Ischemic stroke is the rapid loss of brain function due to disturbance in the blood supply to the brain leading to a loss of function in the affected area.   Studies in rodents have shown that embryonic stem cell (ESC) -derived neural stem cells (NSCs) (Daddi et al, Hicks et aland Ramos-Cabrer et al) and human foetal NSCs (Darsalia et al 2007, Darsalia et al 2011and Kelly et al)can differentiate into neurons leading to some improvement in impaired function in stroke following transplantation into the affected area, however this has not been attempted with cells derived from human induced pluripotent stem cells (hiPSCs).   Now, in a report in Stem Cells, researchers from the group of Zaal Kokaiaat the Lund Stem Cell Center, Swedenhave transplanted long-term self-renewing neuroepithelial-like stem cells, generated from adult human fibroblast-derived iPSCs (hiPS-It-NES), into the stroke-damaged mouse and rat striatum or cortex and show evidence that this is a safe and efficient approach to promote recovery after stroke and can be used to supply the injured brain with new neurons for replacement (Oki, Tatarishvili, Wood,Koch et al).

hiPSC lines were produced by standard retroviral transduction as reported previously (Falk et al),and the hiPSC line selected for use exhibited all expected signs of pluripotency.   Using a previously established protocol (Koch et al), an hiPS-It-NES cell population was established which could be grown long-term in vitro under proliferative conditions without losing stem cell characteristics or differentiation potential, expressed the NSC markers SOX2 and NESTIN, assembled into rosette-like patterns and expressed associated markers PLZF, DACH1 and ZO-1, and could differentiate into neurons and astrocytes following growth factor withdrawal.

Middle cerebral artery occlusion (MCAO) was performed to mimic stroke by inducing a lesion in the dorsolateral aspect of the striatum.   Injection of GFP-marked hiPS-It-NES cells or vehicle-control cells into the striatum of mice after MCAO demonstrated that mice injected with hiPS-It-NES cells did not exhibit any deficits in fine forelimb movement at any time point and performed significantly better than vehicle-treated animals which showed significant impairment in the ability of both grasping and eating food pellets.   However, cell transplantation did not reverse a deficit detected in the corridor test, used to assess sensorimotor impairment caused by striatal damage.   MCAO caused a lesion in the dorsolateral part of the striatum in all mice, and of the surviving grafts (observed in 7 of 12 mice), human cells (HuNu⁺) were mainly found in the striatum, with a few HuNu⁺ cells found in the cerebral cortex and corpus callosum.   The grafts contained around 10% of the initially injected cells (HuNu⁺) and at 10 weeks after transplantation only around 2% of HuNu⁺ cells were proliferative and positive for Ki67.   At 10 weeks, grafts had not survived in 5 out of 12 animals and the volume of the striatum was similar between cell-injected and vehicle-injected mice.   Interestingly, comparison of transplanted mice with and without surviving grafts at this time point showed no significant difference in fine limb control suggesting that the long-term survival of the hiPS-It-NES cells is not required for the functional recovery observed.   At this point, the majority of surviving grafted cells expressed HuD, a marker for young and mature neurons, and these cells (HuNu⁺/HuD⁺) were located in the centre of the graft.   Injection of the retrograde tracer Fluorogold into the globus pallidus (GP), which is the main projection area for striatal medium-sized spiny neurons, ipsilateral to the graft demonstrated that neuronal-type cells derived from the grafted hiPS-It-NES cells projected axons.

The finding that graft long term survival was not required for functional recovery led to the exploration of other mechanisms.   Analysis after 1 week of hiPS-It-NES cell or vehicle transplantation after stroke revealed no differences in the area of cells in the neurogenic subventricular zone which were DCX⁺ (neuroblasts marker) and BrdU⁺ and no evidence of increased activated microglia (Iba1⁺/ED1⁺) or astrocytes (GFAP⁺) in the striatum.   However, it did reveal a larger VEGF⁺ area in mice with hiPS-It-NES cell injections as compared to the vehicle control, and this was confined to astrocytes and blood vessels, which correlates to a previous study which suggested that VEGF secretion by foetal NSCs can improve behavioural response after stroke (Horie et al).   While GFAP⁺ astrocytes were similarly positive between both hiPS-It-NES cell and vehicle transplants, the area of VEGF⁺ astrocytes after hiPS-It-NES cell transplantation was more than two-fold higher as compared to vehicle injected animals.   CD31⁺ blood vessels expressed significantly less VEGF than GFAP⁺ astrocytes (10%) and no differences were apparent between the hiPS-It-NES cell and vehicle injected animals.   Of further interest, hiPS-It-NES cells themselves were also noted to be VEGF⁺.   To understand if VEGF had led to increased vasculogenesis at later time points (9 weeks, where behavioural improvement was obvious), animals were sacrificed and analysed but, interestingly, no differences in vessel length density of CD31⁺ was observed, suggesting that VEGF has another role.

Long term analysis of hiPS-It-NES cells after stroke was next analysed in nude, T-cell-deficient adult rats in which xenotransplanted human cells can survive long-term without immunosuppression.   At 2 and 4 months, grafted cells numbers were ~65% and ~51% of total cells implanted respectively, with proliferative activity (as measured by Ki67 expressing HuNu+ cells) at ~40% at 2 weeks after transplantation but dropping to ~8% and ~1% at 2 and 4 months.   At 2 months, most cells in the graft were DCX⁺ (neuroblasts), and ~66% of cells were GFP⁺NeuN⁺ suggestive of a human neuronal fate, with minimal GFAP⁺ astrocytes (~4%).   By 4 months only a few DCX⁺ cells remained but GFP⁺NeuN⁺ cells remained at a high level (~73%) and GFAP⁺ astrocytes remained at a low level (~6%).   Morphological analysis of the grafted cells and their progeny using GFP and the human cytoplasmic marker SC121 demonstrated that grafted cells had clear morphological characteristics of migrating neuroblasts, mature neurons, or astrocytes but these were more easily detectable in the areas outside the graft core.    It was also noted that GFP⁺ cells with typical neuronal morphology were often not NeuN⁺.   Additional analysis demonstrated GABA⁺ cells were present in the grafted population (~2% and 11% at 2 and 4 months, respectively) while at 4 months a small proportion of HuNu⁺ cells that co-expressed the medium-sized spiny striatal projection neuron marker, DARPP32 was also evident.

Next, transplantation of the hiPS-It-NES cells into the cortex of animals subjected to occlusion of the distal branch of MCA (dMCAO) was analysed.   Cell survival at 2 and 4 months was ~80% and ~60% and the number of cells with proliferative activity was similar to the intrastriatal transplants.   GPF⁺ cells express NeuN (~72%), while ~16% of HuNu⁺ cells were GABA⁺ at 2 months, while GFP and SC121 staining suggested neuronal morphologies.   At 4 months, GFP⁺NeuN⁺ cells remained high (~78%), while GABA⁺ cells decreased (~9%) and the percentage of HuNu⁺DARPP-32⁺ cells was similar to that observed for the intrastriatal grafts.

Finally, the electrophysiological properties of cells generated from the hiPS-It-NES transplanted cells in the striatum or cerebral cortex were analysed by whole-cell patch-clamp recordings in acute brain slice preparations.   A proportion of the cells were found to be functionally mature neurons according to their extraphysiological properties and the majority generated action potentials in response to depolarizing current injection, while spontaneous postsynaptic currents were frequently observed in grafted cells in the striatum.   Overall, extraphysiological data suggested that transplanted neurons generated from iPSC-lt-NES cells receive synaptic input from host neurons and functionally integrate into host brain neural circuitries.

Overall, this data suggests that hiPSC-derived cells are useful for the treatment of stroke; after implantation they generate neurons with mature morphological and electrophysiological properties, send axonal projections throughout the host brain, receive synaptic input from surrounding host neurons, and improve motor recovery in behavioural tests relevant for human stroke.   It also highlights the role for VEGF which the authors suggest allows early and long-lasting behavioural improvement by influencing brain plasticity in the postischemic phase of stroke (Hermann and Zechariah), rather than influencing blood vessel length or density.

References

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