Stem Cell Therapy for Neonatal Hypoxic-Ischemic Encephalopathy: A Systematic Review of Preclinical Studies

Neonatal hypoxic-ischemic encephalopathy (HIE) is an important cause of mortality and morbidity in the perinatal period. This condition results from a period of ischemia and hypoxia to the brain of neonates, leading to several disorders that profoundly affect the daily life of patients and their families. Currently, therapeutic hypothermia (TH) is the standard of care in developing countries; however, TH is not always effective, especially in severe cases of HIE. Addressing this concern, several preclinical studies assessed the potential of stem cell therapy (SCT) for HIE. With this systematic review, we gathered information included in 58 preclinical studies from the last decade, focusing on the ones using stem cells isolated from the umbilical cord blood, umbilical cord tissue, placenta, and bone marrow. Outstandingly, about 80% of these studies reported a significant improvement of cognitive and/or sensorimotor function, as well as decreased brain damage. These results show the potential of SCT for HIE and the possibility of this therapy, in combination with TH, becoming the next therapeutic approach for HIE. Nonetheless, few preclinical studies assessed the combination of TH and SCT for HIE, and the existent studies show some contradictory results, revealing the need to further explore this line of research.


Introduction
Hypoxic-ischemic encephalopathy (HIE) is one of the major causes of neonatal death and long-term disability, leading to chronic motor and cognitive impairments [1]. Several disorders are associated with HIE, namely epilepsy, cerebral palsy [2], attention deficit hyperactivity disorder, seizures, hearing and vision loss, language disorders, and cognitive delay. The different outcomes of this condition can be severe, profoundly affecting the daily life of patients and their families. This condition also represents a major economic burden to the government and caretakers [3].
About a quarter of neonatal deaths worldwide can be attributed to perinatal asphyxia [4]. The estimated incidence of HIE is variable across studies, ranging from 1 to 8 cases per 1000 live births [5]. In developed countries, neonatal HIE incidence is approximately 0.5 to 1 case per 1000 live births [6]; however, the global estimate is highly influenced by the higher incidence found in developing countries [7]. Infants diagnosed Table 1. Studies focusing on the therapeutic potential of umbilical cord blood cells and umbilical cord blood and umbilical cord tissue stem/stromal cells from human origin (except when mentioned otherwise) in animal models for hypoxic-ischemic encephalopathy.

↑ -Yes
Both administration routes led to similar outcomes; decrease in microglial activation, astrogliosis, and invading macrophages.   increase in the number of mature neurons; delayed glial scar formation (12 wpi); increase in cerebral capillary density and cerebral blood flow.       T-cells with pro-inflammatory phenotype, and microglial activation. [43] Abbreviations: ↑ increase or upregulation; ↓ decrease or downregulation; = no significant difference; -not evaluated; dpi-days post insult; EPCs/ECFCs-endothelial progenitor cells/endothelial colony-forming cells; ICV-intraventricular; IP-intraperitoneal; HI-hypoxic-ischemic; MSCs-mesenchymal stem/stromal cells; PD-placenta-derived; ROS-reactive oxygen species; RV-Rice-Vannucci/Rice-Vannucci adaptation; Treg-regulatory T-cells. Figure 1. Mechanisms of action of stem cells identified by the preclinical studies included in this systematic review. Several mechanisms of action that might be mediating the positive functional outcomes observed after SCT in preclinical models of neonatal hypoxic-ischemic encephalopathy (HIE). Stem cell therapy (SCT) was associated with the promotion or upregulation (green arrows) of neuronal stem cells (NSCs) proliferation and differentiation, neurogenesis, cell proliferation, growth factors levels/secretion, angiogenesis, and inhibition or downregulation (orange truncated arrows) of pro-inflammatory cytokines, apoptosis, astrogliosis, microglial activation, and oxidative stress. Also, some studies report stem cell engrafment after SCT, while other report low or no engrafment. Abbreviations: Anti-inflam-anti-inflammatory; BDNF-brain-derived neurotrophic factor; HGF-hepatocyte growth factor; VEGF-vascular endothelial growth factor. Figure 1. Mechanisms of action of stem cells identified by the preclinical studies included in this systematic review. Several mechanisms of action that might be mediating the positive functional outcomes observed after SCT in preclinical models of neonatal hypoxic-ischemic encephalopathy (HIE). Stem cell therapy (SCT) was associated with the promotion or upregulation (green arrows) of neuronal stem cells (NSCs) proliferation and differentiation, neurogenesis, cell proliferation, growth factors levels/secretion, angiogenesis, and inhibition or downregulation (orange truncated arrows) of pro-inflammatory cytokines, apoptosis, astrogliosis, microglial activation, and oxidative stress. Also, some studies report stem cell engrafment after SCT, while other report low or no engrafment. Abbreviations: Anti-inflam-anti-inflammatory; BDNF-brain-derived neurotrophic factor; HGF-hepatocyte growth factor; VEGF-vascular endothelial growth factor.

Umbilical Cord Blood Cells
The mononuclear fraction of the human UCB is a known source of different populations of stem and progenitor cells-hematopoietic stem cells, endothelial progenitor cells, and mesenchymal stem/stromal cells [23]. These cell populations can be identified through their surface marker profile and in vitro growth characteristics: HSCs are non-adherent, positive for CD34 and CD133; EPCs are adherent, positive for CD34, CD133, and CD90, and negative for CD13 and CD44; MSCs are adherent, positive for CD44, CD90, and CD13, and negative for CD34, CD45, and CD133 [23,88] Several preclinical studies used the RV model for HIE [13] to assess treatment efficacy with UCB cells ( Table 1). The experimental protocols applied in these studies were very heterogeneous, with the hypoxic period varying between 0.5 h to 3 h, the administration of UCB cells being conducted from 3 h post insult to 3 weeks post insult, and cell dosage per injection varying from 1.5 × 10 5 cells to 10 8 cells per injection. Different routes of UCB cell administration were also used (intravenous, intraventricular, intra-arterial, intranasal, and intraperitoneal). Nonetheless, most studies report a positive effect of UCB cell treatment in this model for HIE, namely a long-term recovery of the animals' cognitive and motor functions [36][37][38][39][40][41][42][43][44][45][46][47]. Interestingly, two studies reported no improvement in the functional outcome after UCB cell administration [77,86]. However, the lack of a significant effect may be explained by the mild lesion induced, since the animals in this study were subjected to short hypoxic periods (0.5 h or 1 h).
The majority of the studies report a decrease in brain damage after administration of UCB cells, translated in decreased apoptosis [36,37,42,45,72,77,78] and neuronal loss [36,37,39,43,46,81,85,87]. Thus, the positive effects of UCB cells in the animals' functional outcome can be linked to decreased brain damage. Nonetheless, some studies report an improvement of the functional outcome after UCB cell administration without observing a significant decrease in brain damage. Thus, other mechanisms are certainly influencing the long-term recovery observed after UCB cell treatment. Treatment with UCB cells decreased microglial activation [36,37,40,43,44,46,[77][78][79] and astrogliosis [40,42,45,78], levels of proinflammatory cytokines [79] and oxidative stress [77], and increased the expression of growth factors in the central nervous system (CNC) [72], angiogenesis [50,72], neuronal stem cell (NSC) proliferation [85], and the number of mature neurons [42,72]. UCB cell treatment also seems to promote NSC differentiation into mature neuronal cells [81]. A study using an ovine model for HIE at term showed that autologous transplantation of UCB cells might contribute to the restoration of brain metabolic activity by reducing brain lactate levels [78]. Increased lactate levels were previously observed after the HI insult in the same model, correlating with neuronal injury [89].
Recently, Penny et al. (2020) showed that multiple doses of UCB cells administered at different timepoints, targeting the primary, secondary, and tertiary phases of the HI injury, were more effective in improving sensorimotor function and other parameters than a single dose at an earlier timepoint [36].

Mesenchymal Stem/Stromal Cells
Mesenchymal stem/stromal cells are multipotent stem cells present in distinct adult tissues, such as the bone marrow, and neonatal tissues, such as the placenta, umbilical cord tissue, and umbilical cord blood. All MSCs present potential for osteogenic, adipogenic, and chondrogenic differentiation [90], although cells from specific origins may have a stronger bias toward a specific lineage. Interestingly, MSCs isolated from neonatal tissues have improved proliferation, expansion, and transient engraftment capacity [91][92][93][94][95] and are less likely to induce an immune reaction [28]. Several criteria define MSCs, such as plastic adherence when maintained in culture; expression of CD105, CD73, and CD90; lack of expression of CD45, CD34, CD14 or CD11b, CD79a, or CD19, and HLA-DR surface molecules; and differentiation into osteoblasts, adipocytes, and chondroblasts in vitro [96].
Specific in vitro conditions can induce MSCs to differentiate into neuronal-like cells [97]. Some studies showed the differentiation of human MSCs into mature brain cells after a HI insult in the neonatal brain [33,51,55]. However, independent studies reported positive effects after MSC administration in HIE preclinical models, even in the absence of MSC differentiation into mature cell types [32,50,57,58]. An interesting research line would be to investigate the effect of MSC-derived secretome administration in HIE preclinical models. MSC-derived secretome showed to exert neuroprotective effects and/or stimulate paracrine endogenous repair mechanisms [98,99], and the intravenous administration of MSC-derived secretome improved motor recovery in a murine model of spinal cord injury and traumatic brain injury [100].
On the other hand, there is increasing evidence that in vitro MSC-preconditioning before transplantation/infusion enhances the efficacy of MSCs transplantation. There are several types of preconditioning being studied in the context of MSCs transplantation, such as hypoxia, the use of pharmacological agents, mechanotransduction stimuli, genetic engineering, among others [101,102]. For instance, mild-hypoxic preconditioning was shown to enhance the positive effects of MSC transplantation in an in vivo model of ischemic stroke by promoting neuronal repair, improving MSCs homing to the damaged tissues, and decreasing apoptosis [103]. Nonetheless, to the best of our knowledge, no study assessed the potential of preconditioned MSCs on in vivo models of HIE.
Another advantage of using MSCs for SCT is the in vitro expansion of the cells, which allows obtaining a higher number of cells for infusion.

Umbilical Cord Tissue-Derived MSCs
Initially, the scientific community focused on the UCB as a valuable source of stem cells, and the umbilical cord tissue was considered medical waste. However, later in time, researchers realized that it was possible to isolate stem cells, namely MSCs, from various parts of the umbilical cord (e.g., Wharton's jelly, cord lining, and the perivascular region) [104].

Umbilical Cord Blood-Derived MSCs
MSCs are a poorly abundant stem cell population in the UCB; however, it is possible to successfully isolate MSCs from the UCB if the UCB is processed immediately after collection [113]. The UCB-derived MSCs (UCB-MSCs) are also multipotent and can differentiate into mesodermal, endodermal, and, importantly, in the context of HIE, ectodermal lineages [114,115]. Nonetheless, the ability to isolate MSCs from the UCB is not yet well established. Some authors showed the possibility of isolating MSCs from the UCB [116,117], while others disagree [118].

Placenta-Derived MSCs
The placental tissue represents an excellent source of progenitor/stem cells possessing abundant MSCs (PD-MSCs) readily available after birth, which are easily obtained [119,120]. In this review, two studies assessed the effects of PD-MSC administration in the murine model of HIE (Table 2) [52,53]. In one of them [53], PD-MSCs improved the animals' functional outcome and physical appearance while decreasing the extent of brain damage and neuronal morphological changes induced by the HI insult. This treatment also reduced lipid peroxidation and free radical levels, which are hallmarks of HIE. Another study showed that intraventricular administration of PD-MSCs induced an immunomodulatory effect in the RV model for HIE by increasing the generation of regulatory T-cells, thus increasing anti-inflammatory cytokines and reducing pro-inflammatory cytokines in the brain and peripheral blood serum of the RV animal model. These observations were associated with decreased brain damage and improved functional outcomes [52].

Bone Marrow-Derived MSCs
Several studies assessed the effects of bone marrow-derived mesenchymal stem/stro mal cell administration in the murine model of HIE (Table 3).

Endothelial Progenitor Cells
Endothelial progenitor cells can also be isolated from the UCB. This cell type promotes vascular repair and tissue recovery after ischemia through the formation of new blood vessels. EPCs include a subtype of cells, the endothelial colony-forming cells (ECFCs), which have a high proliferating capacity and a specific vasculogenic activity [121].
Regarding HIE, we found three different studies that assessed the effects of EPC/ECFC administration after a neonatal HI insult ( Table 2). These studies reported that the administration of EPCs/ECFCs improved the animal's functional outcome [71]; decreased brain damage [71], which can be linked to the decrease in apoptosis observed in the cortex ipsilateral to the lesion [42,43]; and increased the number of mature neuronal cells [42], cerebral capillary density, and cerebral blood flow [42]. Treatment with this cell population also hampered the inflammatory responses that occur after the neonatal HI insult [42,43].

Therapeutic Hypothermia and Stem Cell Therapy
Although TH is the current standard of care for HIE in term neonates in developing countries, it is not entirely effective in preventing mortality or neurodevelopmental disabilities in HIE patients, especially those diagnosed with severe HIE [6]. Therefore, it is crucial to find safe and effective therapies that will enhance TH's neuroprotective effects and improve the outcome of these patients.
To our knowledge, few preclinical studies assessed the potential of combining TH with SCT [48,49,68] to treat severe HIE. These studies present some contradictory results. Two studies revealed that hypothermia alone did not improve the animals' functional outcome following severe HIE [48,49]; however, hypothermia and MSCs infusion 2 days after insult had a positive effect, improving the animal's functional outcome while decreasing brain damage, cytokine levels, microgliosis, and astrogliosis [48,49]. Interestingly, both studies reported that combined therapy was more effective than MSC administration alone [48,49].
In contrast, a study performed by Herz et al. (2018) showed that animals treated either with TH or MSCs had a better outcome than animals treated with the combined therapy of TH followed by MSC administration 3 dpi [68]. This study revealed that, after HI insult in the neonatal period, only the MSC treatment improved cognitive function and decreased white matter injury, and MSC or TH treatment improved motor function. However, the combined therapy, TH followed later by MSC administration, reversed the protective effects observed with each therapy alone, resulting in increased, long-lasting functional deficits, brain damage, endothelial cells infiltration, peripheral immune cell infiltration, and pro-inflammatory cytokine levels, as well as decreased levels of growth factor expression. One potential mechanism pointed out by the authors is an alteration of the cerebral microenvironment after TH, resulting in a modification of the MSCs phenotype after their administration. This alteration may induce pro-inflammatory cytokine expression and block the expression of growth factors, thus interfering with the rescuing of the injured brain.

Mechanisms of Action of SCT after HI Insult in the Developing Brain
This section summarizes and further discusses the previously identified mechanisms of action of SCT in the preclinical studies included in this systematic review (Figure 1). Several mechanisms of action might be mediating the positive effects observed after SCT in animals subjected to a HI insult in the developing brain. These positive effects are most likely not due to a particular mechanism but due to a synergistic or cumulative effect. Stem cells might exert their action by protecting the brain from injury and enhancing endogenous repair mechanisms.

Secreted Factors and Paracrine Effects
UCB cells and MSCs secrete a wide range of factors that contribute to damaged tissue regeneration, such as angiogenic factors, chemokines, and neurotrophic factors [123][124][125]. Moreover, several studies reported increased levels of these factors after SCT in HIE preclinical models [34,53,[72][73][74]. Thus, it is likely that the beneficial effect of SCT might also be related to the presence of these paracrine factors, or secretome, contributing to neuronal repair, increasing angiogenesis, hampering the inflammatory response, and promoting an anti-apoptotic effect, among others.

Inflammation
SCT was associated with a significant decrease in pro-inflammatory cytokines [42,52,73,79,82]. This effect was further enhanced by combining SCT with TH [48]. Thus, it appears that some positive effects of SCT in HIE preclinical models might be due to the downregulation of the pro-inflammatory cytokines that were augmented after the HI insult in the developing brain.

Angiogenesis
Increased angiogenesis and increased levels of pro-angiogenic factors, such as VEGF and interleukin 8, were reported by two studies after SCT [30,72].

Oxidative Stress
An important hallmark of HIE is oxidative stress, and stem cell administration in a preclinical model was reported to decrease the number of cells positive for oxidative stress markers [77]. Nonetheless, this observation was not accompanied by long-term functional and morphological improvements.

Analysis and Discussion
From the 58 studies included in this systematic review (Supplementary Table S1), we identified 83 different protocols for stem cell therapy in animal models for HIE (Supplementary Table S2). The most common animal model employed was the Rice-Vannucci model, with 88% of the studies choosing this model to induce HI insult in the developing brain (n = 51; Figure 2A). Rodents were predominantly used in the included studies ( Figure 2B), the rat being the most used species (n = 42), followed by mouse (n = 13). As expected, most of the studies (90%) assessed the effect of SCT on histological parameters (n = 52; Figure 3). However, fewer studies assessed the animal's functional outcome following SCT: 64% evaluated the animal's sensorimotor function (n = 37), and only 29% evaluated the animal's cognitive function (n = 17) (Figure 3). The evaluation of the functional outcome after SCT is of great importance for the clinical setting since one of the primary goals is to tackle the cognitive and motor impairments caused by HIE. Additionally, different behavioral tests were used at different time points: 17 different tests were used to evaluate the sensorimotor function, the cylinder rearing test (n = 19) and the rotarod (n = 11) being the most used ones; and three different tests were used to evaluate cognitive function: the Morris water maze (n = 10), the novel object recognition test (n = 5), and the object in place task (n = 2) (Supplementary Table S3). These disparities make a comparison between studies a challenge. Considering only the studies that evaluated sensorimotor function, cognitive function, and/or histological parameters, about 80% of the protocols included in these studies prompted a significant improvement in at least one of these parameters. However, due to the lack of reporting the complete data values for each experimental group (e.g., mean and standard error of the mean), we could not identify which protocol(s) represented the best strategy for neurological and neurobehavioral recovery after a HI insult in the neonatal period. Regarding the sex of the animals ( Figure 2C), 43% of the studies did not report this information (n = 25), 36% used both females and males (n = 21), and 21% used only males (n = 12). None of the included studies investigated the effect of SCT in females alone, nor did a comparative analysis.
As expected, most of the studies (90%) assessed the effect of SCT on histological parameters (n = 52; Figure 3). However, fewer studies assessed the animal's functional outcome following SCT: 64% evaluated the animal's sensorimotor function (n = 37), and only 29% evaluated the animal's cognitive function (n = 17) (Figure 3). The evaluation of the functional outcome after SCT is of great importance for the clinical setting since one of the primary goals is to tackle the cognitive and motor impairments caused by HIE. Additionally, different behavioral tests were used at different time points: 17 different tests were used to evaluate the sensorimotor function, the cylinder rearing test (n = 19) and the rotarod (n = 11) being the most used ones; and three different tests were used to evaluate cognitive function: the Morris water maze (n = 10), the novel object recognition test (n = 5), and the object in place task (n = 2) (Supplementary Table S3). These disparities make a comparison between studies a challenge. Considering only the studies that evaluated sensorimotor function, cognitive function, and/or histological parameters, about 80% of the protocols included in these studies prompted a significant improvement in at least one of these parameters. However, due to the lack of reporting the complete data values for each experimental group (e.g., mean and standard error of the mean), we could not identify which protocol(s) represented the best strategy for neurological and neurobehavioral recovery after a HI insult in the neonatal period.  Focusing on the studies using the RV model in rats (67%, n = 39), the duration of the hypoxic insult-that is, the exposure to 8% O2-ranged between 1 and 4 h (Figure 4), with 50% of the studies applying a hypoxic insult with a duration between 1.5 and 2.5 h (n = 29). Considering these studies, which most likely had a similar lesion extent, we selected the ones conducted in postnatal day-7 rats since they represent 45% of the studies (n = 26) (Supplementary Table S4).  From the 26 studies that established the HIE model using the RV protocol at P7, 19 used cells isolated from neonatal tissues. These 19 studies evaluated 25 protocols (Supplementary Table S5), which included the usage of distinct cell types isolated from Focusing on the studies using the RV model in rats (67%, n = 39), the duration of the hypoxic insult-that is, the exposure to 8% O 2 -ranged between 1 and 4 h (Figure 4), with 50% of the studies applying a hypoxic insult with a duration between 1.5 and 2.5 h (n = 29). Considering these studies, which most likely had a similar lesion extent, we selected the ones conducted in postnatal day-7 rats since they represent 45% of the studies (n = 26) (Supplementary Table S4).  Focusing on the studies using the RV model in rats (67%, n = 39), the duration of the hypoxic insult-that is, the exposure to 8% O2-ranged between 1 and 4 h (Figure 4), with 50% of the studies applying a hypoxic insult with a duration between 1.5 and 2.5 h (n = 29). Considering these studies, which most likely had a similar lesion extent, we selected the ones conducted in postnatal day-7 rats since they represent 45% of the studies (n = 26) (Supplementary Table S4).  From the 26 studies that established the HIE model using the RV protocol at P7, 19 used cells isolated from neonatal tissues. These 19 studies evaluated 25 protocols (Supplementary Table S5), which included the usage of distinct cell types isolated from From the 26 studies that established the HIE model using the RV protocol at P7, 19 used cells isolated from neonatal tissues. These 19 studies evaluated 25 protocols (Supplementary Table S5), which included the usage of distinct cell types isolated from the umbilical cord (blood or tissue) or the placenta: UCB cells (14 protocols), UCB-MSCs (3 protocols), UCT-MSCs (6 protocols), and PD-MSCs (2 protocols) ( Figure 5). practice due to its invasiveness and the risks associated with this procedure. The efficacy of intravenous administration of MSCs was evaluated in a study that compared the administration at two time points and concluded that a single intravenous administration of 5 × 10 5 cells one day post insult was significantly more effective in improving the animal's motor and cognitive function than the same dose administered three days post insult [33]. Nine included studies evaluated the efficacy of intranasal administration of UCB cells [36], UCT-MSCs [29], and BM-MSCs [58,[60][61][62][63]68,80]. All these studies reported histological and functional rescue of animals subjected to a HI insult in the developing brain using this route of administration. Importantly, Donega et al. (2015) showed that intranasal administration of MSCs was long-term effective and safe in mice subjected to neonatal HI [63]. Moreover, intranasal administration appears to have efficacy in low doses of MSCs. A study showed positive histological and functional outcomes even after one administration of 2 × 10 5 UCT-MSCs [29]. The therapeutic window for intranasal administration of MSCs appears to be up to 10 days, even with lower doses [58,63]. On the other hand, Penny et al. (2020) found no significant difference between the effectiveness of intraperitoneal or intranasal administration of UCB cells in the RV model for neonatal HIE [36].
Overall, SCT appears to improve the HIE animal model's sensorimotor and cognitive function and decreases brain damage. Nonetheless, there is a high variability since it used 7 types and sources of stem cells, 18 dosages, 14 time points, and 8 administration routes, suggesting the need for further studies to elucidate the ideal procedure. Regarding UCB cell transplantation, the most used routes of administration were intraventricular injection (4 protocols), intravenous (4 protocols), and intraperitoneal administration (4 protocols) (Supplementary Table S6). The minimal dose of UCB cells eliciting a significant motor function improvement and a decrease in brain damage was 10 6 cells, administered intravenously seven days post insult [45]. In contrast, a different study, which compared the effect of different doses of UCB cells administered intravenously (10 6 , 10 7 , and 10 8 cells) found no improvement of the animal's cognitive function and brain damage with one administration of 10 6 cells [39]. Nonetheless, in this study, UCB cells were administered at a different time point, one day post insult, and the animal's motor function was not evaluated [39].
For MSCs derived from neonatal tissues, the predominant route of administration was intraventricular injection (8 protocols), followed by intravenous (2 protocols) and intraperitoneal administration (1 protocol) (Supplementary Table S7). Although the ICV was the predominant administration route, it has little translatability to the clinical practice due to its invasiveness and the risks associated with this procedure. The efficacy of intravenous administration of MSCs was evaluated in a study that compared the administration at two time points and concluded that a single intravenous administration of 5 × 10 5 cells one day post insult was significantly more effective in improving the animal's motor and cognitive function than the same dose administered three days post insult [33].
Nine included studies evaluated the efficacy of intranasal administration of UCB cells [36], UCT-MSCs [29], and BM-MSCs [58,[60][61][62][63]68,80]. All these studies reported histological and functional rescue of animals subjected to a HI insult in the developing brain using this route of administration. Importantly, Donega et al. (2015) showed that intranasal administration of MSCs was long-term effective and safe in mice subjected to neonatal HI [63]. Moreover, intranasal administration appears to have efficacy in low doses of MSCs. A study showed positive histological and functional outcomes even after one administration of 2 × 10 5 UCT-MSCs [29]. The therapeutic window for intranasal administration of MSCs appears to be up to 10 days, even with lower doses [58,63]. On the other hand, Penny et al. (2020) found no significant difference between the effectiveness of intraperitoneal or intranasal administration of UCB cells in the RV model for neonatal HIE [36].
Overall, SCT appears to improve the HIE animal model's sensorimotor and cognitive function and decreases brain damage. Nonetheless, there is a high variability since it used 7 types and sources of stem cells, 18 dosages, 14 time points, and 8 administration routes, suggesting the need for further studies to elucidate the ideal procedure.

Conclusions and Future Perspectives
We systematically reviewed studies reported in the last 10 years assessing the potential of using cells isolated from the UCB, UCT, placenta, and BM in preclinical animal models for HIE. The positive effects reported included improved functional outcome, both cognitive and motor function, decreased brain damage, translated by a decrease in apoptotic cells and prevention of neuronal loss, microglial activation, astrogliosis, inflammation, and increased angiogenesis and cell proliferation, among others. Thus, stem cell therapy appears to have great therapeutic potential and could become a new therapy for HIE. Nonetheless, there is a high variability regarding the dose of stem cells applied, route, and administration timing. Therefore, it would be critical to perform studies assessing different amounts of stem cells, considering the clinical setting, and determining the optimal time for stem cell administration (e.g., if during the secondary or tertiary phase of the injury) to increase the chance of successful translating stem cell therapy into the clinical practice.
A new possible therapeutic combination would be adding SCT to the current standard of care for HIE, TH, thus improving the effectiveness of TH in treating infants diagnosed with HIE, especially those diagnosed with severe HIE. However, the lack of studies addressing the effect of combining TH with SCT in HIE and the existing heterogeneity in the few studies that were performed until today stresses the importance of exploring this research line.
In conclusion, there is increasing evidence in the literature that SCT could, in combination with TH, be the next standard of care for HIE patients, addressing the lack of effectiveness of therapeutic hypothermia. Infusion of human umbilical cord blood cells was already demonstrated to be safe and feasible in newborns diagnosed with HIE. However, it is still necessary to optimize the protocol for SCT, namely determining the optimal dose, route of administration, and timing, as well as assessing which stem cell types provide the maximal neuroprotection. This is where translational research and animal models become extremely useful, allowing them to explore multiple therapeutic interventions and unravel which ones have the potential to be applied in the clinic.

Literature Search
The literature search was performed using MEDLINE's database, PubMed, and Web of Science on 27 October 2020. Search terms included "hypoxic-ischemic encephalopathy", "stem cells", "umbilical cord cells", "umbilical cord blood cells", and "mesenchymal stem cells", and other synonyms of these words ( Figure 5). Studies in duplicate were manually removed from the search results, as well as studies in languages other than English. Studies published before 2010 were excluded, and only full-text articles were included. Relevant review articles were also manually searched to maximize the inclusion of relevant studies. Two authors screened the abstracts and the full text of the studies independently. Disagreements were resolved by discussion.

Inclusion and Exclusion Criteria
The literature search and the screening processes used are summarized in Figure 6. Only preclinical studies were included in this systematic review, excluding reviews, chapters, clinical studies or case reports, and in vitro studies. Studies were included if in vivo preclinical models of HIE were used, namely occlusion of the carotid artery, middle cerebral artery, uterine artery, or umbilical cord. Studies reporting the effect of stem cells isolated from the umbilical cord, umbilical cord blood, placenta, and bone marrow were included. Other stem cells (e.g., neuronal stem cells, multipotent adult progenitor cells) or stem cells isolated from other adult tissues were excluded. Studies that did not evaluate stem cell efficacy or use modified stem cells, except for tracing and locating the distribution of the cells, were excluded. Studies that assessed the effect of other therapies in combination with stem cell administration were excluded, except if the applied therapy was therapeutic hypothermia since it is the current standard of care for HIE. in combination with stem cell administration were excluded, except if the applied therapy was therapeutic hypothermia since it is the current standard of care for HIE.

Data Extraction
From the included studies, the following data were collected and analyzed: general study design, animal characteristics (animal model, species, sex, age, and weight), the protocol for SCT (source of stem cells, stem cell processing before administration, dose, administration route, timepoint of administration, the number of administrations, and amount of cells/administration), histological techniques and neurobehavioral tests used, and respective outcomes. Supplementary Table S1 summarizes the data extraction for all studies included in this systematic review, and Supplementary Table S2 lists all the protocols for stem cell therapy included in the 58 studies.
Supplementary Materials: The following are available online at www.mdpi.com/xxx/s1. Table S1: Studies included in the systematic review and the extracted data from each study: general study design, animal characteristics (animal model, species, sex, age, weight), the protocol for stem cell therapy (source of stem cells, stem cell processing before administration, dose, administration route, timepoint of administration, number of administrations and amount of cells/administration), histological techniques and neurobehavioral tests used, and respective outcomes; Table S2: Stem cell therapy protocols for animal models of hypoxic-ischemic encephalopathy, evaluated by the 58 studies included in the systematic review; Table S3: Motor and cognitive function tests used in the studies included in this systematic review; Table S4: Distribution of the studies using the Rice-Vannucci protocol to induce hypoxic-ischemic lesion and 1.5-2.5 h of hypoxia regarding the animals' age in days; Table S5: Stem cell types used in protocols that used the Rice-Vannucci animal model with 1.5 to 2.5 h of hypoxia in postnatal day seven rats; Table S6: Different routes of administration for stem cell therapy with umbilical cord blood cells in protocols that used the Rice-Vannucci animal model with 1.5 to 2.5 h of hypoxia in postnatal day seven rats; Table S7: Different routes of administration for stem cell therapy with mesenchymal stem/stromal cells isolated from the umbilical cord blood & tissue or the placenta, in protocols that used the Rice-Vannucci animal model with 1.5 to 2.5 h of hypoxia in postnatal day seven rats.

Data Extraction
From the included studies, the following data were collected and analyzed: general study design, animal characteristics (animal model, species, sex, age, and weight), the protocol for SCT (source of stem cells, stem cell processing before administration, dose, administration route, timepoint of administration, the number of administrations, and amount of cells/administration), histological techniques and neurobehavioral tests used, and respective outcomes. Supplementary Table S1 summarizes the data extraction for all studies included in this systematic review, and Supplementary Table S2 lists all the protocols for stem cell therapy included in the 58 studies.
Supplementary Materials: The following are available online at https://www.mdpi.com/1422-006 7/22/6/3142/s1. Table S1: Studies included in the systematic review and the extracted data from each study: general study design, animal characteristics (animal model, species, sex, age, weight), the protocol for stem cell therapy (source of stem cells, stem cell processing before administration, dose, administration route, timepoint of administration, number of administrations and amount of cells/administration), histological techniques and neurobehavioral tests used, and respective outcomes; Table S2: Stem cell therapy protocols for animal models of hypoxic-ischemic encephalopathy, evaluated by the 58 studies included in the systematic review; Table S3: Motor and cognitive function tests used in the studies included in this systematic review; Table S4: Distribution of the studies using the Rice-Vannucci protocol to induce hypoxic-ischemic lesion and 1.5-2.5 h of hypoxia regarding the animals' age in days; Table S5: Stem cell types used in protocols that used the Rice-Vannucci animal model with 1.5 to 2.5 h of hypoxia in postnatal day seven rats; Table S6: Different routes of administration for stem cell therapy with umbilical cord blood cells in protocols that used the Rice-Vannucci animal model with 1.5 to 2.5 h of hypoxia in postnatal day seven rats; Table S7: Different routes of administration for stem cell therapy with mesenchymal stem/stromal cells isolated from the umbilical cord blood & tissue or the placenta, in protocols that used the Rice-Vannucci animal model with 1.5 to 2.5 h of hypoxia in postnatal day seven rats.

Data Availability Statement:
No new data were created or analyzed in this study. Data sharing is not applicable to this article.

Conflicts of Interest:
The authors declare no conflict of interest.