Modelling heme-mediated brain injury associated with cerebral malaria in human brain cortical organoids

Adriana Harbuzariu,Andrew P Shaw,Keri Oxendine Harp,Mingli Liu,Sidney Pitts,Annette Nti,Jonathan K Stiles,Juan Carlos Cespedes

doi:10.1038/s41598-019-55631-8

Adriana Harbuzariu, Andrew P Shaw + Show 6 more

Open Access

https://doi.org/10.1038/s41598-019-55631-8

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Abstract

Human cerebral malaria (HCM), a severe encephalopathy associated with Plasmodium falciparum infection, has a 20–30% mortality rate and predominantly affects African children. The mechanisms mediating HCM-associated brain injury are difficult to study in human subjects, highlighting the urgent need for non-invasive ex vivo human models. HCM elevates the systemic levels of free heme, which damages the blood-brain barrier and neurons in distinct regions of the brain. We determined the effects of heme on induced pluripotent stem cells (iPSCs) and a three-dimensional cortical organoid system and assessed apoptosis and differentiation. We evaluated biomarkers associated with heme-induced brain injury, including a pro-inflammatory chemokine, CXCL-10, and its receptor, CXCR3, brain-derived neurotrophic factor (BDNF) and a receptor tyrosine-protein kinase, ERBB4, in the organoids. We then tested the neuroprotective effect of neuregulin-1 (NRG-1) against heme treatment in organoids. Neural stem and mature cells differentially expressed CXCL-10, CXCR3, BDNF and ERBB4 in the developing organoids and in response to heme-induced neuronal injury. The organoids underwent apoptosis and structural changes that were attenuated by NRG-1. Thus, cortical organoids can be used to model heme-induced cortical brain injury associated with HCM pathogenesis as well as for testing agents that reduce brain injury and neurological sequelae.

Highlights

Human cerebral malaria (HCM), a severe encephalopathy associated with Plasmodium falciparum infection, has a 20–30% mortality rate and predominantly affects African children
Using flow cytometry (Fig. 1B), we assessed the expression of pluripotency markers {stage-specific embryonic antigen-4 (SSEA-4)[45], sex determining region Y box-2 (SOX-2)[46], and OCTamer-binding transcription factor 3/4 (OCT3/4)}47 at the single-cell level and accounted for the homogeneity of the induced pluripotent cell line[48]
Our results showed that 97.03% of cells were positive for both SOX-2 and SSEA-4 (Fig. 1Ba,d), while 71.2% were positive for OCT 3/4 (Fig. 1Bc) and only 4.6% were positive for SSEA-1 (Fig. 1Bb)

Summary

Introduction

Human cerebral malaria (HCM), a severe encephalopathy associated with Plasmodium falciparum infection, has a 20–30% mortality rate and predominantly affects African children. Brain organoids are self-assembled three-dimensional (3D) aggregates derived from pluripotent stem cells (iPSCs) with cell types and formations that mimic the embryonic human brain[1,2,3,4,5,6,7] They have emerged as innovative model systems that can be used to investigate human brain development, neurologic disease pathogenesis and drug development[8,9,10]. In human HCM26,27, murine ECM28 and in vitro models[29], increased malaria-induced free heme has been shown to elevate the levels of CXCL-10, brain-derived neurotrophic factor (BDNF) and other factors that are tightly correlated with brain injury. The assessment of HCM-associated brain injury in human post-mortem tissues has provided limited cross-sectional data with limited insight into HCM pathogenesis

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