Ischemic Precondition Induced Factor Botch Overexpression In Human Neural Precursor Cells Optimized Cell Based Therapy In Stroke

Background and ObjectiveStroke is the 4th leading cause of death in the world and the leading cause of death and disability in the China. Ischemic neuronal injury that occurs following the loss of oxygen and glucose to the brain leads to a cascade of secondary processes that contribute to the death of neurons, glia, and endothelial cells. The only FDA approved therapy for acute stroke is tissue plasminogen activator (tPA). No proven neuroprotective or neurorestorative therapy for stroke has yet been efficacious. Thus, there is a great need for both innovative neuroprotective and neurorestorative therapies.The ability to differentiate human embryonic stem cells and inducible pluripotent stem cells into different neuronal subtypes provides the opportunity to lay the groundwork for cell based restorative therapies for stroke. It is conceivable that transplanted neural precursor cells could differentiate into new neurons and establish synaptic connections in the stroke damaged host brain leading to restoration of function. Previous studies indicate that human pluripotent stem cell derived neural progenitors (hNPCs) transplanted into animals following stroke develop into neurons leading to functional recovery. A limiting step in transplantation of hNPCs is the propensity of the transplanted cells for excessive proliferation and cell death. Initial attempts to control excessive proliferation and promote differentiation have relied on the use of gamma-secretase inhibitors to impair Notch signaling. The proliferative capacity of neural progenitors is in part regulated by Notch, thus interfering with Notch signaling is a potentially attractive mechanism by which to promote differentiation and limit proliferation. Recently a novel endogenous Notch inhibitor designated Botch has been reported. Notch signaling is substantially impaired in the presence of Botch via inhibition of furin processing of Notch. Accordingly, the effect of Botch expression on transplanted hNPCs was examined in a murine model of stroke. Botch inhibits the proliferation and promotes the survival and differentiation of grafted hNPCs into mature neurons leading to improvements in functional recovery following stroke.Methods and Materials1. We engineered human embryonic stem cells (hESCs) with PiggyBac so the differentiated cells express GFP or dsRed fluorescence. OGD or NMDA precondition was pretreated to dsRed positive hNPCs derived from hESCs, and GFP positive hNPCs were used as non-preconditioned control. We used the double cells system to minimize the individual varieties between different treatment groups. The hNPCs mixture (dsRed+preconditioned hNPCs and GFP+non-preconditioned hNPCs) was injected into the infarcted cortex of mice 1 days post stroke onset. Fluorescence was used to measure the graft volume, and immunostaining was carried out to detect the survival, proliferation and differentiation of the grafts.2. RNA sequence was carried out in hNPCs before and after OGD/NMDA preconditioning. Gene or protein expression of Botch was measured using real-time qPCR or western blot. Immunohistology staining was used for detecting the Botch overexpression effect on hNPCs proliferation and survival. And PI/Hoechst cell death assay was used to verify the neuroprotection effect of Botch.3. The effect of Botch overexpression on hNPCs derived from hESCs was verified using immunohistology staining. At the stage of neural precursor cells, hNPCs were transduced with a lentivirus overexpressing Botch-EGFP. Lentivirus expressing EGFP alone served as a control. Differentiation into cortical neurons representing all the six cortical layers and synapse formation were measured using immunostaining, and maturation of the human neurons derived from hESCs were tested using electrophysiology recording.4. Human NPCs overexpressed Botch-EGFP were transplanted into the infarcted cortex of mice one day after stroke, and hNPCs expressed EGFP transplantatation was used as control. EGFP fluorescence was visualized under confocal to verify the graft volume, the distribution of grafts and the neurite outgrowth of grafts. Immunohisotological staining was used to detect the cortical neuron differentiation and their synapse maturation. Staircase test and Capellini handling test were used to tell the neurological deficits recovery post transplantation.Results1. Ischemic precondition decreases the proliferation of grafted hNPCs derived from hESCs in stroked mice cortexWe observed the graft volume, graft density, proliferation and survival of the grafted dsRed+ preconditioned/GFP+ control hNPCs at 4wks,8wks,12wk and 16wks after transplantation. Although the graft volume was not altered between these two groups, the density within the grafts was significantly lower in OGD or NMDA preconditioned groups as relative to that of their co-transplanted control groups. Different from our expectation, ischemic precondition did not alter the caspase3+cell ratio, but did reduce the Ki67+cell ratio compared with that in the control groups by 4wks post transplantation. Collectively, these indicated that ischemic precondition could inhibit the overgrowth of grafted hNPCs via inhibiting their proliferation.2. Botch is identified from preconditioned hNPCsRNAseq data suggested that Botch mRNA was upregualted after ischemic precondition and this was also confirmed at protein level via western blot. And evidence has been provided that mutation of Botch abolished the neuroprotection provided by ischemic precondition. These suggested that Botch might be one of the key molecules mediating the ischemic precondition effects.3. Botch inhibited the proliferation of hNPCs from hESCs, protected human neurons from ischemic toxicity and the promoted their the neuronal differentiation into cortical neuronsBotch was low expressed at the hNPCs stage as compared with ES, EB or neuron stage. We overexpressed Botch via lentivirus. Botch turned out limiting the proliferation of neural precursor cells and neuroblasts without causing more cell death. Moreover, Botch overexpression provides neuroprotection for human neurons from OGD or NMDA insults. In addition, Botch overexpression promoted mature neuron differentiation, especially differentiation into Tbrq1+ or Ctip2+deep layer cortical neurons and Gad67+inhibitory neurons.4. Botch overexpression limited the overgrowth of grafted hNPCs and promoted their neurite outgrowth and neuronal differentiation. Botch overexpression promoted the skilled motor function improvement after stroke.Grafted hNPCs overexpressed Botch showed smaller graft volume as relative to the control hNPCs. This might be explained by the reduced proliferation in the Botch overexpressed hNPCs. Meanwhile, better cell survival was obtained by Botch overexpression. Botch overexpression did not alter the neural fiber distribution within the brain, but prolonged the outgrowth of grafted NPCs. In addition, Botch overexpression promoted the differentiation into Tbr1+ or Ctip2+ deep layer cortical neurons. Most importantly, Botch overexpressed hNPCs significantly promoted the skilled motor and coordination function improvement after stroke as relative to sham group.Conclusion1. Precondition provided transient proliferation inhibition to the grafted hNPCsOur data suggests that although the grafted survival is not significantly improved by OGD/NMDA precondition, such ischemic precondition could inhibit the proliferation of the grafted cells within the 4wks post transplantation, and thereafter reduces the total number of grafts in the infarcted cortex. Evidence has been provided that hypoxia helped NPCs maintained at a highly proliferating stem cell stage. Our precondition inhibits the proliferation within the first 4wks, maybe due to the transient protein or gene expression induced by precondition.2. Preconditioning induced Botch could inhibit the proliferation of hNPCs and promoted their survival in harsh environment and differentiation into neuronsWe screened the all the genes altered by precondition, and Botch is identified as a promising target. Here our data supports that Botch overexpression inhibits the proliferation of dividing NPCs and promotes their neuronal differentiation. But for non-dividing neurons, Botch actually provides neuroprotection in the situation of ischemia injury. And Botch promotes deep layer cortical neurons and inhibitatory neurons differnentiation. The multifaceted function makes Botch very suitable for optimizing cellular therapy.3. Botch overexpression might optimize the cell based therapy in storkeConsistent with our in vitro data, in vivo transplantation of Botch overexpressed hNPCs showed inhibited overgrowth and better cell survival, neurite outgrowth and cortical neuron differentiation. This perfectly solves the overgrowth and efficacy concern of hNPCs transplantation in stroke. Furthermore, Botch overexpressed hNPCs transplantation rpromoted skilled motor function recovery of stroked animals. This indicated that Botch could enhance the efficiency of cell-based therapy. Taken together, Botch overexpression makes hNPCs transplantation in stroke safer and more efficacious.