Hypoxic-ischemic encephalopathy (HIE) is a leading cause of morbidity and mortality in the adult as well as in the neonate, with limited options for treatment and significant dysfunctionality.

To investigate the safety and preliminary efficacy of allogeneic mesenchymal stem cells (MSCs) in HIE patients.

Patients who had HIE for at least 6 mo along with significant dysfunction and disability were included. All patients were given Wharton’s jelly-derived MSCs at 1 × 10⁶/kg intrathecally, intravenously, and intramuscularly twice a month for two months. The therapeutic effects and prognostic implications of MSCs were evaluated by multiple follow-ups. Functional independence measure (FIM), modified Ashworth, and Karnofsky scales were used to assess any side effects, neurological and cognitive functions, and overall outcomes.

The 8 subjects included in the study had a mean age of 33.25 ± 10.18 years. Mean HIE exposure and mean post-HIE durations were 45.63 ± 10.18 and 19.67 ± 29.04 mo, respectively. Mean FIM score was 18.38 ± 1.06, mean modified Ashworth score was 43.5 ± 4.63, and mean Karnofsky score was 20. For the first 24 h, 5 of the patients experienced a subfebrile state, accompanied by mild headaches due to intrathecally administration and muscle pain because of intramuscularly administration. Neurological and functional examinations, laboratory tests, electroencephalography, and magnetic resonance imaging were performed to assess safety of treatment. Mean FIM score increased by 20.88 ± 3.31 in the first month (P = 0.027) and by 31.38 ± 14.69 in 12 mo (P = 0.012). The rate of patients with an FIM score of 126 increased from 14.58% to 16.57% in the first month and 24.90% in 12 mo.

Multiple triple-route Wharton’s jelly-derived MSC administrations were found to be safe for HIE patients, indicating neurological and functional improvement. Based on the findings obtained here, further randomized and placebo research could be performed.

Occurring due to the disruption of oxygen supply to the brain, hypoxic-ischemic encephalopathy (HIE) is associated with substantial rates of morbidity and mortality in both infants and adult patients[1,2]. Inflammation and neurovascular damage constitute potential warning factors for therapeutic intervention[3]. However, there are inadequate standards and specific measures available for the treatment of HIE. Recently, in addition to supportive therapy and symptomatic treatment, research on the treatment of HIE has focused on the following aspects: hypothermia therapy, neuroprotective agents, etc[4]. Another new therapeutic tool with promising indications for clinical practice is stem cell therapy[5]. There are ever-increasing evidences on mesenchymal stem cells (MSCs), suggesting that they promote the improvement of neurological functions, with significant immunomodulatory effects against neuroinflammatory events[6,7]. Bone marrow-derived MSCs (BM-MSCs) and umbilical cord blood-derived MSCs are now being included in efforts to prevent ischemic brain damage[8]. Although they constitute a major source of MSCs, BM-MSCs are seldom preferred due to the high incidence of viral infection and the low number of cells[9]. On the other hand, fetal-derived MSCs, which are more primitive and have less immune reactivity, have recently been suggested as better alternatives for BM-MSCs. Thus, Wharton’s jelly-derived MSCs (WJ-MSCs), can easily be obtained in abundance and are readily cultured, making them good alternatives[10].

The optimal route of MSC administration remains a question of significance. While efficient and useful for avoiding negative outcomes often encountered in invasive procedures, intravenous (IV) application alone may lead to retainment in other organs, including the liver, the spleen, the kidneys, and the lungs[11]. Multiple-route administration could therefore be a better alternative, with research supporting no side effects by IV and intrathecal (IT) administration[10]. We previously reported multiple triple-route WJ-MSC administrations to be safe and applicable for HIE and cerebral palsy patients[12,13]. Based on the further research on the matter, WJ-MSCs can now be used for the clinical treatment of HIE.

This phase I study aimed to investigate the effects and preliminary estimates of multiple triple-route WJ-MSC administrations. The population of the study consisted of HIE patients with significant dysfunction. Primary outcome was considered safety of application by neurological and functional examinations, laboratory tests, electroencephalography, and magnetic resonance imaging.

The potential disabilities to be caused by HIE can be reduced by acute therapies early after HIE and by restorative therapies during the months or years following HIE for promoting natural repair. However, there has been no specific therapy to yield particular recovery. In addition to single therapies, some options of combinations have been considered, often including moderate hypothermia, to potentially obtain better outcomes[17]. Meanwhile, the regenerative and reparative potentials of stem cells have suggested their transplantation as an alternative in the treatment of neurological disorders (e.g., stroke, spinal cord injury, etc.)[18,19]. Among the sources for these cells are neural stem/progenitor cells derived from fetal tissue, induced pluripotent stem cells, embriyonic stem cells, and MSCs[18]. The latter are multipotent progenitor cells that have a self-renewal property and the ability to differentiate into various mesodermal tissues ranging from bone and cartilage to cardiac muscle[20]. Previously, BM was considered a good candidate as a source of MSCs. However, due to its invasive nature and decreased proliferation and differentiation capacity with advanced age, alternative sources were pursued. Fetal-derived MSCs, which are more primitive and have less immune reactivity, have recently been suggested as better alternatives for BM-MSCs. WJ, which is the primitive connective tissue between the umbilical vessels and the amniotic membrane, protects these vessels from pressure and torsion. During embryogenesis, hematopoietic and mesenchymal cells migrate through the WJ, and some of them become trapped, making this tissue a good source of MSCs[21-23].

Despite the promising findings in favor of stem/progenitor cells in HIE, cellular therapy remains in the early stage for human practice[24]. Miao et al[14] found that UC-MSC given IT yielded functional improvements in 47 patients with various diseases (e.g., spinal cord injury, cerebral palsy, etc.). Some recent studies have also suggested WJ-MSC therapy to be promising for patients with neurological diseases, including stroke[12]. Allogeneic WJ-MSCs are shown to demonstrate significant positive impact, even when given via other routes, including intracerebral[9]. The current study found that multiple (4 doses in total for 2 mo at two-week intervals) triple-route (IV, IT, and IM) implantations of allogeneic WJ-MSCs are associated with safety and potential functional recovery.

To the best of our knowledge, this study is the largest to perform multiple triple-route WJ-MSC administration on HIE patients. Also, this is the first time allogeneic WJ-MSC treatment is evaluated in this patient group[12]. When applied at a dose of 1 × 10⁶/kg IV, IT, and IM, WJ-MSC yielded mild and sparse side effects, seen only in 5 of the patients and lasting only for 24 h.

Chronic stage HIE patients often show continually increasing dysfunction, contrary to the improvement noted in the patients of in this study throughout the first year after the operation. Considering the limited number of choices for functional recovery, chronic stage disease is crucial. Normally, recovering from such dysfunctions progresses in a bimodal form, with spontaneous improvements during the first couple of months, followed by a significant rise in negative conditions within the first year after onset.

Among the findings obtained here, the most significant was the 12-point increase in FIM Motor scores, which could prove substantial if confirmed by further research. Also, at baseline, 14.28% of patients had an FIM Motor score of 91, which increased to 14.70% in the first month and 19.37% in the first year (Table 5).

The potential effects of MSCs on neurological diseases have also been marked in animal studies, and is thought to stem from some of their properties, such as restoring cellular energy, promoting neurogenesis, improving angiogenesis, and dampening inflammatory response. The 1-year sustained recovery in functional indicators demonstrated here is in line with other research on HIE[25].

The patients included in this study also had significant improvement in FIM Cognitive scores over the course of a year, indicating long-term impact on brain functions. While suggesting a promising choice for recovery in chronic stage HIE, these findings still need to be confirmed by further research, preferably of a controlled nature. In addition, such studies could measure the outcomes that are specific to certain modalities, ensuring detailed assessment regarding improvement.

This study had certain strengths in terms of its population, materials, and methods. Considering the targeted patient group, the specificity of the sample was a significant strength, in that the patients constitute a population with major dysfunction and limited options for recovery. Using allogeneic cells thanks to the nature of MSCs does not require immunosuppression and enables some treatment protocols, those that could be largely practiced on HIE patients. Using only 3 passages to harvest cell cultures was another strength, since some of the desired properties of MSCs are negatively affected by higher numbers of cell divisions, which is unavoidable when using more passages. Finally, the safety criteria used here were quite extensive in terms of both time and scope.

The study also had certain limitations, among which the most notable were the dominant focus over safety and the lack of control, as the latter complicates the interpretation of improved results. Though favorable, the findings are possibly influenced by other variables, such as growth factors and anti-inflammatory elements, perhaps even exosomes. Restorative therapies are known to aid desirable recovery outcomes, but were unfortunately lacking here.

Recently, cell therapies, particularly WJ-MSCs, have been paving the way for novel treatment protocols for preventing ischemic brain damage. However, clinical use of stem cell therapy remains conflicted and the root of its efficacy is yet to be fully clarified. Several variables still need to be elucidated, such as the mediators between cells, the optimal type and time of therapy, and the ideal patient characteristics. Thus, future multi-center studies with larger populations are needed to verify the safety and efficacy outcomes obtained here, along with an optimization for the treatment protocol in question.