Nose-to-brain delivery of stem cells in stroke: the role of extracellular vesicles

Abstract

Stem cell transplantation offers a promising therapy that can be administered days, weeks, or months after a stroke. We recognize 2 major mitigating factors that remain unresolved in cell therapy for stroke, notably: (1) well-defined donor stem cells and (2) mechanism of action. To this end, we advance the use of ProtheraCytes, a population of non-adherent CD34+ cells derived from human peripheral blood and umbilical cord blood, which have been processed under good manufacturing practice, with testing completed in a phase 2 clinical trial in post-acute myocardial infarction (NCT02669810). We also reveal a novel mechanism whereby ProtheraCytes secrete growth factors and extracellular vesicles (EVs) that are associated with angiogenesis and vasculogenesis. Our recent data revealed that intranasal transplantation of ProtheraCytes at 3 days after experimentally induced stroke in adult rats reduced stroke-induced behavioral deficits and histological damage up to 28 days post-stroke. Moreover, we detected upregulation of human CD63+ EVs in the ischemic brains of stroke animals that were transplanted with ProtheraCytes, which correlated with increased levels of DCX-labeled neurogenesis and VEGFR1-associated angiogenesis and vasculogenesis, as well as reduced Iba1-marked inflammation. Altogether, these findings overcome key laboratory-to-clinic translational hurdles, namely the identification of well-characterized, clinical grade ProtheraCytes and the elucidation of a potential CD63+ EV-mediated regenerative mechanism of action. We envision that additional translational studies will guide the development of clinical trials for intranasal ProtheraCytes allografts in stroke patients, with CD63 serving as a critical biomarker.

Graphical Abstract

Graphical Abstract.

Stem cells and extracellular vesicles (EVs). The release of CD-63-labeled EVs utilizes the pathway used by high density lipid biogenesis, that is, ApoA-1 cholesterol efflux. CD-63-labeled EVs are lipoprotein structures released from cells that may be enhanced by ApoA-1 involving gene fusion between the transmembrane protein CD-63 and a sequence from ApoA-1. Accordingly, ApoA-1 appears to serve as a receptor to CD63.

Significance statement.

Stem cell therapy has emerged as an experimental treatment for stroke. Postulated mechanisms of action of grafted stem cells include cell replacement and bystander effects. Here, we advance the concept that stem cells secrete extracellular vesicles, which participate in stem cell migration from the periphery to the ischemic brain and improve vasculogenesis, altogether contributing to brain remodeling after stroke. Intranasal delivery of stem cells represents a minimally invasive stroke therapy that may benefit from probing the robust migratory and functional effects of extracellular vesicles.

Current status of stem cell therapy for stroke

Stroke remains as one of the most detrimental diseases that cause significant neurological impairments.^1-3 As the fifth leading cause of death in the United States, stroke has limited treatment options, and the projected growth of vulnerable aging populations makes the search for effective stroke therapy even more urgent. Currently, endovascular thrombectomy and tissue plasminogen activator (tPA) are the only approved acute stroke treatment options. However, the short therapeutic time window and potential inducement of adverse effects limit the use of available acute stroke treatments.^1,4-11 While rehabilitation offers some therapeutic effects, many chronic ischemic stroke victims are unable to fully recover cognitive and motor functions due to loss of brain cells caused by primary and secondary cell death mechanisms.^12,13 A potent treatment that can regenerate lost brain cells is warranted.

Stem cell therapy has emerged as a potential candidate to treat stroke by rescuing ischemic brain cells^14-19 via cell replacement and neurotrophic factor secretion.^20-29 In middle cerebral artery occlusion (MCAO) models in rats, different types of cells, such as mesenchymal stem cells (MSC), exert therapeutic effects by increasing neurotrophic growth factors, and decreasing apoptosis and neurological damage in penumbral lesion areas.^16,30-35 MSCs also increase vascular endothelial growth factor (VEGF) activity and enhance angiogenesis.³² Similarly, MSCs have been advantageous for their accessibility, but their performance is highly dependent on their method of transplantation.^30,36

Similar to acute myocardial infarction, acute cerebral ischemic attacks are followed by large and bursting mobilizations of peripheral blood-derived CD34 + cells at 1–3 days and 7–10 days after the event.³⁷ The extent of the CD34 + cell mobilization significantly correlates with neurological and functional recoveries observed at 1 and 3 months in the NIH Stroke Scale (NIHSS) and modified Rankin Scale (mRS), respectively, therefore being predictive of neurological and functional recovery.³⁷ Indeed, a clinical study that mobilized CD34+ cells via daily subcutaneous injections of granulocyte colony stimulating factor (G-CSF) for 5 consecutive days after ischemic stroke showed functional and structural improvement in some patients.³⁸ When 1–3 × 10⁶ CD34+ cells were injected intraarterially by catheter angiography into the ipsilesional middle cerebral artery within 7 days of stroke onset, all patients showed improvements in mRS and NIHSS score at 6 months.³⁹ Also, when 3–8 × 10⁶ CD34 + cells were injected intracerebrally at ≥6 months after stroke onset in patients with a middle cerebral artery infarct, treated patients experienced significantly greater improvement in NIHSS, mRS, and European Stroke Scale at 12 months post-treatment compared to control patients.⁴⁰ CD34+ cells have been shown to promote angiogenesis via the secretion of paracrine factors such as small EVs containing pro-angiogenic miRNAs.⁴¹

While the success of stem cell therapy relies on the type of stem cell used, the method of transplantation is equally, if not more, important because the route may trigger a myriad of regenerative, as well as degenerative processes. Intracerebral transplantation is known for its high number of stem cells in the lesion area, significant neurological recovery, and lower peripheral side effects.^42,43 However, there are 2 main downsides of intracerebral delivery: (1) is its limited clinical applicability for stroke patients who cannot tolerate direct injection via neurosurgical operations and its potential risks,⁴⁴ and (2) limited area of distribution. Intra-arterial injection offers a less invasive method of delivery for more vulnerable stroke patients. Due to stem cells’ homing capability, indirect injection of stem cells can still penetrate the blood-brain barrier and reach the lesion site.⁴⁵ Intravenous administration also achieves similar therapeutic outcomes as intra-arterial methods by avoiding surgical interventions. Intravenous infusions in phase I/II studies improved functional outcomes over 12 months.⁴⁶ Unfortunately, both methods have lower effectiveness, compared to intracerebral delivery, as fewer cells reach the infarct area. Additionally, intra-arterial and intravenous injections increase the risk of pulmonary embolism and thrombosis as a large number of stem cells accumulate in the lungs and spleen.^45,47,48 Intranasal route has emerged as a new method of stem cell delivery. Because stem cells are administered through the olfactory system, cells can bypass the blood-brain barrier and reach the lesion site.^49,50 Intranasal cell administration in stroke models shows improved cognitive, motor, and sensory functions,⁵¹ highlighting its safety without sacrificing its effectiveness.

Extracellular vesicles (EV) play a notable role in intercellular communications, such as coagulation and immune responses.⁵² Small and large EVs are relevant components of the cells’ secretome, as membrane-structured components are able to exert neurostructural and functional effects in the local and distant environments. Their content is specific to the parental cell and can be characterized by the differential presence of proteins, DNA, lipids, RNA, mRNA, and others. Apoptotic cells release EVs to the extracellular environment, but equally compelling evidence suggests that healthy cells also release EVs to the extracellular environment.⁵³ Tissues of aged mice treated with EVs show lower predicted epigenetic age and metabolome similar to younger mice.⁵³ Small and large EVs may be responsible for the therapeutic effects.^52-54 Small EVs are favored over large EVs in identifying healthy cell activity due to their consistent size and known protein content.⁵⁵ Tetraspanins, such as CD63, are a branch of membrane proteins that congregate into the microdomains of the plasma membrane and their abundance in EVs signifies healthy cell activity.^54,56,57

Lab-to-clinic translational research gaps in knowledge

Several “laboratory-to-clinic” translational challenges have been postulated to mitigate the failure to achieve robust clinical outcomes despite very positive laboratory results. Two major mitigating factors that remain unresolved include (1) well-defined donor stem cells and (2) mechanism of action. To this end, we focused on a newly characterized population of CD34+ cells, called ProtheraCytes, derived from human peripheral blood after G-CSF mobilization, which have been processed under good manufacturing practice and have been tested in a phase 2 clinical trial in post-acute myocardial infarction (NCT02669810) (Figure 1).

Figure 1.

Human CD34+ Cell ProtheraCytes and EVs. Panel A. a, Concentration of VEGF in cell culture supernatants after 9 days of CD34+ cell expansion from 4 healthy donors and 16 patients with acute myocardial infarction, b, No significant difference observed when VEGF concentration was compared between patients and healthy donors, but a significant difference was observed between patients and StemFeed (cultivation medium) (t-test, P = .0007) and healthy donors and StemFeed (t-test, P = 0.0087). c, When the VEGF concentration was normalized by the number of CD34+ cells, we observed that there was no significant difference between the VEGF secreted per cell in healthy donors (4.1 fg/cell) and AMI patients (4.4 fg/cell) (P = .6343). d, VEGF concentration and number of CD34+ cells after expansion. e, Significant correlation between VEGF concentration and the number of CD34+ cells after expansion (Pearson correlation coefficient = 0.7484, P value = .0009). f, In vitro tube formation assay: HUVECs cocultured with ProtheraCytes supernatant have significantly higher tube formation than HUVECS cocultured with StemFeed control (t-test, P = .0082). The pictures display representative brightfield images of tube formation. Scale bar, 20 μm. Panel B. a, The average size of Protheracytes-derived small EVs was determined at 86.7 ± 10.17 nm. b, Small EVs secreted by ProtheraCytes increased over time from around 6000 extracellular vesicles/cell at 30 minutes to 16 000 extracellular vesicles/cell at 2 hours. Protheracytes-derived small EVs express the characteristic exosomal markers CD63, CD81, and CD9 as well as endothelial (CD49e and CD44) and stem cell markers (CD133). Panel C. a, Total RNA from ProtheraCytes (cells) and their small EVs (exosomes) collected from AMI patients (n = 7) were subjected to real time PCR and normalized to small RNA (let-7a). miRNA expression in the small EVs secreted from ProtheraCytes was significantly higher than in ProtheraCytes (Mann–Whitney test). b, Representation of the comparative fold change expression of small EVs vs cells for the indicated miRNAs (n = 7). Panel D. RNA expression levels detected by RNAseq analysis of ProtheraCytes from AMI patients vs healthy donors. Plot bar (FC) of up- and down-regulated genes involved in angiogenesis (a) and vasculogenesis (b). Panel E. a, Representative flow cytometry plots of ProtheraCytes from patients. CD34+ cells were identified within mononuclear cells (MNCs) according to size criteria, reduction of background by stringent exclusion of dead cells, and lineage CD34+ cell phenotype by flow cytometry. b, Histogram represents the mean ± SD of (n = 10). Panel F. a, Schematic representation of the differentiation protocol of ProtheraCytes into endothelial cells. b, Upper panel—bright-field image of ProtheraCytes at day 0 and endothelial differentiation at day 17. Scale bar, 100 μm. Bottom panel immunofluorescence staining showing positive staining for either endothelial marker WWWF, CD31 (PECAM1), VEGFR-2, or VCAM-1 of ProtheraCytes at day 0 and differentiated ProtheraCytes® at day 17. The pictures display representative merged immunofluorescence images. Scale bar, 20 μm. c, Histogram plots showing percentage of positive cells at days 0 and 17 (*P < .05, **P < .01, ***P < .0001). Adapted from Aries et al., Scientific Reports, 2023.⁶⁸

Our preliminary study performed a vis-a-vis comparison of different routes of administration, namely intracerebral, intraarterial, and intranasal delivery of ProtheraCytes in the MCAO model using adult Sprague–Dawley rats. Following optimization of the dose and subacute timing of cell delivery, animals were randomly assigned to receive either ProtheraCytes or vehicle. The significance of our preliminary findings is multi-fold: (1) motor and neurological assays from days 7 to 28 post-stroke revealed significant stable and long-term functional recovery across all three delivery routes of ProtheraCytes compared to vehicle-treated stroke rats; (2) additionally, ProtheraCytes-transplanted stroke rats displayed significantly reduced infarct size and cell loss in the peri-infarct area coupled with enhanced neurogenesis and angiogenesis compared to vehicle-treated stroke rats; (3) mechanistically, ProtheraCytes may secrete growth factors and CD9 + and CD81+ EVs, but in particular CD63+ EVs, which appear to be associated with the observed upregulation of neurogenesis and angiogenesis, and; (4) most importantly, we expanded the therapeutic window up to 3 days after stroke (Figure 2). These findings provide compelling evidence of the safety and efficacy of transplanting ProtheraCytes, including via the minimally invasive intranasal route, in affording robust and stable behavioral and histological therapeutic effects in an animal model of stroke. In line with our long-standing innovative efforts to translate safe and effective treatments from the laboratory to the clinic, we recognize the need to extend our preliminary data showing robust efficacy of ProtheraCytes from 28 days post-stroke to 3 months post-stroke, thereby addressing the chronic nature of stroke secondary cell death. We will also examine therapeutic equivalence between adult and cord blood-derived ProtheraCytes to resolve the issue of ready availability of transplantable cells, allowing transplantation even at the acute stroke stage. A dose escalation study of the selected source will then be conducted in the same MCAO model. Finally, we will further probe the role of CD63+ EVs to mediate the functional effects of transplanted ProtheraCytes. Assessment of the complete expression of different tetraspanin-positive EVs released by ProtheraCytes is equally important since CD63+ EVs might be a fraction of all EVs released by these cells, and that EVs may be functional in the absence of CD63. At least in our reported study,⁵⁸ the expression of CD+ 63 EVs correlated with reduced stroke-induced histological and behavioral deficits. Accordingly, while not discounting the role of other EVs besides CD63+ EVs, our planned experiment of blocking CD63 expression by exposure of ProtheraCytes to anti-CD63 antibodies in culture may provide some insights into the role of EVs in the functional recovery of stroke animals transplanted with ProtheraCytes. To this end, we envision that CD63-based cell product release criterion and treatment biomarker will guide the clinical design of intranasal ProtheraCytes allografts in stroke (Figure 3).

Figure 2.

Panel A shows experimental design. Panel B. ProtheraCytes reduce stroke symptoms. (a) Motor activity revealed by elevated body swing test (EBST). MCAO + cell (ProtheraCytes) groups displayed significantly less asymmetry on days 7, 14, 28 (*P < .05, **P < .01, ****P < .0001). (b) Motor activity revealed by Cylinder Test MCAO cell groups demonstrated significantly more use of impaired forelimb (*P < .05, **P < .01, ***P < .001, ****P < .0001). (c) Motor activity revealed by Grip Strength. MCAO + cell groups presented significantly less impaired paw grasp (*P < .05, **P < .01, ***P < .001). (d) Motor activity revealed by Balance Beam. MCAO + cell showed significantly better motor coordination during beam walks (**P < .01, ***P < .001). Panel C. ProtheraCytes decrease stroke infarct area. Nissl staining for coronal brain sections showing infarct areas of MCAO Cell IA (intraarterial), MCAO Cell IN (intranasal), MCAO Cell IC (intracerebral), MCAO Vehicle Media IA, MCAO Media IN, MCAO Media IC. MCAO Cell groups displayed significantly smaller infarcts (*P < .05, **P < .01, ***P < .001, ****P < .0001). Panel D. ProtheraCytes increase cell survival in peri-infarct zone. Nissl staining for quantitative analysis of live cells in the peri-infarct area for MCAO + Cell IA, MCAO Cell IN, MCAO Cell IC, MCAO Media IA, MCAO Media IN, MCAO Media IC. MCAO cell groups showed significantly more living cells in the peri-infarct region (*P < .05, **P < .01, ***P < .001, ****P < .0001). Scale bar = 50 μm. Panel E. ProtheraCytes dampen inflammation. Iba-1 double stained with CD34 to measure inflammatory activity. MCAO Cell IC had significantly less lba-1 positive cells than MCAO + media IN, MCAO + media IA, and MCAO media IC groups (**P < .01). Treatment groups had significantly more CD34 expression (***P < .001) with MCAO + Cell IC having the highest cell count within the treatment group (*P < .05; ***P < .001). Scale bar 50 µm. Panel F. ProtheraCytes enhance neurogenesis. DCX double stained with CD34 measured neurogenesis differences between groups. All treatment groups MCAO+ Cell IA, MCAO Cell IN, and MCAO Cell IC had significantly higher DCX expression than control groups MCAO + Media IA, MCAO + Media IN, and MCAO + Media IC (**P < .01). Treatment groups had significantly more CD34 expression (**P < .01). MCAO Cell IN displayed significantly lower CD34 expression compared to MCAO Cell IC (*P < .05). Scale bar = 50 µm. Panel G. ProtheraCytes promote angiogenesis. VEGFr1 double stained with CD34 quantified angiogenesis activity. MCAO + Cell IC and MCAO Cell IA had significantly higher VEGFr1 expression than all MCAO Media groups (**P < .01), MCAO Cell IC had significantly more VEGFr1 positive cell counts than MCAO Cell IA (*P < .05) and MCAO + Cell IN (**P < .01). MCAO Cell IN had significantly less angiogenesis activity when compared to MCAO Cell IA (*P < .05). CD34 expression was significantly higher in all treatment groups than in all media groups (**P < .01). Scale bar 50 μm and 10 μm. Panel H. ProtheraCytes trigger expression of extracellular vesicles. CD63 staining was used as a marker to quantify extracellular vesicles and healthy stem cell activity. All treatment groups MCAO + Cell IA, MCAO + Cell IC, and MCAO + Cell IN had significantly higher CD63 positive stain count than control groups MCAO + Media IA, MCAO + Media IN, and MCAO + Media IC (*P < 0.05). CD34 expression was significantly higher in all treatment groups than in all media groups (**P < 0.01). Scale bar = 50 μm. Panel I. ProtheraCytes protect against in vitro stroke model of oxygen glucose deprivation, a, Trypan blue. ANOVA revealed significant treatment effects. Post hoc Bonferroni’s tests revealed significant differences in cell survival, with primary neurons exposed to OGD and co-cultured with ProtheraCytes rescuing against OGD-induced cell death significantly better than primary neurons subjected to OGD. b, MTT. Similarly, ANOVA showed significant treatment effects, with post hoc Bonferroni’s tests detecting significant differences in metabolic activity, again with primary neurons exposed to OGD and co-cultured with ProtheraCytes reducing the OGD-induced metabolic impairment significantly better than primary neurons subjected to OGD. c, CD63. Analysis of exosome marker expression revealed that ProtheraCytes express the exosomal marker CD63, which was not detectable in the standard medium. Statistical significance is depicted as follows: *P < .05; **P < .01; ***P < 0.001. Adapted from Lee et al., Stem Cells Translational Medicine, 2024.⁶⁹

Figure 3.

Stem cell-derived extracellular vesicles promote recovery of ischemic cells. ProtheraCytes confer a novel mechanism of action mediating stem cell-induced therapeutic effects in ischemic stroke. Whereas the long-standing view of stem cell repair of the stroke brain implicates cell replacement, our findings implicate a process of by-stander effects, whereby stem cells release extracellular vesicles (EVs), in particular CD-63-labeled EVs, which appear to target the vasculature and enhance vascular endothelial growth factor levels, while dampening inflammation (Iba-1 cells) and increasing neural cell proliferation (DCX). Altogether, these reparative processes rescue the neurovascular unit via a multi-pronged mechanism involving EV release, angiogenesis and vasculogenesis, anti-inflammation, and neurogenesis. Adapted from Lee et al., Stem Cells Translational Medicine, 2024.⁶⁹

Despite these promising functional outcomes in stroke animals transplanted with ProtheraCytes, outstanding caveats that remain to be examined consist of (1) amplification of ProtheraCytes to achieve clinically relevant supply of transplantable autologous cells requires more than 3 days of in vitro expansion from the time of peripheral blood harvest after G-CSF mobilization, and (2) demonstration of direct causal effect of CD63+ EVs on stroke functional recovery. Accordingly, to further advance the scientific premise and clinical utility of ProtheraCytes, we propose the following translational studies. First, there is a need to test the hypothesis that ProtheraCytes harvested from adult healthy donors and from umbilical cord blood produce equivalent reduction of stroke-induced behavioral and histological deficits, we will intranasally transplant ProtheraCytes at 3 days after MCAO and monitor functional outcomes up to 3 months post-stroke. This study will extend our preliminary data showing robust efficacy of ProtheraCytes up to 28 days post-stroke by testing stable and long-lasting efficacy up to 3 months post-stroke, thereby addressing the chronic nature of stroke secondary cell death. More importantly, if the envisioned results show equivalence between adult and cord blood-derived ProtheraCytes, then this will resolve the issue of ready availability of transplantable cells, allowing allogeneic transplantation even at acute stroke stage. Second, it is also appealing to test the hypothesis that CD63+ EVs mediate the functional effects of transplanted ProtheraCytes. Blocking CD63 expression by exposure of ProtheraCytes to anti-CD63 antibodies in culture prior to transplantation in MCAO stroke rats, or comparing the efficacy of ProtheraCytes supernatant and CD63+ EVs vs standard cultivation medium and CD63-EVs in the in vitro stroke model of oxygen-glucose deprivation may reveal the direct participation of growth factors and CD63+ EVs. If proven that CD63 plays a major role in ProtheraCytes, then CD63 can serve as a cell product release criterion for ProtheraCytes or potency assay for clinical batch release, as well as biomarker of efficacy readout in monitoring stroke patients transplanted with ProtheraCytes (eg, CD63 expression levels in plasma or cerebrospinal fluid). In particular, a potency assay needs to assess the effect of a biological product for a specific application and such a potency assay would predict if a particular EV preparation has the potential to achieve its intended therapeutic effects. To this end, we now have developed a biological measure based on the secretion of VEGF and its validation via the ELISA method.⁵⁹ As a backup potency assay, the measure of the miRNA content inside the exosomes has also been developed demonstrating that these miRNAs are pro-angiogenic and anti-apoptotic.⁵⁹ Altogether, the detection of the high level of CD63 expression in ProtheraCytes corresponded with the biological upregulation of VEGF in parallel with increased proangiogenic and anti-apoptotic miRNAs,⁵⁹ in sum providing a potency assay for ProtheraCytes. Nonetheless, we acknowledge that CD63+ EVs from the exogenously delivered ProtheraCytes may have a very short lifespan, but the downstream VEGF secretion and the pro-angiogenic and anti-apoptotic miRNAs, not just as those exogenously derived but also endogenously triggered by the transplanted ProtheraCytes, may provide a much more robust and long-lasting therapeutic outcomes. Notwithstanding these limitations, both proposed translational studies align with our long-standing campaign to introduce safe and effective stem cell transplant regimen in stroke patients. We envision that CD63-based cell product release criterion and treatment biomarker will guide the clinical design of intranasal ProtheraCytes allografts in stroke.

Clinical translation of intranasal cell transplantation

While the rostral migratory system (RMS) has been widely implicated as the nose-to-brain migratory system in rodents, such olfaction-based system appears only as a structural and functional remnant of the rodent RMS in humans,^60-62 thus may impede the clinical translation of the intranasal peripheral to central homing of stem cells. Here, we hypothesize that CD63 EVs may be anchored to ApoA1, a major lipoprotein, acting as a functional receptor. A few studies have suggested such crosstalk between EVs and lipoproproteins,⁶³ in particular, CD63 and ApoA1⁶⁴ via ligand–receptor interaction and even gene fusion between CD63 and ApoA1.^65,66 That CD63 and ApoA1 are tightly bound to each other has been casually accepted based on most isolation techniques of pure EVs requiring the pivotal elimination of ApoA1 from the blood/serum fraction during the final step of CD63 isolation.^64,67,68 Conversely, ApoA1 increases the release of EVs (or large EVs) from cells,⁶⁵ further illustrating their key and lock interaction. However, beyond this distinct tethering of CD63 to ApoA1, its functional relevance remains unknown. Interestingly, we recently reported that ApoA1 dose-dependently modulates endothelial cell survival in an ALS model,⁶⁹ a disease that manifests a vascular damage reminiscent of the stroke pathology. Still, whether our observed ApoA1-induced endothelial cell survival is mediated by CD63 has yet to be examined. Such unexplored functional relevance of CD63-ApoA1 engagement may mediate stem cell homing and host cell survival, altogether aiding the RMS in guiding intranasally delivered cells to reach and rescue the ischemic brain. This CD63-ApoA1 migration of stem cells if proven true will optimize stem cell therapy for stroke.

Conclusion

Stroke treatments are limited to tissue plasminogen activator and mechanical thrombectomy with narrow therapeutic window, and while stem cell therapy appears to extend this treatment window, critical scientific gaps exist in the field of stem cell therapy for stroke, namely a well-defined stem cell population and a mechanism of action. Moreover, the choice of a stroke model (MCA transient and permanent occlusion/ligation models, blot clot embolic model, photothrombotic model, and models incorporating co-morbidity factors such as hypertension and diabetes) can dramatically influence the effects of a treatment, especially when “aging” is taken into account as age represents the most significant risk factor in stroke context. The aged animals may provide pharmacological insights expected in elderly stroke patients. The aging process is responsible for the promotion of arterial hypertension, hypercholesterolemia, obesity, and diabetes, reduced neurological recovery, and conversion of ischemic penumbra into brain infarction in humans. Even though the shorter lifespan of lab animals compared to humans does not permit to recapitulate risk factor profiles alone, aged rats showed interesting features, such as severe neurological impairment when compared to young rats, reduced neurogenesis in the dentate gyrus in MCAO rats, and stronger astroglial response to stroke injuries. Thus, aged animals represent a reliable choice to model a condition that mainly characterizes elderly people, as risk factor models are able to depict a more consistent pharmacological situation and improve the translation of drugs from bench to bedside.⁷⁰ In advancing the clinical application of ProtheraCytes for stroke, translational experiments along the Stroke Therapy Academic Industry Roundtable and Stem Cell Therapeutics as an Emerging Paradigm for Stroke (STEPS) guidelines are warranted.^71,72 In particular, since stroke is considered an age-related disorder^73,74 and closely associated with co-morbidity factors, such as hypertension^75,76 and diabetes,^77,78 incorporating these clinically relevant iterations into the stroke modeling paradigm will increase the stringent testing of cell therapy in the clinical setting. Additionally, recapitulating the clinical stroke scenario will need to consider the heterogenous stroke patient population who may or may not benefit from tPA and manifest with focal or global ischemia, thereby necessitating the evaluation of cell therapy in multiple stroke models including MCA transient and permanent occlusion/ligation models, embolic blot clot model, and photothrombotic model that partially replicate the stroke pathology.^79-81 Altogether, recognizing these laboratory-to-clinical application caveats will allow the safe and effective translation of ProtheraCytes for stroke patients. By comparing the efficacy of clinical grade ProtheraCytes, a novel non-adherent stem cell population derived from adult human peripheral blood and umbilical cord blood, and processed under good manufacturing practice, and investigating the role of growth factors and CD63+ EVs, these additional translational studies will provide valuable insights into resolving the phenotypic characterization and mechanism underlying the potential benefits of stem cell therapy. We envision that transplantation of ProtheraCytes may significantly impact stroke treatments, specifically in the development of new clinical trials for intranasal stem cell delivery in stroke.

Author contributions

Cesar V. Borlongan: Conceptualization, Funding acquisition, Supervision, Writing. Jea-Young Lee, Francesco D’Egidio, Raphael Guzman: Conceptualization, Writing. Matthieu de Kalbermatten, Ibon Garitaonandia: Conceptualization, Funding acquisition, Writing.

Funding

Research funding from National Institute of Health (NIH). M.de K. and I.G. are affiliated with CellProthera

Conflicts of interest

C.B. declared patents and patent applications on stem cell biology and its therapeutic applications, consultant to a number of stem cell-based companies. The other authors declared no potential conflicts of interest.

Data availability

No new data were generated or analyzed in support of this research.