Safety and efficacy of cell therapies in pediatric heart disease: a systematic review and meta-analysis

Background Adult clinical trials have reported safety and the therapeutic potential of stem cells for cardiac disease. These observations have now translated to the pediatric arena. We conducted a meta-analysis to assess safety and efficacy of cell-based therapies in animal and human studies of pediatric heart disease. Methods and results A literature search was conducted to examine the effects of cell-based therapies on: (i) safety and (ii) cardiac function. In total, 18 pre-clinical and 13 human studies were included. Pre-clinical: right ventricular dysfunction was the most common animal model (80%). Cardiac-derived (28%) and umbilical cord blood (24%) cells were delivered intravenously (36%) or intramyocardially (35%). Mortality was similar between cell-based and control groups (OR 0.94; 95% CI 0.05, 17.41). Cell-based treatments preserved ejection fraction by 6.9% (p < 0.01), while intramyocardial at a dose of 1–10 M cells/kg optimized ejection fraction. Clinical: single ventricle physiology was the most common cardiac disease (n = 9). Cardiac tissue was a frequent cell source, dosed from 3.0 × 105 to 2.4 × 107 cells/kg. A decrease in adverse events occurred in the cell-based cohort (OR 0.17, p < 0.01). Administration of cell-based therapies improved ejection fraction (MD 4.84; 95% CI 1.62, 8.07; p < 0.01). Conclusions In this meta-analysis, cell-based therapies were safe and improved specific measures of cardiac function. Implications from this review may provide methodologic recommendations (source, dose, route, timing) for future clinical trials. Of note, many of the results described in this study pattern those seen in adult stem cell reviews and meta-analyses.

Give the working title of the review. This must be in English. The title should have the interventions or exposures being reviewed and the associated health or social problems.
Safety and efficacy of cell-based/derived therapies in congenital heart disease; a systematic review and meta analysis of pre-clinical and clinical studies 2. Original language title. For reviews in languages other than English, this field should be used to enter the title in the language of the review. This will be displayed together with the English language title. English 3. * Anticipated or actual start date.
Give the date when the systematic review commenced, or is expected to commence. 27/08/2019 4. * Anticipated completion date.
Give the date by which the review is expected to be completed. 01/01/2020 5. * Stage of review at time of this submission.
Indicate the stage of progress of the review by ticking the relevant Started and Completed boxes. Additional information may be added in the free text box provided. Please note: Reviews that have progressed beyond the point of completing data extraction at the time of initial registration are not eligible for inclusion in PROSPERO. Should evidence of incorrect status and/or completion date being supplied at the time of submission come to light, the content of the PROSPERO record will be removed leaving only the title and named contact details and a statement that inaccuracies in the stage of the review date had been identified. This field should be updated when any amendments are made to a published record and on completion and publication of the review. Give a link to the search strategy or an example of a search strategy for a specific database if available (including the keywords that will be used in the search strategies).
MEDLINE PubMed: ((((((((((((((((((((((regenerative[MeSH Terms]) OR regenerative) OR stem cell) OR stromal cell) OR mesenchymal) OR embryonic) OR pluripotent) OR multipotent) OR inducible pluripotent) OR progenitor) OR hematopoietic) OR umbilical cord) OR cord blood) OR c-kit) OR secretome) OR microRNA) OR exosome) OR microvesicle) OR microparticle) OR extracellular vessicle))) AND ((((((((((((((truncus arteriosus) OR transposition of great arteries) OR transposition of great vessels) OR tricuspid atresia) OR hypoplastic right heart syndrome) OR hypoplastic left heart syndrome) OR single ventricle) OR tetralogy of fallot) OR total anomalous pulmonary venous return) OR pulmonary atresia) OR coarctation of aorta) OR interrupted aortic arch) OR doublet outlet right ventricle) OR single inlet ventricle) Alternatively, upload your search strategy to CRD in pdf format. Please note that by doing so you are consenting to the file being made publicly accessible.
Do not make this file publicly available until the review is complete 18. * Human disease modelled.
Give a short description of the disease, condition or healthcare domain being modelled.
Congenital heart disease 19. * Animals/population. Give summary criteria for the animals being studied by the review, e.g. species, sex, details of disease model. Please include details of both inclusion and exclusion criteria.

Inclusion criteria:
Human: Children (newborn-18 years) with congenital heart disease to include adults with history of congenital heart disease receiving cell-based/derived therapies Animal models of congenital heart disease Exclusion criteria: Human: Children without congenital heart disease, Adults without congenital heart disease receiving cellbased/derived therapies, Adult models of heart disease Animal: Animal models without congenital heart disease 20. * Intervention(s), exposure(s).
Give full and clear descriptions of the nature of the interventions or the exposures to be reviewed (e.g. dosage, timing, frequency). Please include details of both inclusion and exclusion criteria.

PROSPERO International prospective register of systematic reviews
Inclusion criteria: The following will be used for both human and animal studies. Regenerative cell-based/derived therapy used to treat congenital heart disease. Regenerative cell therapies will be defined as: mesenchymal, embryonic, multipotent, inducible pluripotent cells, progenitor, hematopoietic, umbilical cord, cord blood, c-kit+, secretome, exosome, microRNA, microvesicles, extracellular vesicles.
Exclusion criteria: The following will be used for both human and animal studies. Non cell-based/derived therapies used to treat congenital heart disease 21. * Comparator(s)/control.
Where relevant, give details of the type(s) of control interventions against which the experimental condition(s) will be compared (e.g. another intervention or a non-exposed control group). Please include details of both inclusion and exclusion criteria.

Inclusion criteria:
Human: Placebo. Children with congenital heart disease who did not receive cell based therapies.
Animal: Animals in experimental models not subject to cell-based/derived therapies for the treatment of congenital heart disease (placebo and sham).

Exclusion criteria:
Human: Children without congenital heart diseases Animal: Animals not modeling congenital heart disease 22. * Study designs to be included.
Give details of the study designs eligible for inclusion in the review. If there are no restrictions on the types of study design eligible for inclusion, or certain study types are excluded, this should be stated. Please include details of both inclusion and exclusion criteria.

Inclusion criteria:
Clinical trials, cohort, case reports Exclusion criteria: Articles not assessing outcomes of interest 23. Other selection criteria or limitations applied.
Give details of any other inclusion and exclusion criteria, e.g. publication types (reviews, conference abstracts), publication date, or language restrictions.

PROSPERO International prospective register of systematic reviews
Review articles, book chapters, abstracts will be excluded. No restrictions placed based on publication date or language.
Give detail of the outcome measures to be considered for inclusion in the review. Please include details of both inclusion and exclusion criteria.
Inclusion criteria: The following outcome measures will be used for both human and animal studies Cardiac function, as measured by: -Right/Left ejection fraction-End diastolic volume-End systolic volume-Tricuspid annular plane systolic excursion-Fractional area change-Fractional shortening Safety: -Mortality -Adverse events with administration (fever, rash, infection, hemodynamic instability, arrhythmia, etc) Exclusion criteria: Animal or human studies not assessing safety or efficacy (as defined above) after cell-based/derived.

N/A.
This question does not apply to systematic reviews of animal studies for human health submissions.
26. * Study selection and data extraction.
Procedure for study selection Give the procedure for selecting studies for the review, including the screening phases (title and/or titleabstract and/or full-text), the number of researchers involved, and how discrepancies will be resolved.
Study selection: a) Two investigators (J. Martinez & S. Zoretic) will independently screen all the abstracts/full texts for the inclusion criteria. b) Differences of opinion in either phase that cannot be resolved by discussion will be resolved by consulting a third investigator (A. Moreira).

Prioritise the exclusion criteria
Multiple exclusion criteria may apply to an abstract/paper, which can cause discrepancies between reviewers in the reason for exclusion recorded. To avoid this, it is helpful to prioritize the exclusion criteria (e.g. 1) not an animal study; 2) not a myocardial infarction model, etc.) and record the highest ranking applicable criterion as the reason for exclusion. Please sort the exclusion criteria defined in questions 19 to 24. If applicable, do so for each screening phase. 1) in-vitro studies2) studies not including cell-based/derived therapies 3) Human studies not including congenital heart disease models 4) Animal models without congenital heart disease5) Animal or human studies not assessing safety or efficacy (as defined above) after cell-based/derived. 6) Adults without congenital heart disease receiving cell-based/derived therapies 7) Adult models of heart disease 8) Review articles, book chapters, abstracts Page: 6 / 12

PROSPERO International prospective register of systematic reviews
Methods for data extraction Describe methods for data extraction, including the number of reviewers performing data extraction, extraction of data from text and/or graphs, whether and how authors of eligible studies will be contacted to provide missing or additional data, etc.
Data extraction: Study design, methodology, patient demographics, clinical diagnoses, cell characteristics (source, dose, frequency and delivery), cardiac imaging parameters, laboratory values, publication details (author, year, funding, etc), follow up data a) Two investigators (J. Martinez & S. Zoretic) will independently screen all the abstracts/full texts for the inclusion criteria. b) Differences of opinion in either phase that cannot be resolved by discussion will be resolved by consulting a third investigator (A. Moreira). Data will be extracted from text, tables and figures (webplot digitizer). For missing data, will contact authors. Data will be recorded via excel spreadsheet.
Data to be extracted: study design Specify the data to be extracted related to characteristics of the study design, e.g. controlled versus crossover, number of experimental groups, etc.
Humans: Number of children in experimental +/-control group, number of experimental groups, phase of clinical trial, cell-based/derived therapies parameters (dose, frequency, route, etc), cardiac assessments (echo, MRI, CT, biomarkers), time points for data collection Animals: Number of animals in experimental +/-control group, number of experimental groups, cellbased/derived therapies parameters (dose, frequency, route, etc), cardiac assessments (echo, MRI, CT, biomarkers), time points for data collection Data to be extracted: animal model Specify the data to be extracted related to characteristics of the animal model, e.g. species, sex of the animals, etc.
Number of animals in experimental and control groups, power calculation reported, method(s) to induce congenital heart disease, animal species/strain, age, gender, weight and immune status.

Data to be extracted: intervention of interest
Specify the data to be extracted related to characteristics of the intervention of interest, e.g. dose, timing, etc.
Cell type, tissue source, dose, mode of delivery, frequency, timing, passage number Data to be extracted: primary outcome(s) Define the primary outcome measure(s). For each outcome measure, specify in which format data will be extracted, including the eligible units of measurement, and data type (continuous/dichotomous). A description of any other manipulation or transformation of the extracted data that is planned may be included.
The following outcomes will be assessed in both animal and human studies Data to be extracted: secondary outcome(s) Define the secondary outcome measure(s). For each outcome measure, specify in which format data will be extracted, including the eligible units of measurement, and data type (continuous/dichotomous). A description of any other manipulation or transformation of the extracted data that is planned may be included.
n/a Data to be extracted: other Specify any other data or study characteristics to be extracted, e.g. bibliographical details, such as author, year and language.
Author, year, funding, title, language, contact author email, journal 27. * Risk of bias and/or quality assessment.
State whether and how risk of bias and/or study quality will be assessed. Assessment tools specific for preclinical animal studies include SYRCLE's risk of bias tool and the CAMARADES checklist for study quality

PROSPERO International prospective register of systematic reviews
Method for risk of bias and/or quality assessment Give the procedure for the risk of bias and/or quality assessment, including the number of reviewers involved, their contribution, and how discrepancies will be resolved.
Two separate reviewers will assess risk of bias for each study. Discrepancies will be resolved by senior author.

Planned approach
For each outcome measure, specify whether a quantitative or narrative synthesis is planned and how this decision will be made.
Quantitative synthesis will be preferred method for reporting information, however if 4 studies are assessing a particular outcome we will conduct a narrative explanation as, too few studies will be available to conduct meta-analysis.
If a meta-analysis is planned, please specify the following:

Effect measure
For each outcome measure, specify the effect measure to be used (e.g. mean difference, odds ratio etc.).
Animal studies: standardized mean difference Human studies: odds ratio Effect models For each outcome measure, specify the statistical model of analysis (e.g. random-effects or fixed-effect model).

Random-effects model
Heterogeneity Specify the statistical methods to assess heterogeneity (e.g. I², Q). For further guidance please refer to the introduction and practical guide to pre-clinical meta-analysis.

I²
Other Specify other details of the meta-analysis methodology (e.g. correction for multiple testing, correction for multiple use of control group). n/a 29. * Analysis of subgroups or subsets.

Subgroup analyses
Give any planned exploration of subgroups or subsets within the review. 'None planned' is a valid response List other places where the systematic review protocol is registered. The name of the organisation and any unique identification number assigned to the review by that organisation should be included.  Alternatively, upload your published protocol to CRD in pdf format. Please note that by doing so you are consenting to the file being made publicly accessible.
No I do not make this file publicly available until the review is complete Please note that the information required in the PROSPERO registration form must be completed in full even if access to a protocol is given.

Dissemination plans.
Give brief details of plans for communicating essential messages from the review to the appropriate audiences.
The manuscript will be submitted to a loading journal in field. In addition, a report will be submitted to the funder (Parker B Francis foundation).
Do you intend to publish the review on completion?
Give words or phrases that best describe the review. Separate keywords with a semicolon or new line.
Regenerative medicine, cell-based/derived therapies, stem cells, congenital heart disease, human, clinical trials, animal studies 37. Details of any existing review of the same topic by the same authors.
Give details of earlier versions of the systematic review if an update of an existing review is being registered, including full bibliographic reference if possible.
Previous manuscript focusing on cell-based/derived therapies as a treatment for right ventricular dysfunction is currently being considered for publication.

* Current review status.
Review status should be updated when the review is completed and when it is published. Please provide anticipated publication date Review_Ongoing 39. Any additional information.
Provide any further information the review team consider relevant to the registration of the review.

Details of final report/publication(s).
This field should be left empty until details of the completed review are available. Give the full citation for the final report or publication of the systematic review.
Give the link to the published review. Specify (a) the number of reviewers per screening phase and (b) how discrepancies will be resolved a) Two investigators (J. Martinez & S. Zoretic) will independently screen all the abstracts/full texts for the inclusion criteria. b) Differences of opinion in either phase that cannot be resolved by discussion will be resolved by consulting a third investigator (A. Moreira).

Define all inclusion and exclusion criteria based on:
23. Type of study (design) Specify (a) the number of reviewers assessing the risk of bias/study quality in each study and (b) how discrepancies will be resolved a) Two investigators (J. Martinez & S. Zoretic) will independently screen all the abstracts/full texts for the inclusion criteria. b) Differences of opinion in either phase that cannot be resolved by discussion will be resolved by consulting a third investigator (A. Moreira)

38.
Define criteria to assess (a) the internal validity of included studies (e.g. selection, performance, detection and attrition bias) and/or (b) other study quality measures (e.g. reporting quality, power) X By use of SYRCLE's Risk of Bias tool 4 □By use of SYRCLE's Risk of Bias tool, adapted as follows: □By use of CAMARADES' study quality checklist, e.g 22 □By use of CAMARADES' study quality checklist, adapted as follows: □Other criteria, namely: Collection of outcome data 39.
For each outcome measure, define the type of data to be extracted (e.g. continuous/dichotomous, unit of measurement) All outcome measures will be expressed through study units of measure, values expressed as continuous measures will be recorded as means +/-SD, SEM or median +/-IQR 40.
Methods for data extraction/retrieval (e.g. first extraction from graphs using a digital screen ruler, then contacting authors) Extraction from text, tables, and figures (GetData graph digitizer 2.26) Contact authors in case of missing data 41.
Specify (a) the number of reviewers extracting data and (b) how discrepancies will be resolved a) Two investigators (J. Martinez & S. Zoretic) will independently screen all the abstracts/full texts for the inclusion criteria. b) Differences of opinion in either phase that cannot be resolved by discussion will be resolved by consulting a third investigator (A. Moreira) Data analysis/synthesis 42.
Specify (per outcome measure) how you are planning to combine/compare the data (e.g. descriptive summary, meta-analysis) For sufficient data, we will conduct a meta-analysis for eligible studies. If insufficient data to measure outcomes, we will provide a descriptive summary of study results

43.
Specify (per outcome measure) how it will be decided whether a metaanalysis will be performed A minimum of 4 articles for the same outcome is required. High heterogeneity is expected between studies due to differences in the study designs. We will perform a metaregression analysis to investigate sources of heterogeneity. If a meta-analysis seems feasible/sensible, specify (for each outcome measure):

44.
The effect measure to be used (e.g. mean difference, standardized mean difference, risk ratio, odds ratio) Continuous outcomes will be analysed using standardized mean differences (95% CI) 45.
The statistical model of analysis (e.g. random or fixed effects model) Random effects model 46.
The statistical methods to assess heterogeneity (e.g. I 2 , Q) I 2

5-6
Information sources 7 Describe all information sources (e.g., databases with dates of coverage, contact with study authors to identify additional studies) in the search and date last searched.

5
Search 8 Present full electronic search strategy for at least one database, including any limits used, such that it could be repeated.

5
Study selection 9 State the process for selecting studies (i.e., screening, eligibility, included in systematic review, and, if applicable, included in the meta-analysis).

5
Data collection process 10 Describe method of data extraction from reports (e.g., piloted forms, independently, in duplicate) and any processes for obtaining and confirming data from investigators.

5-6
Data items 11 List and define all variables for which data were sought (e.g., PICOS, funding sources) and any assumptions and simplifications made.

5-6
Risk of bias in individual studies 12 Describe methods used for assessing risk of bias of individual studies (including specification of whether this was done at the study or outcome level), and how this information is to be used in any data synthesis.

Study selection
17 Give numbers of studies screened, assessed for eligibility, and included in the review, with reasons for exclusions at each stage, ideally with a flow diagram.

9
Study characteristics 18 For each study, present characteristics for which data were extracted (e.g., study size, PICOS, follow-up period) and provide the citations.

9, 11
Risk of bias within studies 19 Present data on risk of bias of each study and, if available, any outcome level assessment (see item 12).

13-14
Results of individual studies 20 For all outcomes considered (benefits or harms), present, for each study: (a) simple summary data for each intervention group (b) effect estimates and confidence intervals, ideally with a forest plot.

9-13
Synthesis of results 21 Present results of each meta-analysis done, including confidence intervals and measures of consistency.

9-13
Risk of bias across studies 22 Present results of any assessment of risk of bias across studies (see Item 15).

DISCUSSION
Summary of evidence 24 Summarize the main findings including the strength of evidence for each main outcome; consider their relevance to key groups (e.g., healthcare providers, users, and policy makers).