Enhanced proliferation and neuronal differentiation of adult brain

stroke model of Nestin-GFP mice

Adult Nestin-GFP Transgenic mice (3-5 months) were subjected to 60 minutes transient right MCAo by the intraluminal suture method. Animals with similar behavior scores on day 1 were randomly assigned to 5 groups: a vehicle PBS control, MSC, MSC/Ngn1, MSC+HGF and MSC/Ngn1+HGF ( n=3-4 Per group). 28 days after MCAo, animals were stereotactically transplanted with 3x10⁵ cells diluted in 6ul PBS into the right striatum. As a vehicle control, 6ul of PBS was also injected using Hamilton Syringe. For the detection of proliferating cells after transplantation, a thymidine analog, BrdU was intraperitoneally(50mg/kg) injected from day 31 to 36 after MCAo. On day 36 after MCAo, animals were sacrificed by trans-cardiac perfusion. Brain samples were processed in paraffin and 5 micrometer thick coronal sections at the level of the striatum were used for immunohistochemical analysis. GFP and DCx immunohistochemistry reveals the increased number of GFP+ neural stem cells and DCx+ neuroblasts were significantly higher in the striatum of MSC/Ngn1+HGF transplanted chronic stroke model of Nestin-GFP ( Fig 24 A-C). Further to confirm where this increase in GFP and DCx Positive cells is due to the increased proliferation of Nestin-GFP+ neuroblasts and DCx+

neuroblasts, double immunohistochemical analysis against GFP and BrdU or

GFP and DCx was performed. GFP and Brdu immunostaining reveal the increased number of GFP and BrdU double-positive cells in the striatum of MSC/Ngn1+HGF cell treated chronic stroke model of Nestin-GFP mice compared to other treatment groups (Fig 25 A-D). No significant difference in the number of GFP and BrdU double-positive cells was observed in the cortex of MSC/Ngn1+HGF treated animals compared to other groups. Arrows indicate the cells positive for both GFP and BrdU (25 B). We further characterized these GFP and BrdU double-positive cells increased by MSC/Ngn1+HGF cells in chronic stroke striatum by immunohistochemical method. Double immunofluorescence images of GFP & DCx, GFP & GFAP and GFP & NG2 immunostaining from the striatum of MSC/Ngn1+HGF treated chronic stroke Nestin-GFP mice depicts that these Nestin-GFP+ cells start to express neuroblast marker DCx while losing the expression of NG2 in them. None of the Nestin-GFP cells expressed astroglial marker GFAP. (Fig 27) Similarly, the quantification of DCx and BrdU double-positive cells showed that MSC/Ngn1+HGF cells significantly increased the number of proliferating neuroblasts in the striatum of chronic stroke animals (Fig 26).

Fig. 24. MSC/Ngn1+HGF treatment during chronic stroke increases Neural progenitor cells in the ipsilateral striatum (A) Schematic diagram showing the experimental scheme in chronic stroke Nestin-GFP mice. (B) Light microscopy Images of Nestin-GFP+ neural progenitors as detected by GFP immunostaining in the striatum of chronic stroke models after the treatment of PBS, MSC, MSC/Ngn1, MSC+HGF or MSC/Ngn1+HGF cells. (C) Light microscopy Images of neuroblasts as detected by Doublecortin (DCx) immunostaining in the striatum of chronic stroke models after the cell treatment.

Fig 25. MSC/Ngn1+HGF cell treatment during chronic stroke increases the proliferation of Nestin-GFP progenitors cells in the ipsilateral striatum. A.

Schematic diagram of experimental design. B.GFP and Brdu immunostaining reveals the increased neurogenesis in the striatum of MSC/Ngn1+HGF cell treated chronic stroke model of Nestin-GFP mice compared to other treatment groups. Arrows indicate the cells positive for both GFP and BrdU. (C, D) Total number of GFP and BrdU double positive cells detected per mm² striatal and cortical area in the brain of MSC/Ngn1+HGF, MSC+HGF, MSC/Ngn1, MSC and PBS treated chronic stroke model of Nestin-GFP mice model ( n=3-4 animals per group, Data are means ± SEM; *p<0.01vs. PBS; # P<0.01 vs MSC ;

$ P<0.01 vs MSC/Ngn1 ; ! P<0.01 vs MSC+HGF cells.

Fig 26. MSC/Ngn1+HGF cell treatment during chronic stroke increases the proliferation of DCx+ neuroblasts in the ipsilateral striatum (A) DCx and Brdu immunostaining reveal the increased number of DCx and BrdU double-positive cells in the striatum of MSC/Ngn1+HGF cell treated chronic stroke model of Nestin-GFP mice compared to other treatment groups. Arrows indicate the cells positive for both DCx and BrdU. (B). Low magnification image showing the region of interest (striatum) for quantification analysis (C) Total number of DCx and BrdU double-positive cells detected per 200X high power field in the striatal area in the brain of MSC/Ngn1+HGF, MSC+HGF, MSC/Ngn1, MSC and PBS treated chronic stroke model of Nestin-GFP mice model (n=3-4 animals per group, Data are means ± SEM; *p<0.05; # P<0.01

Fig 27. MSC/Ngn1+HGF cells enhance the neuronal differentiation of Nestin-GFP+ cells in the ipsilateral striatum. A. Double immunofluorescence image showing Nestin-GFP signal is completely overlappd with NG2 expression in normal adult Nestin-GFP mice. B. Low magnification image of GFP&DCx and GFP&GFAP double immuno-staining showing the region of interest for characterizing Nestin-GFP+ cells in the brain of MSC/Ngn1+HGF treated chronic stroke model of Nestin-GFP mouse model. C. Double-immunofluorescence images from the striatum of MSC/Ngn1+HGF treated chronic stroke Nestin-GFP mice reveal that striatal Nestin-GFP+ cells start to express neuroblast marker DCx while losing the expression of NG2 in them.

None of the Nestin-GFP cells expressed astroglial marker GFAP. These DCx+

cells are proliferating in nature as depicted by BrdU staining.

Part E: NG2CreERTM::RosaFloxedTdTomato double transgenic reporter Mice as a Model System to study parenchymal neurogenesis from NG2 cells in chronic stroke

1. Characteristics of NG2 positive progenitor cells in the parenchyma/non-canonical niche of the normal brain.

To explore the role of brain parenchyma (Striatal and cortical) derived NG2+ progenitors in the post-stroke neurogenesis and behavior recovery observed in MSC/Ngn1+HGF treated chronic stroke brain, we utilized NG2-CreERTM::Rosa-Floxed-TdTomato double transgenic (Fig 29A,B) and Nestin-GFP:NG2-CreERTM: Rosa-Floxed-TdTomato triple transgenic mice (Fig 28 A,B) for chronic stroke modelling. Administration of tamoxifen in normal adult (P30) NG2-CreERTM::Rosa-Floxed-TdTomato double transgenic mice drove TdTomato expression ubiquitously in the brain (Fig 29 B). Double immunofluorescence analysis revealed that most of the TdTomato+ cells in the cortex and striatum were positive for expression of NG2 and Olig2, classical markers of oligodendrocyte progenitor cells (Fig.29 C-F). The Cre recombination efficiency in NG2 and Olig2+ cells was almost 60 % in our P30 mice after 5 days of tamoxifen injection (Fig 29 C, D). Similarly, In Nestin-GFP:NG2-CreERTM: Rosa-Floxed-TdTomato triple transgenic mice, TdTomato and GFP expression were colocalized in NG2 cells in the brain parenchyma (Fig 28 D). We observed that most of the Nestin-GFP+ cells in the striatum and cortex were positive for TdTomato but were negative for the

expression of GFAP, a classical marker of the type B SVZ NSCs. Interestingly, TdTomato expression was not seen in the canonical neural stem cell niches, i.e., SVZ and SGZ. Nestin-GFP cells in these canonical niches expressed GFAP being negative for TdTomato expression (Fig 29 D).

Fig 28. NG2-Cre targets Nestin-GFP cells in Non-canonical stem cell niche, i.e, cortex and striatum. A. Schematic representation of transgenically targeted fate-mapping strategy to generate Nestin-GFP:NG2-TdTomato double reporter mice. NG2-CreERTM mice were crossed with Rosa-TdTomato mice and offspring were further crossed with GFP mice to obtain

Nestin-GFP:NG2-TdTomato double reporter mice. B. Experimental scheme. C.

Fluorescence image showing TdTomato and GFP expression in the sagittal brain section of Postnatal day 30 NG2-TdTomato mice after injection of tamoxifen. D.

Distribution of TdTomato and GFP expression in the canonical NSC niche (SVZ and subgranular zone of dentate gyrus) and Non-canonical NSC niche (Cortex and Striatum). GFAP immunostaining was performed to distinguish Type B classical NSCs expressing Nestin-GFP from Non-classical (Brain parenchymal) Nestin-GFP+/NG2+/GFAP- NSCs. (white arrows: GFP positive cells colocalized with Type B NSC marker, GFAP. Red arrows: GFP positive cells colocalized with NG2-TdTomato in cortex and striatum)

Fig 29. Cre recombination efficiency in Double transgenic mice. A. NG2-CreERTM mice were crossed with Rosa-TdTomato mice and offspring were injected with Tamoxifen for 5 days to obtain NG2-TdTomato reporter mice. B.

Fluorescence image showing TdTomato expression in the sagittal brain section of Postnatal day 30 NG2-TdTomato mice after injection of tamoxifen for 5 consecutive days. C, D. Quantification of Percentage of TdTomato cells per NG2 and Olig2+ cells in cortex and striatum. Distribution of TdTomato

expression and NG2 immunoreactivity in the cortex and striatum respectively. E, F. Distribution of TdTomato and Olig2 expression in cortex and striatum respectively. The percentage of NG2 and Olig2 immunoreactive cells co-expressing TdTomato reporter protein in brain parenchyma is shown in G and H respectively.

2. In-vivo differentiation of NG2-TdTomato cells in adult mouse brain parenchyma.

We then analyzed the temporal fate map analysis of TdTomato+ cells in P30 NG2-CreERTM::Rosa-Floxed-TdTomato double transgenic mice after 5 days of tamoxifen injection and sacrificed 1 week later (Fig 30A). We analyzed whether these TdTomato+ cells give rise to astrocytes, neurons, and oligodendrocytes in cortex and striatum of normal adult mice. We found no evidence that NG2-TdTomato cells could give rise to GFAP+ astrocytes and NeuN+ neuronal cells in these locations. However, most of the TdTomato cells co-expressed the oligodendrocyte marker APC1 in both cortex and striatum (Fig 30 B). These results suggest that NG2 cells in normal adult mouse brain give to oligodendrocytes but not neurons and astrocytes at least in the dorsal cortex and striatum.

Fig 30. NG2 Progenitors Give Rise to Oligodendrocytes in adult brain Cortex and striatum. A. Experimental scheme to show the in-vivo differentiation of NG2 cells in NG2-TdTomato reporter mice. B. Immuno-staining against the markers of astrocyte (GFAP), Neuron (NeuN) and Oligodendrocyte (APC1) along with TdTomato expression depicts NG2 progenitors do not give rise to neurons and astrocytes but Oligodendrocytes (white arrows) in cortex and striatum.

3. Characterization of NG2-TdTomato cells in vitro.

To verify whether NG2 progenitors can act as multipotent stem cells in-vitro, we isolated TdTomato cells from P30 NG2CreERTM-TdTomato reporter mice after injection of tamoxifen for 5 days. Mice were sacrificed and the brain was carefully isolated. The brain was dissociated with Milteyni Biotech’s Adult brain dissociation kit to obtain a single-cell suspension. Cells were plated in the clonal density in the presence of bFGF and EGF. After 7-10 days in culture. NG2-TdTomato cells formed primary neurospheres which upon dissociation and replating gave rise to secondary neurospheres, which could then be re-passaged several times and frozen, indicating self-renewal (Fig 31). The NG2-TdTomato cells in Secondary and tertiary neurospheres expressed progenitor cell markers Nestin, NG2 and Olig2 showing that these cells act as progenitor cells in-vitro as well (Fig 32).

Fig 31. NG2-TdTomato form neurospheres in-vitro. (A) Adult (P30+5) NG2-TdTomato reporter mice were sacrificed and the brain was carefully isolated. The brain was dissociated with Milteyni Biotech’s Adult brain dissociation kit to obtain single cells. Cells were plated in the clonal density in the presence of bFGF and EGF. B. NG2-TdTomato cells formed primary neurospheres within 10 days in vitro. C. Dissociation of neurospheres into single cells and replating gave rise to secondary neurospheres, which could then be repassage several times and frozen, indicating self-renewal.

Fig 32. NG2-TdTomato cells consistently express progenitor markers in-vitro. Secondary and tertiary neurospheres were dissociated into single cells and plated to characterize by immunocytochemical analysis. Immunostaining against progenitor cell markers Nestin, NG2 and Olig2 showed that TdTomato cells were positive for the expression of these progenitor cells specific proteins (white arrowheads).

Part F: Role of conditioned media from HGF overexpressing MSCs and MSC/Ngn1 cells in in-vitro model of Neurogenesis and angiogenesis.

1. Effects of Conditioned media from HGF overexpressing MSCs on proliferation and neuronal differentiation of NG2 cells.

To further examine the pro-neurogenic effects of HGF in adult mouse brain-derived NG2 progenitors, neurosphere formation assay was carried using TdTomato cells obtained from P30 NG2CreERTM-TdTomato mice after 5 days of tamoxifen injection. Secondary or tertiary neurospheres were dissociated and cultured in the presence of Serum-Free conditioned medium (CM) obtained from the culture of MSC, MSC/Ngn1, MSC+HGF, and MSC/Ngn1+HGF cell culture. Non-conditioned serum-free media was used as a control. Cells were analyzed for the expression of Ki67, a marker of cell proliferation after 16 hours by immunocytochemistry. Compared to control, CM from MSC and MSC/Ngn1 significantly increased the number of Ki67 positive NG2 cells. Interestingly this effect was further enhanced by conditioned media from HGF overexpressing cells (Fig 33 A, B). Similarly, when we cultured NG2-TdTomato cells in the presence of 10% serum-containing CM from HGF overexpressing MSC and MSC/Ngn1 cells, the neuronal differentiation of these NG2-TdTomato cells was greatly enhanced. The Tuj1 and TdTomato double Positive cells were frequently observed in CM from HGF overexpressing cells (Fig 34 A-F). The Tuj1 Fluorescence intensity was also significantly higher in Conditioned media from HGF overexpressing MSC and MSC/Ngn1 cells (Fig 34 G).

Fig 33. Conditioned media from HGF over-expressing MSC and MSC/Ngn1 cells enhance the proliferation of NG2-TdTomato cells in vitro. Secondary and tertiary neurospheres were dissociated into single cells and plated in PDL

coated cover glasses. NG2-TdTomato cells were incubated for 16 hours in Conditioned media (in Serum-free media) and were fixed with formalin for assessment of proliferation by Ki67 immunocytochemistry. Fresh MSC culture media was used as a control. A. Ki67 immunocytochemical analysis depicts that the MSC conditioned media significantly increased the number of Ki67/TdTomato cells compared to control. Conditioned media from HGF overexpressing MSCs and MSC/Ngn1 cells gave a higher number of Ki67/TdTomato cells than MSCs and MSC/Ngn1 cells. B. Quantification of Ki67 cells per 200X field in different experimental groups.

Fig 34. Conditioned media from HGF over-expressing MSC and MSC/Ngn1 cells enhances the neuronal differentiation of NG2-TdTomato cells in vitro. Secondary and tertiary neurospheres were dissociated into single

cells and plated in PDL coated cover glasses. Conditioned media (in 10%

serum-containing media) from MSC, MSC/Ngn1, MSC+HGF, and MSC/Ngn1+HGF cell culture was obtained 48 hours of confluent culture. NG2-TdTomato cells were incubated for 3 days in conditioned media and were fixed with formalin for assessment of Tuj immunocytochemistry. Fresh MSC culture media was used as a control. A. Low magnification immunofluorescence image showing Tuj1 immunoreactivity in MSC/Ngn1+HGF conditioned media treated NG2-TdTomato cells (B-D). Tuj1 immunocytochemical analysis depicts that the MSC conditioned media significantly increased the number of Tuj1/TdTomato cells compared to control. Conditioned media from HGF overexpressing MSCs and MSC/Ngn1 cells gave a higher number of Tuj1/TdTomato cells than MSCs and MSC/Ngn1 cells suggesting enhancement of neuronal differentiation of NG2-TdTomato cells in-vitro. B. Quantification of average Tuj1 fluorescence intensity cells per mm² Region of interest in different experimental groups.

2. Effects of conditioned media from HGF overexpressing MSCs on in-vitro angiogenesis of bEND.3 cells.

The conditioned media obtained from HGF overexpressing MSC/Ngn1 and MSC cells stimulated tube forming ability of bEND.3 cells in a system of in-vitro angiogenesis. Total tube length and total mesh area were significantly higher in the HGF over-expressing cell groups than their naïve counterparts (Fig 35). However compared to control, conditioned media from MSCs and MSC/Ngn1 cells also enhanced the tube forming ability of bEND.3 cells (Fig 35 B, C).

Fig 35: HGF over-expressing MSC and MSC/Ngn1 cells enhance in-vitro angiogenesis (A) Light microscopy images showing in-vitro tube formation result of bEND.3 cells in the presence of conditioned media from MSC, MSC/Ngn1, MSC+HGF, and MSC/Ngn1+HGF cell culture. (B) HGF over-expressing MSC and MSC/Ngn1 cell-conditioned media increased the total mesh area (B) and tube length (C) in invitro angiogenesis assay. (*, p<0.05 Vs -ve; #, P<0.05 Vs M; $, P<0.05 Vs MN, One-way anova)

DISCUSSION & CONCLUSION

In Preclinical animal studies, Stem cell therapy has been proven to be a potential therapeutic option mostly in the acute phase of the stroke. stem cell transplantation is shown to be a safe and effective therapy for stroke. Despite extensive pre-clinical and clinical studies in the stroke arena, very little progress has been achieved to ameliorate the persisting behavioral impairments in the chronic phase of the stroke. Thus, National Institutes of Health (NIH) and FDA representatives formulated research guidelines called Stem Cells as an Emerging Paradigm in Stroke III (STEPS III) which recommends researchers to focus on more advanced stages of clinical testing, as well as the testing of cell therapies in a broader stroke population including chronic stroke.

Our previous study (Kim, Yoo et al. 2008) proposed the scientific basis for a requirement of neural induction of MSCs by overexpressing Neurogenin 1 for better treatment of neurological dysfunction in acute stroke. We tested whether Neurogenin 1 overexpressing MSCs cells exert similar therapeutic effects in the chronic phase of stroke, i.e when the cells are transplanted 1 month after the ischemic stroke. Our findings show that overexpression of Neurogenin1, Ngn1 alone in mesenchymal stem cells is not enough to achieve therapeutic benefits in chronic stroke. However, Concomitant overexpression of Hepatocyte growth factor, HGF, and Ngn1 in MSCs improved behavior functions and brain tissue integrity in chronic stroke brain. This therapeutic effect was associated

with the augmentation of neuro-reparative mechanisms like angiogenesis, neurogenesis, and anti-fibrosis in the injured brain. The spatiotemporal analysis showed that in the chronic phase of ischemic stroke, there is the presence of growth inhibitory glial scar forming a fine border between ischemic core and normal viable brain tissue. Double immune-histochemical studies showed that GFAP-positive reactive astrocytes in the glial scar expressed a high amount of extracellular matrix-like CSPGs. GFAP and ED1 positive cells along with extracellular matrix molecules formed a layer around the ischemic lesion, suggesting the presence of inhibitory glial scar in the chronic stroke brain. The glial scar is positioned in such a way that it forms a border around the injury site and acts as a neuroprotective barrier to evading inflammatory cells in the acute inflammatory phase. However, scar formation has been shown to inhibitory for the long term behavior recovery in the chronic phase of stroke (Hettiaratchi, O'Meara et al. 2019). Reactive Astro-gliosis along with CSPGs has been shown to inhibit neuroplasticity by inhibiting axonal outgrowth and cellular migration by secreting non-permissive molecules and exerting a physical barrier for the migration of neural progenitors towards the ischemic core (Siebert, Conta Steencken et al. 2014). Studies have shown that the dissolution of the glial scar with chondroitinase ABC enhances the behavior recovery and neuroplasticity in chronic stroke brain(Hill, Jin et al. 2012, Soleman, Yip et al. 2012, Gherardini, Gennaro et al. 2015). So, in our study, the inefficiency of MSC/Ngn1 in chronic

stroke treatment might have arisen from this complex nature of the chronic stroke.

Shimamura et al. reported that intracisternal gene transfer of the HGF gene alone in the chronic stroke rat model enhanced the learning and memory

Shimamura et al. reported that intracisternal gene transfer of the HGF gene alone in the chronic stroke rat model enhanced the learning and memory