Stem cell‐based interventions for the prevention and treatment of intraventricular haemorrhage and encephalopathy of prematurity in preterm infants

Olga Romantsik; Alvaro Moreira; Bernard Thébaud; Ulrika Ådén; David Ley; Matteo Bruschettini

doi:10.1002/14651858.CD013201.pub3

Stem cell‐based interventions for the prevention and treatment of intraventricular haemorrhage and encephalopathy of prematurity in preterm infants

Authors' declarations of interest

Version published: 15 February 2023 Version history

https://doi.org/10.1002/14651858.CD013201.pub3

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Abstract

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Background

Germinal matrix‐intraventricular haemorrhage (GMH‐IVH) and encephalopathy of prematurity (EoP) remain substantial issues in neonatal intensive care units worldwide. Current therapies to prevent or treat these conditions are limited. Stem cell‐based therapies offer a potential therapeutic approach to repair, restore, or regenerate injured brain tissue. These preclinical findings have now culminated in ongoing human neonatal studies. This is an update of the 2019 review, which did not include EoP.

Objectives

To evaluate the benefits and harms of stem cell‐based interventions for prevention or treatment of GM‐IVH and EoP in preterm infants.

Search methods

We used standard, extensive Cochrane search methods. The latest search was April 2022.

Selection criteria

We attempted to include randomised controlled trials, quasi‐randomised controlled trials, and cluster trials comparing 1. stem cell‐based interventions versus control; 2. mesenchymal stromal cells (MSCs) of type or source versus MSCs of other type or source; 3. stem cell‐based interventions other than MSCs of type or source versus stem cell‐based interventions other than MSCs of other type or source; or 4. MSCs versus stem cell‐based interventions other than MSCs. For prevention studies, we included extremely preterm infants (less than 28 weeks' gestation), 24 hours of age or less, without ultrasound diagnosis of GM‐IVH or EoP; for treatment studies, we included preterm infants (less than 37 weeks' gestation), of any postnatal age, with ultrasound diagnosis of GM‐IVH or with EoP.

Data collection and analysis

We used standard Cochrane methods. Our primary outcomes were 1. all‐cause neonatal mortality, 2. major neurodevelopmental disability, 3. GM‐IVH, 4. EoP, and 5. extension of pre‐existing non‐severe GM‐IVH or EoP. We planned to use GRADE to assess certainty of evidence for each outcome.

Main results

We identified no studies that met our inclusion criteria. Three studies are currently registered and ongoing. Phase 1 trials are described in the 'Excluded studies' section.

Authors' conclusions

No evidence is currently available to evaluate the benefits and harms of stem cell‐based interventions for treatment or prevention of GM‐IVH or EoP in preterm infants. We identified three ongoing studies, with a sample size range from 20 to 200. In two studies, autologous cord blood mononuclear cells will be administered to extremely preterm infants via the intravenous route; in one, intracerebroventricular injection of MSCs will be administered to preterm infants up to 34 weeks' gestational age.

PICOs

Population

Intervention

Comparison

Outcome

The PICO model is widely used and taught in evidence-based health care as a strategy for formulating questions and search strategies and for characterizing clinical studies or meta-analyses. PICO stands for four different potential components of a clinical question: Patient, Population or Problem; Intervention; Comparison; Outcome.

See more on using PICO in the Cochrane Handbook.

Plain language summary

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Stem cell‐based therapies for brain injury in newborns born preterm

Review question

Do stem cell‐based therapies save the lives or improve the long‐term development of preterm newborns who have or may develop bleeding to the brain (intraventricular haemorrhage) or grey matter damage?

Background

Newborns born too early ('preterm'), especially babies born before 28 weeks of pregnancy, sometimes develop bleeding to the brain. Babies with less severe bleeding may make a full recovery or may have only mild problems. For other babies with more serious bleeding, or with grey matter damage, this may lead to death or to problems later in life. The brain is growing very fast during the second and third trimester of pregnancy. Nerve cells move throughout the brain to destination regions, where they become mature and make connections with each other in order to proceed with the information. In addition, some nerve fibres are surrounded by a specific insulating layer, called myelin, and the process of such insulation – myelinisation – starts around 24 weeks of pregnancy. If the baby is born preterm these processes in the brain may be altered and there is a higher risk for long‐term abnormal neurodevelopment. This condition is called 'encephalopathy of prematurity'. For instance, some of these babies develop intellectual disabilities, behavioural problems, concentration difficulties, socialisation problems, and cerebral palsy. Currently, no approaches are available to prevent or treat bleeding to the brain or grey matter damage.

What did we want to find out?

The aim of this review was to assess whether stem cell‐based therapies could reduce death and improve the long‐term development of newborns born too early. During cell stem‐based therapy, stem cells are given to the baby, for instance, through injection. These stem cells may have come from humans or animals and may have been taken from cord blood, bone marrow, or other parts of the body. These cells then repair the brain cells that have been damaged by bleeding or grey matter damage.

What did we do?

We searched medical databases for clinical trials looking at stem cell‐based therapies for brain injury in newborns born too soon.

Key results

We were unable to include any studies in our review. We did identify nine studies, but we excluded them because of the way they were designed, which meant that their results could not answer our review question.

How current is the evidence?

This review updates and expands our previous review which was published in 2019. The evidence is current to April 2022.

Authors' conclusions

Implications for practice

No evidence is currently available to evaluate the benefits and harms of stem cell‐based interventions for treatment or prevention of germinal matrix‐intraventricular haemorrhage (GM‐IVH) or encephalopathy of prematurity (EoP) in preterm infants. We identified three ongoing studies, with a sample size range of 20 to 200. In two studies, autologous cord blood mononuclear cells will be administered to extremely preterm infants via the intravenous route; in one, intracerebroventricular injection of MSCs will be administered to preterm infants up to 34 weeks' gestational age.

Implications for research

Future challenges in summarising data on regenerative cells for neonates with, or at risk for, GM‐IVH or EoP can be divided into four central themes.

Cell therapy: does cell therapy provide therapeutic benefit? Which cell and tissue source confers efficacy? What dose and route of delivery optimises benefit? How frequently should therapy be administered? Is there synergism when more than one cell therapy is used?
Patient selection: which patients should be treated? When should they be treated (i.e. preventive or rescue)? Does sex or ethnicity (or both) impact therapeutic potential?
Cell processing: should cells be used fresh, or can they be frozen and thawed? What viability of cells is acceptable for transplantation? How many passages of the cells are tolerable? Which assay(s) deliver the best in vivo results? What is/are the mechanisms by which regenerative cells work for neonatal brain injury?
Outcome measure: should the focus be on prevention of intraventricular haemorrhage/EoP or progression of disease? On functional outcome or imaging studies? Or on a combined outcome of death or ventriculoperitoneal surgical intervention?

Ideally, these questions ought to be addressed in rigorous preclinical studies to guide the design of human studies. Focusing on answering these questions may prevent large, costly, labour‐intense human studies that may result in variable results. In summary, optimising regenerative cells in small and large animals will provide the groundwork for future clinical trials.

Background

Description of the condition

Neonatal brain injury

Neonatal brain injury in preterm infants includes germinal matrix‐intraventricular haemorrhage (GMH‐IVH) and encephalopathy of prematurity (EoP), which are discussed below.

Germinal matrix‐intraventricular haemorrhage

Preterm birth remains the major risk factor for developing GMH‐IVH. GMH‐IVH occurs in 25% of very low birth weight (VLBW) infants (NCT01825499), and complications of GMH‐IVH, including periventricular haemorrhagic infarction, posthaemorrhagic ventricular dilation (PHVD), cerebellar haemorrhagic injury, and periventricular leukomalacia (PVL), affect neonatal morbidity, mortality, and long‐term neurodevelopmental outcomes (Sherlock 2005).

Although improvements in perinatal care since the late 1970s have led to significant reduction in the incidence of GMH‐IVH in preterm infants (from 50% in the late 1970s to current rates of 15% to 25%) (Hamrick 2004; Horbar 2002; Philip 1989), GMH‐IVH remains a substantial issue in neonatal intensive care units worldwide. Survival of extremely preterm infants has increased drastically to as high as 85% to 90% (EXPRESS 2009; Ishii 2013). Moreover, the survival rate of preterm infants at the lower gestational age, who are highly susceptible for the development of GMH‐IVH and its sequelae, has increased notably since the late 1970s. Approximately 45% of preterm infants with birth weight below 750 g are affected by some degree of GMH‐IVH, and up to 35% of these haemorrhages are categorised as severe (Wilson‐Costello 2005). Thus, it seems that we have observed a plateau in the incidence of GMH‐IVH (Horbar 2002; Horbar 2012). Approximately 50% to 75% of preterm survivors with GM‐IVH (any grade) develop cerebral palsy, cognitive impairment, PHVD, or a combination of these conditions, with serious sequelae for neurodevelopmental outcomes (Luu 2009). Moreover, around 25% of non‐disabled survivors develop psychiatric disorders and problems with executive function (Indredavik 2010; Nosarti 2007; Whitaker 2011). Hence, GMH‐IVH as well as its neurological and psychiatric consequences continues to be a noteworthy public health concern worldwide.

In general, the diagnosis of GMH‐IVH is made during the first week of life; approximately 50% of these haemorrhages are seen during the first 24 hours of life, and as much as 90% of GMH‐IVH is diagnosed within the first 96 hours of life. Up to 40% of these bleeds will increase in size during the first days of life (Volpe 2008). The incidence of antenatal GM‐IVH is unclear, although an estimate for intracranial bleeding of 1 in 10,000 pregnancies has been suggested (Vergani 1996). Antenatal foetal intracranial haemorrhage may occur spontaneously or in association with various maternal or foetal conditions. Predisposing maternal conditions include platelet and coagulation disorders, medications (warfarin), illicit drugs (cocaine), seizures, smoking, trauma, amniocentesis, and febrile disease; foetal conditions include twin–twin transfusion, demise of a co‐twin, hydrops fetalis, congenital tumour, and fetomaternal haemorrhage (Kutuk 2014). GM‐IVH may undergo spontaneous resolution or, especially for grade 3 and 4, may lead to development of PHVD.

Pathophysiology of germinal matrix‐intraventricular haemorrhage

Both low‐grade and high‐grade GMH‐IVH may affect cerebellar growth, resulting in reduced cerebellar volume and impaired white matter and motor tract microstructure (Morita 2015; Sancak 2016; Sancak 2017; Srinivasan 2006; Tam 2009; Tam 2011). The cerebellum is the fastest‐growing portion of the brain; its volume increases fivefold from 24 weeks' postmenstrual age to 40 weeks' postmenstrual age (Volpe 2009a). During this period, extravasation of haemoglobin due to GMH‐IVH into cerebrospinal fluid and further haemolysis of free haemoglobin may result in deposition of haemosiderin on the cerebellar surface, disturbing normal development of the cerebellar cortex (Agyemang 2017; Fukumizu 1995; Koeppen 2008; Messerschmidt 2005). It is well recognised that the cerebellum plays a crucial role not only in motor function but also in many higher‐order cognitive and affective functions, such as executive function, working memory, and emotional processing (van Overwalle 2014; Volpe 2009a). Thus, preventing GMH‐IVH would also help to preserve cerebellar integrity.

The aetiology of GMH‐IVH is multifactorial, complex, and heterogeneous (Romantsik 2019a). An intrinsic fragility of the germinal matrix (GM) vasculature predisposes patients to haemorrhage, and fluctuation in cerebral blood flow (CBF) induces the rupture of vasculature (Ballabh 2014). The association between CBF fluctuation and onset of GM‐IVH in ventilated neonates with respiratory distress syndrome (RDS) during the first day of life was suggested by Perlman and colleagues (Perlman 1983). One subsequent study by the same group showed that elimination of CBF fluctuation by neuromuscular paralysis (pancuronium) resulted in reduction in GM‐IVH (Perlman 1985). The question remained whether the fluctuations in CBF were due to breathing against the ventilator, which could be explained by increased pleural pressure fluctuations. Two studies confirmed that CBF fluctuations were related to RDS extent and pleural pressure fluctuations, and that those could be damped by mechanical ventilation (Mullaart 1994; Perlman 1988). It has been suggested that loss of cerebral autoregulation, which is important for maintaining constant CBF, may predispose preterm infants to haemorrhagic and ischaemic cerebral injury (Boylan 2000; Tsuji 1998). However, ensuing studies on impaired autoregulation were not predictive of the subsequent development of GM‐IVH; nevertheless impaired autoregulation was correlated with higher mortality (Soul 2007; Wong 2008). Vaginal delivery, low Apgar score, severe RDS, pneumothorax, hypoxia, hypercapnia, seizures, patent ductus arteriosus, infection, and other conditions seem to increase primarily the fluctuations in CBF, and thus represent important risk factors for development of GM‐IVH (Ballabh 2014). If platelet or coagulation disorders are associated, the homeostasis mechanisms are impaired, which might accentuate the haemorrhage. The GM is located inside an arterial end zone, resulting in a direct interconnection with the deep galenic venous system of the brain (Nakamura 1990; Pape 1979), thereby exposing this particular area to arterial ischaemia‐reperfusion insults and to venous congestion (Pape 1979; Takashima 1978). The immature deep galenic system is highly susceptible to venous congestion and stasis, thus playing a central role in the advancement of GMH‐IVH and its sequelae (Pape 1979; Volpe 2008).

Encephalopathy of prematurity

The term 'EoP' was introduced by Volpe in 2005 to describe the injury to the white and grey matter in the preterm infant (Volpe 2005). The injury to the neuronal/axonal unit, affecting the brain white matter, thalamus, basal ganglia, cerebral cortex, and cerebellum, leads to cognitive, behavioural, or socialisation deficits in 25% to 50%, and major motor deficits (e.g. cerebral palsy) in 5% to 10% of extremely preterm infants (Morsing 2022; O'Reilly 2020; Volpe 2009b). Due to the increased survival of extremely preterm infants (Ishii 2013; Norman 2019; Younge 2017), the issue of EoP becomes really substantial with a significant contribution to the burden of disability. Cognitive and behavioural abnormalities without any major motor impairment are dominant neurodevelopmental sequelae following preterm birth. Traditionally, neonatal brain injury was referred to as either cerebral lesion or tissue loss (Volpe 2009c). However, since the 1990s, thanks to advancements in neonatal neurology, neuroradiology, and developmental neurobiology, the mapping out the pathophysiology of brain injury and the role of maturation‐dependent factors made it possible to identify specific brain regions and cell types that are vulnerable to injury (Volpe 2009b; Volpe 2009c; Volpe 2009a). Thus, EoP reflects in both primary brain injury and in mechanisms related to brain development. The most fragile perinatal period seems to be between gestational weeks 22 to 32, in which the final stages of neurogenesis, neuronal migration, differentiation, maturation, synaptogenesis, and initial cortical myelination take place (Fleiss 2020; Volpe 2009c). These neurodevelopmental events are highly vulnerable to endogenous and exogenous insults, such as infection/inflammation (often the onset is antenatal), hyperoxia, hypoxia‐ischaemia, and excitotoxicity (Volpe 2009b). Currently, no treatment is available.

Due to the lack of specific clinical signs in the neonatal period, the diagnosis of EoP relies on neuroimaging studies, such as cerebral ultrasound and magnetic resonance imaging (MRI), and neurophysiological studies, such as electroencephalogram. In older infants and children who developed EoP, the neurodevelopmental impairment may include co‐ordination problems, cognitive difficulties, visual impairment, and behavioural and socialisation problems. Neuroimaging may be helpful to predict neurodevelopmental disabilities to some extent; however, some problems will become more evident at school age.

Pathophysiology of encephalopathy of prematurity

The multifactorial pathophysiology of EoP includes systemic infection/inflammation, hypoxia‐hyperoxia‐ischaemia, and alteration in glucose metabolism (as hypo‐/hyperglycaemia), which results in toxicity to pre‐oligodendrocytes, neurons, and axons caused by free radicals, cytokines, and unbalance in excitatory/inhibitory neurotransmitters. It involves both cerebral white and grey matter. There is a spectrum of white matter injury (WMI) varying from diffuse non‐destructive injury to necrotic lesions known as PVL. While damage to white matter in PVL is characterised by the loss of axons, oligodendrocytes, glia, and interstitial neurons, leading to a myelination failure and retrograde degeneration of axons resulting in neuronal loss in the cerebral grey matter, the diffuse white matter injury (dWMI) is characterised by selective degeneration of preoligodendrocytes, leading to altered myelination due to differentiation failure of preoligodendrocytes to oligodendrocytes (Back 2018). Axons and interstitial neurons seem to be spared in dWMI, however, some experimental data suggest that larger calibre axons may be susceptible to hypoxic‐ischaemic injury during the later stages of myelination of white matter (Alix 2012). Altogether, dWMI is characterised not only by hypomyelination but also by the aberrant organisation of myelin fibres.

Along with WMI, there is increased recognition of grey matter involvement in the pathogenesis of EoP. Several MRI studies in preterm infants demonstrated the volumetric reduction of both cortical grey matter and deep grey matter with diffusivity alterations in thalamocortical networks (Ball 2012; Ball 2015; Boardman 2006; Makropoulos 2016). These clinical data are supported by animal studies (Crum 2017; Dean 2011; Dean 2013). Furthermore, preterm infants might show an alteration in cerebral cortex folding and gyrification, such as decreased cortical surface, and fewer and shallower sulci (Engelhardt 2015; Kersbergen 2016; Long 2018; van Essen 2019; Zhang 2015). Reduced cortical folding appears to be associated with adverse neurodevelopment further in childhood (Kersbergen 2016). On a cellular level, a reduction in neurons and increased neuronal death in cerebral grey matter in newborns with PVL has been demonstrated (Andiman 2010; Haynes 2013; Kinney 2012; Ligam 2009), whereas in newborns with dWMI neuronal number seems to be not affected. However, subtle changes in interneurons are reported, such as the alteration in a population of different subtypes of interneurons (Lacaille 2019; Panda 2018; Stolp 2019). These pathological findings reported in preterm infants postmortem are confirmed by experimental data in animal models of EoP (Ardalan 2019; Canetta 2016; Lacaille 2019; Panda 2018; Stolp 2019; Vaes 2020). Furthermore, the complexity of dendritic arborisation and synapse formation – which are essential for proper connectivity in the brain – seems to be reduced for certain populations of interneurons (Dean 2011; Stolp 2019).

Description of the intervention

Advances in stem cell research have highlighted the therapeutic potential of regenerative cells. At the forefront of these cell‐based therapies is the mesenchymal stem/stromal cell (MSC). MSCs are the most commonly used regenerative cells in clinical trials due to their relatively safe profile, ease of isolation and propagation, and pleiotropic effects in preventing/restoring organ damage (Trounson 2015). The minimum criteria for defining MSCs include adherence to plastic in standard culture conditions; expression and lack of specific surface markers; and multipotent differentiation potential along the osteogenic, chondrogenic, and adipogenic lineages (Dominici 2006). Although MSCs are ubiquitously used in regenerative studies, other cell‐based therapies are beginning to receive attention. For instance, mononuclear cells (MNC), oligodendrocyte progenitor cells (OPCs), neural stem cells (NSC), haematopoietic stem cells, and inducible pluripotent stem cells (iPSC) have demonstrated efficacy in animal models of brain injury (see Table 1; Phillips 2013; Pimentel‐Coelho 2012). Furthermore, one ongoing clinical trial using autologous human umbilical cord blood for neonatal asphyxia contains a heterogeneous mixture of these cells (MSCs, haematopoietic stem cells, and other MNCs) (NCT02612155). Although iPSCs are typically obtained from skin cells, they can also be retrieved from blood or MSCs to become a specialised cell, such as a NSC or an oligodendrocyte (Cai 2010).

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Table 1. Types of regenerative cells

Cell type	Source	Rationale	Mechanism of action	Preclinical/clinical results	References
MSC	Human umbilical cord tissue/blood; rodent/human bone marrow	Safe and feasible in phase 1 RCT for bronchopulmonary dysplasia Low immunogenicity (low MHC II), easily obtainable, rapid expansion Autologous/allogeneic administration Paracrine release of trophic factors	Paracrine release of IGF‐1, EGF, VEGF, BDNF Immunomodulatory: regulate T‐cell, B‐cell function, and production of inflammatory cytokines Mitochondrial transfer	Nerve fibre remyelination and axonal regeneration; improve behavioural/motor tests Enhance neural cell proliferation, survival, function Decrease infarct size	Ahn 2016; Boshuizen 2018; Chopp 2002; Hsu 2016; Islam 2012; Liu 2010; Murphy 2013; Park 2016
MNC	Human umbilical cord blood	Readily collected and large supply in cord blood with high plasticity Safe and feasible in phase 1 RCT for hypoxic‐ischaemic encephalopathy Low immunogenicity (minimal HLA matching) Paracrine release of trophic factors Autologous/allogeneic administration	Increase expression of BDNF, NGF, VEGF, GDNF Activate prosurvival Akt pathway Decrease TNF‐α and increase IL‐10 gene expression Reduce CD4+ T‐cell infiltration Regulate hedgehog signalling	Decrease neuronal apoptosis, astrogliosis, inflammation Improve oligodendrocyte survival; induce axonal growth Improve neurobehavioural outcomes	Aridas 2016; Cotten 2014; Fan 2005; McDonald 2018; Pimentel‐Coelho 2012; Rowe 2010; Wang 2013
OPC	Rodent/human ESC; human NSC derivation	Differentiate into oligodendrocytes (cells highly susceptible to hypoxic‐ischaemic injury) Remyelinate injured axons	Diffuse biodistribution along white matter tracts and differentiate into myelin sheath‐producing oligodendrocytes	Promote myelin sheath formation and NSC proliferation, inhibit apoptosis Motor recovery following CNS injury	Chen 2015; Gopagondanahalli 2016; Kim 2018; Manley 2017; Niimi 2018; Xu 2015
NSC	Human foetal striatum; human ESC; human iPSC	Differentiate into cells necessary for brain repair, including neurons, astrocytes, and oligodendrocytes Paracrine release of trophic factors Low immunogenicity and tumorigenicity	Immunomodulation Paracrine secretion of BDNF, VEGF, and EGF Attenuate NF‐κB signalling Upregulate glutamate transport	Stimulate survival and migration of endogenous NSCs and neurons Reduce inflammation and reactive oxygen species production Improve axonal growth, motor function; decrease infarct size	Daadi 2016; Huang 2018; Ji 2015; Mine 2013
HSC	Umbilical cord blood	Paracrine release of neurotrophic factors Multipotent capacity and ability to transdifferentiate into neuronal cells Autologous/allogeneic administration	Reduce microglial cells and T lymphocytes Secrete VEGF, HGF, IGF‐1	Decrease infarct size and maintain cerebral blood flow Enhance axonal growth Ameliorate neuronal apoptosis and postischaemic inflammation	Schwarting 2008; Tsuji 2014; Verina 2013
iPSC	Skin fibroblasts, umbilical cord tissue, amniotic tissue	Autologous administration Differentiation into multiple neural lineage cells Low immunogenicity	Differentiate into functional neural cells (electrophysiological properties) Decrease infiltration of MPO+ neutrophils and CD11b+ microglia VEGF expression and organelle transfer	Improve survival and sensorimotor function Establish axonal connections Inhibit inflammation, neural apoptosis, and glial scar formation	Cai 2010; Hsu 2016; Oki 2012; Pluchino 2013; Qin 2015; Tornero 2013

BDNF: brain‐derived neurotrophic factor; CNS: central nervous system; EGF: epidermal growth factor; ESC: embryonic stem cell; GDNF: glial cell‐line‐derived neurotrophic factor; HGF: hepatocyte growth factor; HLA: human leukocyte antigen; HSC: haematopoietic stem cell; IGF‐1: insulin‐like growth factor‐1; IL: interleukin; iPSC: inducible pluripotent stem cell; MHC: major histocompatibility complex; MNC: mononuclear cell; MSC: mesenchymal stem cell; NF‐κB: nuclear factor kappa beta; NGF: nerve growth factor; NSC: neural stem cell; OPC: oligodendrocyte progenitor cell; RCT: randomised controlled trial; TNF: tumour necrosis factor; VEGF: vascular endothelial growth factor.

Aside from cell type, tissue source and laboratory processing may affect the efficacy of the regenerative cell. The perinatal period offers an opportune time to collect umbilical cord tissue/blood, amniotic fluid, or placental tissue (Garcia 2014; Parolini 2014; Sanberg 2014; Taghizadeh 2014). These sources are considered medical waste and therefore are easily available in a non‐invasive manner and offer reduced immunogenicity and higher differential potential compared to other sites (Batsali 2013; Parolini 2008). The therapeutic potential of regenerative cells may also be influenced by passage number. A passage consists of removing cells from a culture flask and plating them into more culture flasks. Fewer passages are preferred, as multiple passages may impair cell function (Bellayr 2014; Wagner 2008). It is interesting to note that cord blood from preterm infants contains a greater quantity of immature haematopoietic progenitor cells and MSCs that have higher proliferative capacity compared with term infants (Podesta 2015).

Although regenerative cells are characterised by low immunogenicity (Gebler 2012), autologous transplantation is likely to be associated with lower risk for infection and immune rejection. However, allogeneic transplantation may offer significant practical advantages.

How the intervention might work

EoP may result in long‐term neurocognitive dysfunction, and behavioural and socialisation problems (Volpe 2009b; Volpe 2009c). Despite the burden of disabilities caused by EoP no treatment is available. One of the hallmarks of EoP is hypomyelination and arrest of oligodendrocyte maturation. In several rodent models of EoP mimicking dWMI, MSCs (administered either intranasally or intraperitoneally) migrate into the damaged regions of the brain, restore oligodendrocyte maturation and improve myelination, attenuate neuroinflammation, resulting in the better functional outcome on neurobehavioral tests (Mueller 2017; Oppliger 2016; Vaes 2021). Furthermore, in vitro studies demonstrated that MSCs express several growth factors, such as insulin‐like growth factor‐1 (IGF‐1), epidermal growth factor (EGF), leukaemia inhibitory factor (LIF), interleukin (IL)‐11 following ex vivo exposure to dWMI (Vaes 2021). These factors are important for oligodendrocyte's maturational process. Additionally, in the sheep foetal model of dWMI, induced by either transient umbilical cord occlusion or intra‐uterine lipopolysaccharide (LPS) infusion, it has been shown that intravenous infusion of MSCs resulted in a reduction of oligodendrocytes loss, reduced demyelination, and microgliosis (Jellema 2013; Li 2016; Paton 2018), and diminished neuroinflammatory response (Li 2016; Paton 2018; Paton 2019). The other important aspect of EoP is a subtle injury to cerebral grey matter (Fleiss 2020; Volpe 2009b). γ‐Aminobutyric acid (GABA)‐ergic interneurons are rapidly developing during the third trimester in human pregnancies and this process continues several months after birth (Lim 2018). It is believed that maturational arrest and function of interneurons may be the underlying cause of behavioural deficits, such as attention deficit disorders, autism spectrum disorder, and socialisation problems. These problems tend to be important in children born extremely preterm (Fleiss 2020; Marin 2012). The only animal study published so far on the effect of MSC treatment on interneuron development in EoP model demonstrated partial recovery in social behaviour and restoration of parvalbumin‐positive interneurons, vasoactive intestinal polypeptide (VIP)‐positive cells in the cortex, and somatostatin (SST)‐positive cells in the hippocampus (Vaes 2020). Despite the few animal studies available at present stem cells seem to be a promising treatment strategy for EoP.

Preterm infants with intraventricular haemorrhage (IVH) are at higher risk for white matter damage resulting in cerebral palsy and cognitive‐behavioural disorders (Luu 2009). Currently, no treatment is available for white matter damage following IVH. It has been suggested that the haemorrhage originates in the GM and evolves into IVH with further rupture of the ventricular ependyma (Ballabh 2014). Periventricular tissue is characterised by high presence of preoligodendrocytes, which progressively differentiate to mature oligodendrocyte. This process is particularly active between weeks 23 and 35 of gestation (Back 2001; Khwaja 2008). Upon injury, OPCs migrate to the area of damage and differentiate to oligodendrocyte to restore myelination (Bonfanti 2017). However, following a neurological insult, the remyelination potential of resident OPCs is exhausted (van Velthoven 2012). In a rat model of hypoxic‐ischaemic brain injury, OPCs (differentiated from mouse embryonic stem cells (ESC)) migrated to the site of injury, differentiated into myelin‐producing oligodendrocyte, dampened neuronal apoptosis, and stimulated endogenous NSC proliferation (Chen 2015). Animal studies suggest that both acute hypoxic‐ischaemic insult and IVH may arrest maturation of the pro‐oligodendrocytes, resulting in hypomyelination of brain white matter and subsequent loss of brain grey matter (Dummula 2011; Segovia 2008). NSCs residing in the GM are therefore particularly vulnerable in preterm infants at risk of GMH (Dawes 2016; Fuentealba 2012). Currently, no strategies to prevent WMI are known.

The first use of stem cells for intracranial bleeding was proposed in adult animals: NSCs derived from mouse ESCs were transplanted into 10 adult rats with collagenase‐induced intracerebral haemorrhage (Nonaka 2004). Subsequent studies focused on the neuroprotective effects of MSCs. In the same animal model of intracerebral haemorrhage, administration of umbilical cord‐derived MSCs resulted in nerve fibre remyelination and axonal regeneration, and better neurological recovery compared to vehicle‐treated animals (Liu 2010). Similar findings were discovered after NSC administration in non‐human primates with spinal injury (Rosenzweig 2018). The effects of umbilical cord blood MSCs and MNCs have been explored in small and large animals with severe IVH (Ahn 2015; Aridas 2016). In rat pups with severe IVH, administration of both intracerebral and intravenous MSCs resulted in attenuation of PHVD, better myelination, and improvement on behavioural tests (Ahn 2015). Subsequent studies showed that early MSC administration resulted in greater brain damage recovery (Park 2016). Even a low‐grade GM‐IVH may impair normal oligodendrogenesis by significantly reducing the number of oligodendrocytes in the cortex, whereas postnatal neurogenesis per se did not seem to be affected (Dawes 2016).

Regenerative cells do not exert their therapeutic benefit by engrafting into the host tissue, but rather through the release of paracrine effects (Hodgkinson 2016). They promote axon and dendrite growth by secreting mitogenic growth factors (brain‐derived neurotrophic factor (BDNF), stromal cell‐derived factor‐1 (SDF‐1), nerve growth factor (NGF)) known to enhance proliferation, migration, and differentiation of native neuronal progenitor/stem cells (Chen 2008; Chopp 2002; Gage 2013; Murphy 2013). Moreover, cell‐based therapies can attenuate inflammation (IL‐1β, interferon‐γ (IFNγ)) by modulating the function of immune cells, such as T and B cells, macrophages, and dendritic cells (Boshuizen 2018; Iyer 2008). They enhance angiogenesis (vascular endothelial growth factor (VEGF), IGF‐1), regulate reactive oxygen species production, and may even transfer organelles to injured cells (i.e. mitochondria) (Boshuizen 2018; Chen 2008; Islam 2012; Murphy 2013). Hence, regenerative cells are emerging as potential treatment strategies for complex diseases such as GM‐IVH with subsequent impaired brain development.

Why it is important to do this review

This review is important to conduct as regenerative cells might be an effective intervention for prevention and treatment of GM‐IVH and EoP, which are amongst the most severe morbidities in preterm infants. To date, US clinical trials studying the safety, feasibility, or efficacy (or a combination of these) of regenerative cells have been registered for bronchopulmonary dysplasia (BPD), hypoxic‐ischaemic encephalopathy, hypoplastic left heart syndrome, and GM‐IVH. The most widespread tissue used to derive regenerative cells is the umbilical cord (Mitsialis 2016; Yoon 2016). Although MSCs dominate as the cell type, a few trials are evaluating neural progenitor cells, MNCs, and placenta/cord blood cells (NCT02434965; NCT02854579; NCT02999373).

The efficacy and safety of regenerative cell administration have been assessed in several systematic reviews and meta‐analyses. For instance, one meta‐analysis across different disciplines evaluated the safety of MSCs (eight studies including 321 adults), finding no association between acute infusional toxicity, organ system complications, infection, death, or malignancy (Lalu 2012). However, the risk of potential tumourigenicity related to MSC‐based interventions needs to be further elucidated (Barkholt 2013). The Cochrane Review entitled "Stem cell transplantation for ischaemic stroke" included three small trials in adults (Boncoraglio 2019). For newborns, one Cochrane Review has been conducted on MSC for prevention and treatment of BPD amongst preterm infants (Pierro 2017). Early‐phase trials have been conducted (or are underway) on the use of MSCs or cord blood cells (or both) for BPD, severe IVH (NCT02274428), and hypoxic‐ischaemic encephalopathy (Cotten 2014).

Objectives

To evaluate the benefits and harms of stem cell‐based interventions for prevention or treatment of GM‐IVH and EoP in preterm infants.

Methods

Criteria for considering studies for this review

Types of studies

We included randomised controlled trials (RCTs), quasi‐RCTs, and cluster trials. We excluded cross‐over trials.

Types of participants

Prevention studies:

extremely preterm infants (less than 28 weeks' gestation), 24 hours of age or less, without ultrasound diagnosis of GM‐IVH or EoP.

Treatment studies:

preterm infants (less than 37 weeks' gestation), of any postnatal age, with ultrasound diagnosis of GM‐IVH (GM‐IVH treatment studies). We included infants with ventricular shunt;
preterm infants (less than 37 weeks' gestation), of any postnatal age, with diagnosis of EoP (e.g. cystic PVL, dWMI, grey matter injury) (EoP treatment studies).

Types of interventions

For each of the population described above, we conducted the following comparisons.

Comparison 1: stem cell‐based interventions (any type) compared to control (placebo or no treatment)
Comparison 2: use of MSCs of type (e.g. number of doses or passages) or source (e.g. autologous versus allogeneic, bone marrow versus cord) versus MSCs of other type or source
Comparison 3: use of stem cell‐based interventions other than MSCs of type (e.g. MNCs, OPCs, NSCs, haematopoietic stem cells, iPSCs) or source (e.g. autologous versus allogeneic, bone marrow versus cord) versus stem cell‐based interventions other than MSCs of other type or source
Comparison 4: MSCs versus stem cell‐based interventions other than MSCs

We included all types of transplantation regardless of cell source (bone marrow, cord blood versus Wharton's jelly, placenta, adipose tissue, peripheral blood), type of graft (autologous or allogeneic), route of administration (intravenous or intraventricular, e.g. in infants with PHVD and ventricular shunt), and dose. We excluded stem cell‐derived cerebral organoids (Di Lullo 2017).

Although procedures to increase placental transfusion such as delayed cord clamping and cord milking are potential sources of (limited numbers of) stem cells, a separate Cochrane Review has addressed these interventions (Rabe 2019).

Characteristic of these interventions are specified in Subgroup analysis and investigation of heterogeneity.

Types of outcome measures

Primary outcomes

All‐cause neonatal mortality (mortality less than 28 days of age)
Major neurodevelopmental disability: cerebral palsy, developmental delay (Bayley Mental Developmental Index (Bayley 1993; Bayley 2006) or Griffiths Mental Development Scale (Griffiths 1954) assessment greater than two standard deviations (SDs) below the mean), intellectual impairment (intelligence quotient (IQ) greater than two SDs below the mean), blindness (vision less than 6/60 in both eyes), or sensorineural deafness requiring amplification (Jacobs 2013). We planned to separately assess data on children aged 18 to 24 months and those aged three to five years
For prevention trials: GM‐IVH: any grade; severe IVH grade 3 to 4 (for prevention studies only) (Papile 1978)
For prevention trials: EoP (Volpe 2005)
For GM‐IVH treatment trials: extension of pre‐existing non‐severe GM‐IVH (e.g. grade 1 to 2) to severe IVH (e.g. grade 3 to 4) (yes/no) (Papile 1978). Although the timing for neuroimaging might differ between trials, within each trial the intervals schedule must be identical in the intervention and control groups. If both ultrasound and MRI are available for the same infants, we plan to refer to the modality with the highest IVH grade

Secondary outcomes

All‐cause mortality before first hospital discharge
Cerebellar haemorrhage on brain ultrasound in the first month of life (yes/no; Graça 2013)
Cystic PVL on brain ultrasound in the first month of life
MRI abnormalities at term equivalent age (yes/no), defined as white matter lesions (i.e. cavitations; Rutherford 2010), punctate lesions (Cornette 2002), GM‐IVH (Parodi 2015), or cerebellar haemorrhage (Limperopoulos 2007)
Need for ventricular shunt in the first three months of life (yes/no)
Duration of hospital stay (days)
BPD/chronic lung disease: 28 days (NIH 1979); 36 weeks' postmenstrual age (Jobe 2001); 'physiological definition' (Walsh 2004)
Patent ductus arteriosus (that has been treated with cyclo‐oxygenase inhibitor or surgery)
Necrotising enterocolitis (NEC; defined as Bell's stage II or greater) (Bell 1978)
Retinopathy of prematurity (ROP) in infants examined (all stages and severe (stage 3 or greater)) (ICROP 1984)
Tumour formation, any type, any location, detected by MRI or computed tomography (to assess risk of tumourigenicity of donor MSCs)
Immune rejection or any serious adverse event (certain, probable, or possible according to the World Health Organization (WHO) probability scale). We planned to consider post‐hoc analyses for any unexpected adverse effects reported by these studies
Each component of the composite outcome 'major neurodevelopmental disability' (see Primary outcomes)

Search methods for identification of studies

Electronic searches

The Cochrane Sweden Information Specialist revised search strategies for this iteration of the review to incorporate additional terminology necessary due to the inclusion of EoP. They searched the following databases in April 2022; we did not use date, language, or publication type limits:

Cochrane Central Register of Controlled Trials (CENTRAL), Issue 4, 2022;
PubMed (1946 to 11 April 2022);
Embase (Embase.com, Elsevier), (1947 to 11 April 2022);
CINAHL (Cumulative Index to Nursing and Allied Health Literature) (EBSCOhost, 1982 to 11 April 2022).

Full search strategies are available in Appendix 1; search strategies used for the previous version of this review are in Appendix 2.

We searched the following clinical trials registries for ongoing and recently completed trials:

ClinicalTrials.gov (clinicaltrials.gov);
WHO International Trials Registry and Platform (ICTRP) (trialsearch.who.int/Default.aspx).

Searching other resources

We reviewed the reference lists of all identified articles for relevant articles not identified in the primary search. We searched for errata or retractions from included studies published in full text on PubMed (www.ncbi.nlm.nih.gov/pubmed).

Data collection and analysis

We used the standard methods of Cochrane Neonatal, as described below.

Selection of studies

Two review authors (MB, OR) independently screened title/abstracts and the full text of potentially relevant studies included based on title/abstract review. At any point in the screening process, disagreements between review authors were resolved by discussion or a third review author (DL). We independently assessed the eligibility of studies by filling out eligibility forms designed in accordance with the specified inclusion criteria. We excluded studies published only in abstract form unless the final results of the trial were reported and all necessary information could be ascertained from the abstract or from study authors, or both. We reviewed studies for relevance by assessing study design, types of participants, interventions provided, and outcome measures reported. We resolved disagreements by discussion and, if necessary, by consultation with a third review author (DL). We provided details of excluded studies in the Characteristics of excluded studies table, along with reasons for exclusion. We planned to contact trial authors if details of primary trials were unclear.

We recorded the selection process in sufficient detail to complete a PRISMA flow diagram (Moher 2009), and Characteristics of excluded studies tables.

Data extraction and management

Two review authors (MB, OR) planned to independently extract data using a data extraction form integrated with a modified version of the Cochrane Effective Practice and Organisation of Care Group data collection checklist (Cochrane EPOC Group 2017). We planned to pilot the form within the review team using a sample of included studies.

We planned to extract the following characteristics from each included study.

Administrative details: study author(s); published or unpublished; year of publication; year in which study was conducted; presence of vested interest; details of other relevant papers cited.
Study: study design; type, duration, and completeness of follow‐up (e.g. greater than 80%); country and location of study; informed consent; ethics approval.
Participants: sex, birth weight, gestational age, number of participants.
Interventions: initiation, dose, and duration of MSC administration.
Outcomes as mentioned above under Types of outcome measures.

We planned to resolve disagreements by discussion. We described ongoing studies identified by our search, when available, detailing the primary author, research question(s), methods, and outcome measures, together with an estimate of the reporting date, and we report them in the Characteristics of ongoing studies table.

Should any queries have arisen, or in cases for which additional data were required, we planned to contact study investigators/authors for clarification. Two review authors (MB, OR) planned to use the Cochrane statistical tool for data entry (RevMan Web 2022). We planned to replace any standard error of the mean (SEM) with the corresponding SD.

Assessment of risk of bias in included studies

Two review authors (MB, OR) planned to independently assess risk of bias (low, high, or unclear) of all included trials using the Cochrane RoB 2 tool for the following domains (Higgins 2021).

Sequence generation (selection bias).
Allocation concealment (selection bias).
Blinding of participants and personnel (performance bias).
Blinding of outcome assessment (detection bias).
Incomplete outcome data (attrition bias).
Selective reporting (reporting bias).
Any other bias.

We planned to resolve any disagreements by discussion or by consultation with a third review author (AM). See Appendix 3 for a more detailed description of risk of bias for each domain.

Measures of treatment effect

We planned to use risk ratios (RRs), risk differences (RDs), number needed to treat for an additional beneficial outcome (NNTBs) or number needed to treat for an additional harmful outcome (NNTHs) for categorical variables, and mean differences (MDs) for continuous variables. We planned to replace any within‐group SEM reported in a trial with its corresponding SD using the formula SD = SEM × √n, where n is the number of participants. We planned to report 95% confidence intervals (CIs) for each statistic.

Unit of analysis issues

We planned to include all RCTs and quasi‐RCTs in which the unit of allocation was the individual infant. If we had found any cluster RCTs, we planned to adjust analysis for the designed effect using the method stated in the Cochrane Handbook for Systematic Reviews of Interventions (Higgins 2021).

Dealing with missing data

We planned to obtain a dropout rate for each study. If we had found a significant dropout rate (e.g. greater than 20%), we planned to contact the study author(s) to request additional data. We planned to perform a sensitivity analysis to evaluate the overall results with and without inclusion of studies with a significant dropout rate. If a study had reported outcomes only for participants completing the trial or only for participants who followed the protocol, we planned to contact study author(s) to ask them to provide additional information to facilitate an intention‐to‐treat analysis; in instances when this would not be possible, we planned to perform a complete‐case analysis.

Assessment of heterogeneity

We planned to assess clinical heterogeneity by comparing the distribution of important participant factors between trials and trial factors (randomisation concealment, blinding of outcome assessment, loss to follow‐up, treatment type, co‐interventions). We planned to assess statistical heterogeneity by examining the I² statistic (Higgins 2021), a quantity that describes the proportion of variation in point estimates that is due to variability across studies rather than to sampling error.

We planned to interpret the I² statistic as follows, as described by Higgins 2003.

Less than 25%: no (none) heterogeneity.
25% to 49%: low heterogeneity.
50% to 74%: moderate heterogeneity.
75% or greater: high heterogeneity.

We planned to consider statistical heterogeneity to be substantial when the I² statistic was 50% or greater. In addition, we planned to use the Chi² test of homogeneity to determine the strength of evidence that heterogeneity was genuine. We planned to explore clinical variation across studies by comparing the distribution of participant‐important factors amongst trials and trial factors (randomisation concealment, blinding of outcome assessment, loss to follow‐up, treatment types, and co‐interventions). We planned to consider a threshold of P less than 0.1 as an indicator of whether there was heterogeneity (genuine variation in effect sizes).

Assessment of reporting biases

We planned to examine the possibility of within‐study selective outcome reporting for each study included in the review. We planned to search for trial protocols of included trials through electronic sources such as PubMed, ClinicalTrials.gov, and the WHO ICTRP to assess whether outcome reporting was sufficiently complete and transparent. We planned to investigate publication using funnel plots if we could include 10 or more clinical trials in the systematic review (Egger 1997; Higgins 2021).

Data synthesis

We planned to perform statistical analyses according to the recommendations of Cochrane Neonatal (neonatal.cochrane.org/en/index.html), using RevMan Web 2022. We planned to analyse all infants randomised on an intention‐to‐treat basis. We planned to analyse treatment effects in the individual trials. We planned to use a fixed‐effect model to combine the data. For all meta‐analyses, we planned to synthesise data using RR, RD, NNTB, NNTH, MD, and 95% CI. We planned to analyse and interpret individual trials separately when we judged meta‐analysis to be inappropriate.

Subgroup analysis and investigation of heterogeneity

We planned to perform the following subgroup analyses.

For MSC trials:

gestational age: extremely preterm infants (less than 28 weeks' gestation), very preterm infants (28 to 31+6 weeks' gestation);
chronological age: less than 3 days, 3 days or greater;
co‐intervention: with/without cooling;
MSC source: bone marrow, cord blood versus Wharton's Jelly, placenta, adipose tissue, peripheral blood;
type of graft: autologous or allogeneic;
preconditioned (yes, no);
fresh or frozen and thawed;
MSC dose: less than 2 × 10⁷/kg; 2 × 10⁷/kg or greater;
number of doses: multiple or single administration; and
passage number (i.e. removing cells from a culture flask and plating them into more culture flasks, see Description of the intervention): less than three; three to six; greater than six.

For other cell‐based interventions:

gestational age: extremely preterm infants (less than 28 weeks' gestation), very preterm infants (28 to 31+6 weeks' gestation);
chronological age: less than three days, three days or greater;
co‐intervention: with/without cooling;
cell source: bone marrow, cord blood, peripheral blood, placenta;
type of graft: autologous or allogeneic;
fresh or frozen and thawed; and
number of doses: multiple or single administration.

Sensitivity analysis

We planned to conduct sensitivity analyses to explore the effects of the methodological quality of trials, checking to ascertain whether studies with high risk of bias might overestimate the effects of treatment. Differences in study design of included trials might affect the results of the systematic review. We planned to perform a sensitivity analysis to compare the effects of MSCs in truly randomised trials as opposed to quasi‐randomised trials.

Summary of findings and assessment of the certainty of the evidence

We planned to use the GRADE approach to assess the certainty of evidence for the following (clinically relevant) outcomes (Schünemann 2013):

all‐cause neonatal mortality (mortality less than 28 days of age);
major neurodevelopmental disability: cerebral palsy, developmental delay (Bayley Mental Developmental Index or Griffiths Mental Development Scale assessment greater than two SDs below the mean), intellectual impairment (IQ greater than two SDs below the mean), blindness (vision less than 6/60 in both eyes), or sensorineural deafness requiring amplification;
severe IVH grade 3 to 4, for prevention studies;
all‐cause mortality before first hospital discharge;
EoP (Volpe 2005), for prevention studies;
ROP in infants examined (stage 3 or greater);
immune rejection or any serious adverse event (certain, probable, or possible according to the WHO probability scale).

Two review authors (MB; OR) planned to independently assess the certainty of evidence for each of the seven outcomes above. We planned to consider evidence from RCTs as high certainty but to downgrade the evidence one level for serious (or two levels for very serious) limitations based upon the following: design (risk of bias), consistency across studies, directness of evidence, precision of estimates, and presence of publication bias. We planned to use GRADEpro GDT to create a summary of findings table to report the certainty of evidence.

The GRADE approach results in an assessment of the certainty of a body of evidence for one of four grades.

High certainty: we are very confident that the true effect lies close to that of the estimate of the effect.
Moderate certainty: we are moderately confident in the effect estimate; the true effect is likely to be close to the estimate of the effect, but there is a possibility that it is substantially different.
Low certainty: our confidence in the effect estimate is limited; the true effect may be substantially different from the estimate of the effect.
Very low certainty: we have very little confidence in the effect estimate; the true effect is likely to be substantially different from the estimate of effect.

Results

Description of studies

We have provided results of the search for this review update in the study flow diagram (Figure 1).

Figure 1

Results of the search

The search identified 723 references (673 from databases; 50 from trial registries). After removing 119 duplicates, 604 references were available for screening. We excluded 592 references based on title/abstract and reviewed 12 full‐texts (nine full studies; three ongoing studies). We identified no studies for inclusion in this review, but the three ongoing studies may prove eligible for a subsequent update (ACTRN12619001637134; NCT02890953; NCT04440670). For details of excluded studies see Characteristics of ongoing studies table.

Included studies

We identified no trials that matched our inclusion criteria.

Excluded studies

We identified nine studies and excluded them all at full‐text screening: five were not randomised trials (Ahn 2018; Kotowski 2017; NCT02274428; NCT03696745; Yang 2018), two studied cerebral palsy (NCT01763255; NCT03087110), one used intratracheal administration (Ahn 2017), and one was withdrawn (NCT01121328) (see Characteristics of excluded studies table).

Ahn 2017 enrolled nine extremely preterm newborns at high risk of developing BPD. The mean gestational age was 25.3 (SD 0.9) weeks and the mean birth weight was 793 (SD 127) g. All newborns received MSCs intratracheally at a mean age of 10.4 (SD 2.6) days after birth. The first three enrolled infants received MSCs at a low dose (1 × 10⁷ cells/kg in 2 mL/kg of saline) and the remaining six newborns received MCSs at a high dose (2 × 10⁷ cells/kg in 4 mL/kg of saline). The MCSs were administered in two fractions similarly to surfactant administration. An historical case‐matched comparison group of 14 infants was used. All 9 infants were discharged alive and followed up to 24 months of corrected age. None of the infants required extra oxygen at discharge. One of nine infants died at six months' corrected age due to Enterobacter cloacae sepsis. No adverse events were reported in the remaining eight surviving infants at two years of follow‐up. The mean rehospitalisation rate in the MSC group was 1.4/participant because of respiratory infections during the two‐year follow‐up. The bodyweight growth of the infants in the MSCs group was significantly higher compared to the age‐matched comparison group, while there were no differences in the length/height and head circumference. None of the infants in the MCSs group was diagnosed with cerebral palsy, blindness, hearing deficiency requiring a hearing aid, or developmental delay.

Ahn 2018 enrolled nine newborns (three girls, six boys) with a mean gestational age of 26 weeks (birth weight 440 g to 1310 g at 7 to 15 postnatal days) and diagnosis of severe IVH. The intervention consisted of intraventricular transplantation via an anterior fontanelle tap, using a 24‐gauge catheter guided by cranial ultrasound, and the following sedation with intravenous fentanyl. The first three newborns received a low dose of ex vivo cultured allogeneic, unrelated, human umbilical cord blood‐derived MSCs (5 × 10⁶ cells/kg), and the next six received a double dose (1 × 10⁷ cells/kg). Transplantation was performed seven days after the diagnosis of IVH. All nine infants had IVH grade 4. No serious adverse effects or dose‐limiting toxicities attributable to MSC transplantation were identified. No infants died during the study period. Five of nine infants required shunt derivation. The follow‐up assessment (two years of age) of these nine preterm infants is ongoing (NCT02673788).

Kotowski 2017 included preterm newborns (less than 32 weeks' gestational age) who received immediate cord clamping and required blood transfusion for anaemia. The intervention consisted of autologous umbilical cord blood transfusion (five newborns) versus allogeneic red blood cell transfusion (15 newborns). There were no differences in neonatal morbidities (ROP, RDS, NEC); GM‐IVH displayed a trend towards improvement. Umbilical blood infusions appeared to be feasible and without relevant adverse effects, with trends towards hypernatraemia and acidaemia in the autologous cord blood group. A total of 22 plasma proteins were significantly different amongst cord blood recipients compared to controls (i.e. insulin‐like growth factor‐binding protein (IGFBP)‐2, IGFBP‐3, platelet‐derived growth factor A and B (PDGF‐AB), IGF‐1).

NCT01121328 was withdrawn (no recruitment). This study was initially designed to test the feasibility of collection, preparation, and infusion of autologous umbilical cord blood in the first 14 days after birth in preterm infants with gestational age less than 35 weeks. The primary outcome was the need for mechanical ventilation during the first month of life. The secondary outcomes were survival until 12 months of age; neurodevelopment at six, 12, and 18 months of age; and postnatal growth.

NCT01763255 is an ongoing RCT with a parallel assignment, which included eight children, aged 4 to 12 years with quadriplegic cerebral palsy. The intervention group children receive an intrathecal injection of bone marrow‐derived CD133 cells and the control children undergo regular follow‐up. The bone marrow‐derived CD133 cells are injected twice: at the diagnosis of quadriplegic cerebral palsy and six months later. The primary outcome is a motor and sensory dysfunction within six months; and unconsciousness and fever within 48 hours post‐transplantation. The secondary outcomes are motor, balance, and spasm improvement within six months.

NCT02274428 is an ongoing phase 1 clinical trial on MCSs (Pneumostem) treatment in preterm infants (23 to 34 weeks' gestational age) diagnosed with IVH grade 3 to 4. Nine newborns with a single assignment group are enrolled in the study. All infants are included within seven days after the diagnosis of IVH grade 3 to 4. The route of administration and the dose of MCSs are not reported. The primary outcome of the study is unexpected death or anaphylactic shock within six hours after MCSs transplantation; the secondary outcome is death or hydrocephalus requiring shunt operation at first discharge up to one year after birth.

NCT03087110 is an ongoing study of a single intravenous infusion of cord blood cells (greater than 1 × 10⁷ cells/kg) of matched sibling donor to a child diagnosed with any type of cerebral palsy. Twelve children aged one to 16 years are enrolled in a single group assignment study. The primary outcome is safety, defined by the number of participants with abnormal clinical assessment or laboratory values (or both) within 12 months. The secondary outcomes are changes in motor and cognitive functions at three months, changes in quality of life at 12 months, and detection of circulating fraction of donor's DNA at three months.

NCT03696745 is an ongoing non‐randomised open‐label clinical trial that recruiting newborns born between 28 and greater than 37 weeks affected by hypoxic‐ischaemic encephalopathy. It is planned to recruit 200 newborns, and they will be assigned either to the intervention group (receiving autologous umbilical cord blood stem cells at 5 × 10⁷ cells/kg; up to four infusions), or to the placebo group (0.9% sodium chloride). The primary outcome is safety, defined as the rate of adverse events during and within 24 months of infusion. The secondary outcome is neurodevelopmental function at four to six months and nine to 12 months of age.

Yang 2018 enrolled preterm newborns (none less than 28 weeks). Autologous volume and red blood cells reduced non‐cryopreserved umbilical cord blood infusion by 5 × 10⁷ cells/kg. The sample included 15 newborns (28+2 to 34+1 weeks). All survived, and there was no significant change in safety measures after infusion.

Ongoing studies

We identified three relevant studies on the clinical trials registries for ongoing or recently completed trials (ACTRN12619001637134; NCT02890953; NCT04440670). On 25 March 2022, we contacted the study authors to request an update on the recruitment status of the infants. See Characteristics of ongoing studies table.

ACTRN12619001637134 is single‐centre safety and feasibility study on extremely preterm infants (i.e. born before 28 weeks' gestation). Autologous via the intravenous route at a dose of 25 to 50 million MNCs/kg bodyweight of live cells are administered. Primary outcomes are feasibility ("access to sufficient cord blood at collection and MNCs following processing") and safety ("adverse events directly related to autologous MNCs administration in the first few days after cell administration"). Secondary outcomes include neurodevelopmental outcome, cord blood cell characteristics, and cytokine responses to cell administration in transplanted infants to 36 weeks' corrected age. Dr Malhotra replied that nearly half of participants are recruited (planned sample size: 20 preterm infants); likely to be completed by the end of 2022/early 2023.

NCT02890953 is a phase 2 RCT that is planning to recruit 22 preterm infants with gestational age 23 to 34 weeks, postnatal age within 28 days, and IVH grade 3 to 4. Exclusion criteria include newborns with severe congenital abnormalities; antenatal brain haemorrhage; asphyxia or hypoxic‐ischaemic encephalopathy; chromosome anomalies; severe congenital malformation; concurrent severe congenital infection; C‐reactive protein greater than 30 mg/dL; severe sepsis or shock; severe IVH; and history of participating in other clinical trials. Infants will be randomised to direct intracerebroventricular injection of either MSCs (Pneumostem) or normal saline. The primary outcome will be death or shunt operation; secondary outcomes will include volume ratio of ventricle to whole brain on brain MRI and death.

NCT04440670 is a multicentre RCT enrolling 200 extremely preterm infants, randomised to either intravenous autologous cord blood MNCs infusion (5 × 10⁷ cells/kg) or placebo (normal saline) within 24 hours after birth. The primary outcome is survival without BPM at 36 weeks of postmenstrual age or discharge home; the secondary outcomes include mortality rate, another common preterm complication rate, respiratory support duration, the length and cost of hospitalisation, and long‐term outcomes after two years' follow‐up postinfusion.

Studies in other neonatal populations

The use of stem cell‐based interventions for hypoxic‐ischaemic encephalopathy and BPD is reported in other Cochrane Reviews (Bruschettini 2020; Pierro 2017).