research Chair.

MTA is funded by a Canadian Institutes of Health Research (CIHR) post-

doctoral fellowship in Knowledge Translation.

Appendix 1

Exploratory MEDLINE search – Ebscohost interface

S3 (MH "Mesenchymal Stromal Cells") AND (MH

"Bronchopulmonary Dysplasia") Limiters – Animals

13

S2 (MH "Mesenchymal Stromal Cells") 16,385

S1 (MH "Bronchopulmonary Dysplasia") 3,175

Appendix 2: Data Collection Form

Category Specific Items

Study Characteristics Study title, authors, language, date of

publication,

journal published, sponsorship, country

of publication, email address

Animal Model Animal species, strain, age, sex, and

weight, presence of co-morbid illnesses

BPD Model Model & Method to mimic BPD: chronic

hyperoxia, mechanical ventilation, chemical

induction (for example Bleomycin,

thalidomide, Su-5416 and fumagillin), or

inflammatory mediators (for example LPS,

TGF-, TNF-)

Severity of BPD

Intervention and comparison Time, dose and route given, description of

preparation and suspension of MSCs and

controls, use of any co-interventions

Outcomes Primary outcome of improved lung histology

as determined by lung morphometry (mean

linear intercept, radial alveolar count,

secondary septae). Secondary outcomes of

survival, lung function, exercise capacity,

lung fibrosis, lung inflammation, pulmonary

hypertension, pulmonary vessel density,

safety as determined by tumor formation at

predetermined time points, weight gain and

long-term outcome

Risk of Bias assessement Using SYRCLE’s Risk of Bias tool for animal

studies containing 10 entries related to

selection bias, performance bias, detection

bias, attrition bias, reporting bias and other

biases.

Quality of reporting In accordance to ARRIVE guidelines

Other Funding source, confict of interest

statement, treatment effect demonstrated

in more than one model replication by

independent group, and presence of a priori

sample size calculation.

Appendix 3

STUDY ELIGIBILITY FORM STUDY ELIGIBILITY FORM FACTORS ASSESSMENT COMMENTS

TYPE OF STUDY

1. Is the study described as Preclinical?

NB: Preclinical can be in vivo animals or non-human

Yes Unclear No

Exclude

2. Is the study described as interventional?

Yes Unclear No

Exclude

3. Is there a control group? Yes Unclear No

Exclude

PARTICIPANTS

4. Is it an animal model of BPD? Yes Unclear No

Exclude

5. Is it a “neonatal” animal model?

Yes Unclear No

Exclude

Subgroups available?

INTERVENTIONS

6. Were comparison groups treated with Mesenchymal

Stromal Cells (MSC) or MSC-Conditioned media (MSC-

CM) in one group and control intervention in other

group?

NB: study can have 3 arms, if so please cross “Yes“ and

state it as comments.

Yes Unclear No

Exclude

6. Intervention with Mesenchymal Stromal Cells (MSC)

or MSC-Conditioned media (MSC-CM) does NOT involve

co-treatment?

Yes Unclear No

Exclude

OUTCOMES

7. Did the study report pre-specified outcomes? Yes Unclear No

Exclude

FINAL DECISION

1 X “No” = EXCLUDE

1 X “Unclear” = UNCLEAR

ORGANISATIONAL ASPECTS EX IN

REF ID Reviewer, Date Checked by

Author, Year

Journal/Source Study ID Not Reported /

Country of origin

Publication type

Fulltext

Abstract

Book chapter

internal progress report

other (please specify)

Other relevant

publications in DE-form

Decision pending

Check references

Use for discussion /

Fate EX without listing

EX with listing

Other (please specify)

Notes / Short description

REASONS FOR EXCLUSION OF STUDY FROM REVIEW (PLEASE SPECIFY according to

protocol

Methods

No Intervention Not MSC/ MSC-CM No Control Other

Population

Not BPD

Human

Invitro Age

Subgroups available?

Outcomes

No relevant outcomes assessed

No data for relevant subgroup extractable

Other

Duplicate publication / Other

NONE Included

CURRENT STATUS: (NAME OF REVIEWER + DATE)

Question to clinician

Question to author

Status verified with study investigators or sponsors: Yes / No

Enter name of the source (e.g. PI, sponsor, etc.) ___________________

Contact address:

3

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