Allogeneic Cell Therapy Applications in Neonates: A Systematic Review

Abstract Background Neonatal cell therapy applications are increasing; however, data on allogeneic cell therapy are limited. Objective To summarize evidence on allogeneic cell therapy in term and preterm neonates. Methods Cochrane Central Register of Controlled Trials, Embase, Ovid Medline, and various registries were searched for studies investigating the safety, feasibility, and efficacy of allogeneic cell therapy in neonates. Two authors independently selected the articles, extracted data, and assessed the risk of bias. Results Twelve published (153 infants) and 21 ongoing studies were included. These studies predominantly sourced allogeneic cells from umbilical cord blood (UCB). Mesenchymal stromal cells (MSCs) were the main cell type used (134 of 153 infants); others included UCB-derived total nucleated cells (TNCs) and human amnion epithelial cells (hAECs). Applications included bronchopulmonary dysplasia (BPD; 113 infants), Krabbe disease (13 infants), intraventricular haemorrhage (10 infants), perinatal arterial ischemic stroke (10 infants), hypoxic-ischaemic encephalopathy (6 infants), and necrotizing enterocolitis (1 infant). Nine out of 12 studies did not report any serious adverse events (SAEs) related to cell administration. Three studies reported SAEs, such as graft versus host disease (GVHD) in 5 infants (UCB-derived TNCs for Krabbe disease); and transient cardiorespiratory compromise in 1 infant (hAECs for BPD). Data on efficacy outcomes were limited. Conclusion The safety and feasibility of allogeneic cell therapy applications in neonates are available, mainly from the use of MSCs. Further safety data for other cell types are required, and the risk of GVHD in different settings needs to be determined. Efficacy studies are largely lacking for all cell types. Protocol Registration The protocol was registered with PROSPERO (registration number CRD42023397876), the international prospective register for systematic reviews (https://www.crd.york.ac.uk/PROSPERO).


Introduction
Cell therapy is a potential paradigm shift in neonatal medicine.In particular, it is a promising therapy to reduce the complications of prematurity and perinatal asphyxia. 1,2It involves administering biological living cells to prevent or reverse the disease process and normalize the structure and function of organs and tissues by reducing inflammation and promoting endogenous repair of damaged or diseased cells or tissues. 3Cell therapy is commonly used to treat cancers and hematological disorders, but its use in neonates for regenerative and immunomodulatory applications remains a relatively new field with many unanswered questions. 4,5reclinical studies have demonstrated the potential role of cell therapies to prevent or treat pathology associated with prematurity, including bronchopulmonary dysplasia (BPD), 6 preterm brain injury, 7 necrotizing enterocolitis (NEC), 8 and other conditions, such as perinatal asphyxia, 9 perinatal arterial ischaemic stroke (PAIS), 10 and congenital heart disease. 11hese preclinical studies demonstrate that, depending on the cell type and timing of administration, cell therapy may modulate tissue injury via an anti-inflammatory, [12][13][14] anti-apoptotic, 15 paracrine, 16 or angiogenic effects 17 or, in the case of some stem cells, may replace damaged cells. 18These benefits are also shown in clinical studies investigating the role of cell therapy in several neonatal diseases, with a few studies showing that it could be promising 19,20 ; however, the data are not robust. 21ll therapies are broadly categorized as autologous (using one's own cells) or allogeneic (cells from others).While it is unknown whether there are differences between these 2 types of cell therapy, research is ongoing to examine the safety and efficacy of both types of cell therapies in various neonatal conditions.Research on allogeneic cell therapy has been gaining momentum, 22,23 given that autologous cell therapy is not feasible for many infants, as complicated or preterm births are not usually anticipated, and in some babies, the target cell dose required may not be achievable. 24owever, allogeneic cell therapy is not without challenges.Allogeneic cell therapies are expensive, and some require extensive logistical support.As cells in allogeneic cell products are obtained from healthy donors, there is a risk of immunological reactions, such as graft-versushost disease (GVHD).Furthermore, due to the recognition of donor cells by the immune system, donor cells can be rapidly removed from the system before they can offer any therapeutic or regenerative benefits.Hence, it is important to understand whether allogeneic cell therapy in the neonatal period is safe, feasible, and efficacious.The aim of this systematic review was to examine the published evidence that currently exists regarding the safety, feasibility, and efficacy of allogeneic cell therapy applications for newborn infants.

Methods
The systematic review was conducted as per Preferred Reporting Items for Systematic Reviews and Meta-analyses reporting guideline.The study protocol was registered with PROSPERO (registration number CRD42023397876), the international prospective register for systematic reviews (https://www.crd.york.ac.uk/PROSPERO).

Search Strategy
Literature search was conducted using prespecified search terms within the following databases: Cochrane Central Register of Controlled Trials (CENTRAL), Embase, and Ovid Medline from inception through October 2022 without any language restriction.In addition, reference lists and citations of included studies and relevant reviews were also searched.

Type of Studies
All published and ongoing human clinical studies using allogeneic cells in neonates with or without a control arm and evaluating the feasibility, safety, or efficacy were included.Allogeneic cells were considered as biological living cells with broad or specific regenerative or immunomodulatory potential obtained from a donor and intended for administration into a genetically distinct related or unrelated recipient.

Population
Term or preterm neonates with or without neonatal morbidities.

Interventions
Studies using allogeneic cell therapy for any neonatal condition, irrespective of source, route of administration, and dosing, during the first 4 weeks of life in full-term neonates or until 4 weeks of corrected age (44 weeks' postmenstrual age) in preterm neonates were included.

Outcomes
The following outcomes were evaluated: 1. Safety, including any issues related to infusion or any complications, such as infection, anaphylaxis, and GVHD, as defined by authors.2. Feasibility, including the ability to carry out therapy once eligibility is confirmed, as defined by authors.3. Clinical outcomes, as defined by authors, included death, neurodevelopmental impairment, cerebral palsy, brain injury (intraventricular haemorrhage (IVH), white mat-ter injury, or seizures), BPD, retinopathy of prematurity, NEC, and a composite of death or any morbidities.

Study Selection, Data Abstraction, and Assessment of the Methodological Quality
A.R. and A.M. independently read the titles and abstracts of the final list of records assimilated using Covidence systematic review software, Veritas Health Innovation, Melbourne, Australia, available at www.covidence.org.They also independently reviewed the shortlisted full-text articles for inclusion.Furthermore, A.R. and D.L. independently extracted the relevant information from included studies.In addition, A.R. and D.L. independently assessed the methodological quality using the Cochrane Risk of Bias tool (version 2) for randomized studies, 25 modified New Ottawa Scale for nonrandomized studies, 26 and another tool for studies with no comparator group. 27Finally, discrepancies were resolved by discussion and consensus.

Published Studies
Details of included studies are summarized in Table 1.
Of the 12 studies, 5 were phase I studies with no control arm, 29,33,36,39,40 3 were case reports, 30,34,38 and the remainder were phase II randomized controlled trial, 20 cohort study (phase I clinical trial with historic case-matched controls), 28,35 descriptive study, 31,37 and a case series. 32These studies included 153 term and preterm infants worldwide who received allogeneic cell therapy in the neonatal period.

Risk of Bias Assessment of Included Studies
Methodological assessment for each study is provided in the Supplementary material.A complete methodological evaluation was not performed for one report where only the abstract was available. 36[32][33][34][37][38][39][40] Five of these 9 studies were deemed at low-risk, 29,31,33,37,39,40 and the remainder were at moderate (3 studies) 30,32,38 and high (1 study) 34 risk of bias.
The phase II randomized trial 20 and the cohort study 28,35 were at low risk of bias.
Allogeneic Cells: Indication, Source, Dose, and Route Allogeneic cells were used in human neonatal trials for various disease conditions, most commonly being BPD (5 studies, 113 infants) (Fig. 2).Other conditions include Krabbe disease (2 studies, 13 infants), 31,37 IVH (2 studies, 10 preterm infants), 29,34 PAIS (1 study, 10 infants), 33 hypoxic-ischaemic encephalopathy (HIE) (1 study, 6 infants), 36 and NEC (1 report, 1 infant). 30Most studies used mesenchymal stromal cells (MSCs) (134 of 153 infants in 9 studies) derived from UCB (5 studies), 20,28,29,34,35,40 umbilical cord tissue (2 studies), 30,36 and bone marrow (2 studies), 32,33 with a cumulative dose between 2 and 20 million MSCs per kg 20,28,29,32,34,35,40 or 45 to 50 million cells total. 33Other studies used UCB-derived total nucleated cells (TNCs) 31,37 of 30 million per kg of body weight and human amnion epithelial cells (hAECs) 39 of 1 million per kg of body weight.35,40 In one report, cells were administered via intravenous and intraventricular routes.37 In both reports, the authors mentioned a favorable natural history of disease and neurological outcomes in infants who received cell therapy in the neonatal period compared with the infants who received cell therapy in the postneonatal period. Howevr, Escolar et al reported that among newborn infants, 57% (4/7) experienced acute GVHD of grade I-II severity, while 42% (3/7) developed chronic GVHD. 37urthermore, Allewelt et al reported death in 16.6% (2/12) and spasticity in 80% (8/10) infants at 5 years.31 In another report on Krabbe disease, Lesnik et al reported a term infant who underwent allogeneic cell therapy and developed GVHD and steroid-induced hypertrophic cardiomyopathy, which were managed appropriately with medical therapies with complete resolution at 8 months. 38 It is  iportant to note that the purpose of cell therapy in these reports differs from rest of other reports in that the intended therapeutic approach is transplantation.In these reports, transplantation procedure was preceded by myeloablative conditioning and followed by post-transplant immune suppression for prophylaxis against GVHD.

IVH
One study reported that intraventricular injection of allogeneic UCB-MSCs at a dose of 5 to 10 million cells per kg in preterm infants with severe IVH was safe, as demonstrated by the absence of immediate serious adverse events and lack of dose-limiting toxicity. 29In addition, none of the infants died or had serious adverse events related to the therapy.The authors also deemed the treatment feasible.In another report by Bozkaya et al, a preterm infant had complete resolution of severe IVH with a normal neurological outcome and no deficits at 2 years following a dose of intraventricular and intravenous allogeneic UCB-MSCs in the neonatal period. 34ther neurological disorders: Baak et al utilized a single dose of intranasal allogeneic BM-MSCs for 10 infants with PAIS within 7 days of diagnosis and showed it as safe and feasible with all infants surviving at 3 months and without any unexpected cerebral abnormalities on magnetic resonance imaging. 33Similarly, another report by Cotten et al shows the safety of intravenous allogeneic umbilical cord tissue-derived MSCs in infants with perinatal HIE, with all infants surviving at 6 months. 36However, the authors have yet to report the long-term neurodevelopmental outcomes in these infants at 12 to 16 months of age.

Pulmonary Conditions (5 Studies)
Three studies used intratracheal UCB-MSCs, 20,28,35,40 one study used intravenous bone marrow-MSCs, 32 and another used intravenous hAECs 39 for infants at risk of BPD or with BPD.The study using intravenous hAECs in extremely preterm infants with BPD showed it as safe and feasible, but one infant had transient cardiorespiratory compromise during cell administration secondary to presumed pulmonary embolism, 39 which did not occur in other babies after the change of protocol, including use of an inline filter and infusion over a longer period of time.Furthermore, a study by Powell  reported intratracheal administration of UCB-MSCs as safe and feasible in 12 extremely preterm infants requiring mechanical ventilation with no serious adverse events related to the therapy. 40Similarly, another study by Chang et al reported the safety and feasibility of intratracheal UCB-MSCs in extremely preterm infants. 35The authors also compared treated infants with matched historical controls and noted no differences in clinical outcomes, except for differences in the severity of BPD between the groups.Intratracheal UCB-MSCs administration was associated with higher mild BPD but lower severe BPD.In addition, the authors provide neurodevelopmental follow-up data in another report, which showed no differences in clinical outcomes. 28Finally, one phase II randomized controlled trial (RCT) comparing intratracheal UCB-MSCs versus placebo found no difference in clinical outcomes, including BPD.But, in the 23-24 weeks GA subgroup, lower severe BPD was found in the intratracheal UCB-MSCs group. 20

Summary of Findings
Our systematic review summarizes the data from 12 studies that have assessed the safety, feasibility, and efficacy of allogeneic cell therapy in 153 term and preterm infants worldwide.In addition, it provides the details of 21 ongoing or planned studies using allogeneic cell therapy in 1020 neonates.Most studies used MSCs (134 of 153 infants) derived from UCB, 20,28,29,34,35,40 umbilical cord tissue, 30,36 and bone marrow, 32,33 whereas others used UCB-derived TNCs 31,37 and hAECs. 39The most common indication was bronchopulmonary dysplasia (113 infants).Nine out of 12 studies did not report any serious adverse events related to the therapy and described the therapy as safe and feasible.Other studies reported serious adverse events, such as GVHD (UCBderived TNCs for Krabbe disease) in 5 infants 31,37,38 and transient cardiorespiratory compromise (hAECs) in one infant. 39he data on efficacy outcomes from comparative studies were similar except for reduced severe BPD with allogeneic cell therapy in 2 studies. 20,35

Study Implications
Our systematic review highlights some evidence of the safety and feasibility of allogeneic cell therapy in the neonatal period.However, the findings should be interpreted carefully as data are derived from a few studies evaluating cell therapy in only 153 neonates.Moreover, the cells used in the studies predominantly consist of MSCs (134 of 153 infants) with the remaining being UCB-TNCs and hAECs; hence, these findings should not be generalized to other allogeneic cells.
In addition, it is crucial to acknowledge that while MSCs and hAECs were administered as specific cell types, UCB-TNCs involve a scenario where multiple cell types are present.Furthermore, it is important to note that GVHD occurred exclusively with UCB-TNCs, despite prior myeloablative conditioning, highlighting the potential variability of immunologic reactions depending on the specific type of allogeneic cells administered.This finding emphasizes the need for accurate description and characterization of the immunologic reactions based on the cell types employed.Although MSCs were the predominant cell type studied, their usage has exhibited heterogeneity in terms of cell source (umbilical cord tissue, UCB, and BM) and indications (BPD, IVH, PAIS, and HIE).In addition, it is acknowledged that the efficacy studies conducted on MSCs have been limited in scope, with 2 studies reporting a reduction in the severity of BPD with allogeneic cells.But, these differences were found in the subgroup and exploratory analyses and should be interpreted cautiously. 20,35Finally, it is important to note that both published and ongoing research on allogeneic cell therapy is focused on preterm lung disease, with few studies focusing on other relevant conditions, such as brain injury.Hence, further research is warranted to establish and replicate the safety and efficacy of diverse allogeneic cells in different neonatal conditions, involving a larger cohort of neonates.

Comparison with Previous Research
To date, this is the first systematic review to scrutinize evidence specifically on allogeneic cell therapy in the neonatal period.][33][34][35][36]40 Similar to our study, a recent systematic review by Paton et al focusing on allogeneic UCB-derived TNCs or mononuclear cell therapy in children and adults reported no safety concerns or GVHD. 64In contrast, our study identified GVHD in 4 out of 7 patients in one study using UCB-derived TNCs. 37It is important to note that all these patients received myeloablative conditioning, and the purpose of cell therapy was transplantation for Krabbe disease.Whereas the indications for allogeneic cell therapy in the review by Paton et al were neurological conditions such as autism, cerebral palsy, stroke, and traumatic brain injury, 6 of 10 studies did not use immunosuppressive prophylaxis. 64herefore, it is important to study the safety of these cell types in the neonatal age group in different settings and determine the true risk of GVHD in this cohort.

Complexities in Allogeneic Cell Therapy Research
All except 2 studies included no controls.The 2 studies with a control population were small and were not powered to detect meaningful differences in clinical outcomes. 20,35But, we acknowledge that well-designed small trials may be required to assess and ensure safety before carrying out large, well-powered clinical trials for therapeutic efficacy.In addition, we recognize that even small trials in this field are very challenging, as they require significant effort in arranging the therapy, including processing the blood, relevant HLAmatching, storing and transporting cells, complying with infection control measures and governance policies, and adhering to the highest standards, as is the case for any stem cell transplantation.Furthermore, significant funding is also required to carry out these trials, which is often challenging.Our search has determined that there are 21 ongoing studies for the use of allogeneic cells for neonatal morbidities, with dates indicating that many planned studies are slow to begin and may not reach completion.For example, an RCT investigating neural progenitor cells and paracrine factors in 120 HIE infants has been underway since 2013 and is yet to be completed. 42The trial was registered on the clinicaltrials.gov website in 2016, with no updates since then.

Strengths and Limitations
Our systematic review has several strengths.It followed the PRISMA reporting guideline and systematically summarized the evidence from published research on allogeneic cell therapy in term and preterm neonates.The search was comprehensive and included several databases and clinical trial registries.The systematic review also highlighted ongoing research and identified critical gaps and the need for further studies.Nonetheless, limitations must be acknowledged.The limitations largely arise from the design of the studies included in this systematic review.Included studies were few, had enrolled a small cohort of patients, had methodological limitations, were primarily single-arm studies, and lacked long-term outcome data.Furthermore, the safety and feasibility of other allogeneic cells are limited, as MSCs were predominantly used.In addition, as in any other cell therapy research, it is challenging to make distinct conclusions as different cells were used for different conditions with different dosing and timing.Finally, no meta-analysis was conducted due to a lack of homogeneous data from 2 or more studies.

Conclusions
Results from this systematic review summarize the curevidence on the safety, feasibility and efficacy of allogeneic cell therapy in neonates.This primarily involved studies examining the administration of MSCs from different sources, highlighting some evidence regarding the safety and feasibility.However, the review notes that data on efficacy outcomes are very limited, in both the short and long term.Therefore, further research on a broader population of allogeneic cells is required, particularly emphasizing their efficacy related to the predominant morbidities that require therapeutic intervention in the neonatal period.

Funding
No specific funding was sought for this project.Dr Razak receives a doctoral scholarship from Monash University and Lions Cord Blood Foundation.Professors Miller and Malhotra receive funding from the National Health and Medical Research Council of Australia.Professor Malhotra and Dr McDonald receive funding from Medical Research Future Fund of Australia.The funding body played no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.

Figure 1 .
Figure 1.Study flow diagram outlining stages of search results and filtering process (as per Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines).

Figure 2 .
Figure 2. Types of allogeneic cells used and their indications in the neonatal period.

Table 1 .
Characteristics of included studies.

Table 2 .
Characteristics of ongoing studies.