Next-generation regenerative therapy for ischemic stroke using peripheral blood mononuclear cells

Masato Kanazawa,Itaru Ninomiya,Yutaka Otsu,Masahiro Hatakeyama

Stroke is the second leading cause of death and the third leading cause of disability worldwide after heart disease.Researchers predict that stroke deaths and permanent disabilities will increase worldwide by the year 2050.Single-target therapies may be insufficient,because ischemic cerebral injury involves several mechanisms.Cell-mediated therapies are ideal,because they target multiple cell types to enhance protection and recovery.Sources for cell therapy include bone marrow-derived mesenchymal and embryonic stem cells.Although clinical trials on the administration of autologous mesenchymal stem cells have shown improved functional outcomes after ischemic stroke,the number of these cells is limited.Therefore,it requires time to obtain sufficient functionally mature cells for treatment.Simple and abundant cell sources,such as peripheral blood cells,are suitable for clinical applications.

We have shown that primary microglia (Kanazawa et al.,2017) and peripheral blood mononuclear cells (PBMCs) (Hatakeyama et al.,2019;Otsu et al.,2023) preconditioned with 18 hours of oxygenglucose deprivation (OGD-PBMC) assume a polarprotective phenotype.Αlthough the administration of microglia and PBMC under normoxic conditions(without any stimuli) did not result in functional recovery after cerebral ischemia,OGD-microglia and OGD-PBMCs prompted functional recovery(Figure 1).The following therapeutic mechanisms were considered to be involved in the effects of OGD-microglia and PBMC administration: (i) the protective switch to induction of angiogenesis and anti-inflammatory polarization,(ii) the protective switching induced by upregulation of peroxisome proliferator-activated receptor-gamma and hypoxia-inducible factor-1alpha (HIF-1α),and decreasing exosomal miR-155-5p levels after OGD in PBMCs,(iii) the paracrine effects of vascular endothelial growth factor (VEGF) and decreasing exosomal miR-155-5p-induced upregulation of VEGF via the HIF-1α axis,(iv) upregulation of VEGF and transforming growth factor-beta by cellcell communications in host resident cells such as microglia,and (v) angiogenesis and axonal outgrowth in the brain.The alteration of resident cells in the neurovascular unit by secretomes,including exosomes,is one of the mechanisms of action of this cell therapy.Even in mesenchymal stem cells,hypoxia enhances the secretion of exosomal microRNAs (miRNAs) and cytokines to promote cell regeneration (Haupt et al.,2023).Cell therapies with enhanced cellular protection from any stimulus are nearing clinical application.

Direct reprogramming is a technique that transforms one cell type into another,directly altering the fate of cells without involving pluripotent stem cells.This is achieved through the control of transcription factors via genetic intervention or drugs (Moris et al.,2016;Wang et al.,2021).Over the past decade,this technology has attracted considerable attention as a new regenerative medicine technology.Direct reprogramming differs from conventional therapies in its ability to not only remove unwanted cells or replenish necessary cells,but also enable the conversion of unwanted cells into necessary cells.Additionally,the potential to create target cellsin vivo,instead of directly administering cells,is expected to mitigate the risks of immune rejection,cellular viability,and tumorigenesis,which are common challenges in cellular medicine.Theoretically,the direct reprogramming of somatic cells into neurons represents a promising regenerative therapy for neurological diseases.Many of these phenomena,which rewrite once-determined cell fates,involve acquired modifications (epigenetics)to DNA and histones by transcription factors/transcription-coupled factors.The discovery of the reprogramming process,which is an initialization process,has changed the idea that cell fate is unidirectional,such as descending a mountain(Waddington’s epigenetic landscape),and this direct reprogramming has drawn attention to the fact that cell fate can diverge in multiple directions(Moris et al.,2016).Notable examples include the transformation of glial cells,fibroblasts,and T cells into neurons (Wang et al.,2021).

Chemical direct reprogramming of macrophages into neurons is a promising therapy for ischemic stroke.In the acute phase of cerebral infarction,blood monocytes infiltrate the infarcted lesion and differentiate into macrophages,which are involved in inflammation and protection of the brain tissue.Moreover,we reported an increasing number of stage-specific embryonic antigen-3-positive stem cells in PBMCs after cerebral ischemia and OGD (Hatakeyama et al.,2019;Otsu et al.,2023).Monocytes and macrophages transdifferentiate into neurons in response to various stimuli.Theoretically,it is possible to convert these macrophages to neuronsin vivoby administering specific low-molecular-weight compounds to induce neural regeneration.Based on this evidence,Ninomiya et al.(2023)investigated the transformation of monocytes and macrophages into neurons through chemical direct reprogramming and examined the therapeutic effects of these chemical compounds in animal experiments.

Ninomiya et al.(2023) identified six compounds—CHIR99021,dorsomorphin,forskolin,isoxazole 9 (ISX-9),Y27632,and DB2313—that when cocultured with monocyte-derived macrophages,successfully transformed them into neuronlike cells expressing the neuronal markers TUJ1,DCX,and MAP2.Although the reprogramming of monocytes was not efficient,these compounds have been reported to effectively transform macrophages into neurons.Gene expression analysis revealed enhanced expression of nervespecific genes and suppressed expression of macrophage-specific genes.The absence of DB2313 did not lead to the transformation of macrophages into neurons.Intraperitoneal administration of these compound cocktails in mice with cerebral infarction induces regeneration of the cerebral cortex and enhanced neurological function.In particular,DB2313 was essential for macrophage differentiation and transformation.In the context of neuronal transdifferentiation,the inhibition of thePU.1gene was efficient in macrophages.Overexpression ofPU.1efficiently reprogramed neural stem cells into monocytes(Forsberg et al.,2017).We speculated that DB2313 plays a central role in the inhibition of lymphoid and myeloid cell fates.IfPU.1expression persists,macrophages tend to remain in a steady state as they strive to maintain their nature,thereby making successful neural induction unlikely to occur.The outcome emphasizes the significance of master gene dependency in determining cellular fate.Furthermore,the direct administration of these compounds was suggested to inducein vivodirect programming into neurons.This suggests the potential for their application in novel regenerative medicine using a combination of chemicals.

Despite the promising advancements in chemical direct reprogramming,further studies are required to investigate the potential effects of drugs.First,it is essential to address,issues such as the transformation efficacy and neuronal purity were low (approximately 30%).Hypoxic stimulation is one of the candidates for improving the transformation efficacy and purity (Figure 1;Nakamura et al.,2021).Several studies have shown that culturing cells under hypoxic conditions enhances cellular reprogramming by upregulating the expression of the transcription factor,HIF,inducing epigenetic effects (H3K4me3 and H3K27me27),and altering the chromatin topography.Interestingly,bone marrow hematopoietic or mesenchymal stem cell maintains their pluripotency and proliferative capabilities through the action of HIF-1α(Huang et al.,2018).For application in ischemic stroke,hypoxia might be a suitable factor for reprogramming.The modulation of miRNΑ is also a candidate for improving transformation efficacy(Figure 1).Exosomal miR-155-5p inhibition in blood mononuclear cells triggers a polarized protective state by upregulating VEGF and stagespecific embryonic antigen-3 through the HIF-1α axis (Otsu et al.,2023).Αlthough not resulting in complete reprogramming,miRNAs can mediate changes in cell fate.Demethylation of H3K27me3 is essential for inducing direct cardiac reprogramming.Moreover,the combination of miR-1,miR-133,miR-208,and miR-499 decreases H3K27me3 levels in the promoter region of cardiac transcription factors and increases the efficacy of reprogramming (Dal-Pra et al.,2017).Mir-34a knockout mice exhibited significantly increased reprogramming efficiency in mouse embryonic fibroblasts,which regulate somatic reprogramming downstream of p53 (Choi et al.,2011).miRNAs may improve reprogramming efficacy.Methods to increase the transformation efficacy should be investigated.Second,the longterm effects of small-molecule administration are unclear.However,the long-term systemic effects of the six compounds remain unknown.Macrophages are abundant in the body;however,we did not observe any abnormalities,such as neural differentiation or tumorigenesis.Thus,ISX-9 is a synthetic promoter of adult neurogenesis that triggers the differentiation of adult neural stem/precursor cells.ISX-9 activates multiple pathways including transforming growth factor-β induced epithelial-mesenchymal transition signaling and Wnt signaling at different stages of cardiac differentiation.This may induce abnormal angiogenesis.Moreover,DB2313 plays a key role in inhibiting lymphoid and myeloid cell fates.However,the long-term effects of small molecule administration must be carefully evaluated.

Regarding immunological concerns,autologous cells are safer than allogeneic cells.Moreover,techniques for the isolation and preparation of autologous PBMCs have been established.Therefore,our results are promising for clinical applications.Moreover,hypoxic stimulation can be applied to stem cells to enhance their protective effects.While future studies are required to provide evidence for OGD-PBMC therapy and the concept of cell transdifferentiation,PBMCs may be applied for cell-based therapies.These adaptive responses have facilitated the development of regenerative therapies for ischemic stroke.In conclusion,these new insights about PBMCs attribute novel potential therapeutic targets for ischemic stroke.

This work was supported by the JSPS KAKENHI Grant-in-Aid for Scientific Research(Grant number:21K19441,22H03183)(to MK),and Early-Career Scientists(Grant number:21K15185)(to IN)and(Grant number:20K16485)(to MH).

Masato Kanazawa*,Itaru Ninomiya,Yutaka Otsu,Masahiro Hatakeyama

Department of Neurology,Brain Research Institute,Niigata University,Niigata,Japan

*Correspondence to:Masato Kanazawa,MD,PhD,masa2@bri.niigata-u.ac.jp.

https://orcid.org/0000-0001-6337-8156(Masato Kanazawa)

Date of submission:October 31,2023

Date of decision:December 1,2023

Date of acceptance:December 21,2023

Date of web publication:January 31,2024

https://doi.org/10.4103/NRR.NRR-D-23-01784

How to cite this article:Kanazawa M,Ninomiya I,Otsu Y,Hatakeyama M(2024)Next-generation regenerative therapy for ischemic stroke using peripheral blood mononuclear cells.Neural Regen Res 19(11):2341-2342.

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