Cell surface marker expression and multilineage differentiation of Adeno-

To evaluate the effects of adenoviral transduction on the MSC phenotype, cell surface marker expression was examined. As shown in Fig. 13, Adeno-HGF transduced MSCs and MSC/Ngn1 cells displayed similar phenotype profiles. Ad-HGF transduced MSCs and MSC/Ngn1 cells were positive for CD105 and CD90 while being negative for HLA-DR, CD34, and CD45 at similar proportion to Adeno-HGF non-transduced cells. Similar to our previous study, MSCs expressing Ngn1 did not differentiate into osteo and adipogenic lineage. However, Adeno-HGF transduction did not alter the osteogenic differentiation of MSCs (Fig 14). Both osteocytic cultures of MSC and MSC+HGF cells exhibited mineralization nodules over the MSC monolayer that stained positively with Alizarin S, confirming successful osteocyte differentiation (Figs. 14A). Similarly, to the results from Adeno-GFP transduced MSCs the majority of Adeno-HGF transduced MSCs lacked the capacity for adipogenic differentiation as shown by the absence of lipid vacuoles, which were readily apparent in untransduced counterparts (Fig 14B).

Fig 13. Surface antigen expression profiles of Adeo-HGF transduced MSCs and MSC/Ngn1 cells. MSCs and MSC/Ngn1 cells transduced with HGF-expressing adenovirus were examined for MSC specific surface marker expression at 2 days post-transduction. Isotype antibodies served as a negative control.

Fig. 14. Mesodermal differentiation potential of Ad-HGF transduced MSCs and MSC/Ngn1 cells. (A) Osteogenic differentiation was assessed by alizarin red S staining to visualize calcium deposits (B) Adipogenesis was monitored for intracellular lipid accumulation by oil-red O staining. Negative control included cells growth in non-differentiation media. i.e., Fresh MSC growth media.

Part C: Therapeutic effects of HGF overexpressing MSC/Ngn1 cells in chronic stroke.

1. Improved functional recovery and tissue integrity with MSCs/Ngn1+HGF

Data described above (Fig. 6) demonstrated that both MSC/Ngn1 or MSC cells did not exert therapeutic benefits in the chronic phase of ischemic injury. Thus, we next investigated whether over-expression of HGF in MSC or MSC/Ngn1 cells could improve behavioral functions in the chronic stroke brain.

All the ischemic animals recovered spontaneously in behavior from day 2 to day 30 when animals had stable residual behavior. There was no significant difference in the rota-rod performance and adhesive removal time among/between all the groups at 1 month after ischemic injury. At 1 week to 8 weeks after cell transplantation, no significant differences in behavior functions were detected in MSC and MSC/Ngn1 treated groups compared to PBS treated group. Over-expression of HGF in MSC/Ngn1 cells was associated with improvement in behavioral deficits as assessed by Adhesive removal test and Rotarod test from 2 weeks of cell transplantation and was sustained at least until 8 weeks after transplantation (Fig 15 A, B). This improvement of behavior in HGF over-expressing MSC/ngn1 cells transplanted group was associated with a reduction in infarct volume as assessed by MRI (Fig 16 A, B). However, Over-expression of HGF alone in MSC cells was not associated with enhanced behavior recovery and reduction of infarct volume.

Fig 15. Enhanced functional recovery in the chronic stroke animals treated with MSC/Ngn1+HGF cells. Sensorimotor recovery was assessed by Adhesive removal (A) and rotarod (B) test starting from day 1 until 12 weeks after ischemia. Treatment with MSC/Ngn1+HGF significantly reduced the time to remove the adhesive patch and increased the duration of stay in the rotarod drum starting from 2 weeks of transplantation (6 weeks). The data were collected from 5 animals per group and presented as mean±SD. (*,p<0.05; **, p<0.01 compared to PBS control)

Contributed by Dr. YSW

Fig 16. Improved brain tissue integrity of chronic stroke brain by MSC/Ngn1+HGF cells. The brain tissue integrity of each animal was assessed by magnetic resonance imaging (MRI) starting from the week of cell transplantation (4 weeks) till 12 weeks (A) and results are summarized as mean relative infarct volume±SD (B). Statistically significant differences between the groups were determined by ANOVA. (*, p< 0.5 compared to PBS control)

Contributed by Dr. YSW

2. Augmentation of neuro-regenerative mechanisms by MSC/Ngn1+HGF cells in chronic stroke.

We next investigated whether transplantation of MSC/Ngn1+HGF cells into chronic stroke brain could improve vascular remodeling. Compared with control groups, animals treated with MSC/Ngn1+HGF cells showed a significant increase in the number of lectin+ microvessels in peri-infarct regions of striatum and cortex. The vascular density defined by the number of lectin+

vessels per 0.05 mm² ROI was increased by more than twofold in MSC/Ngn1+HGF treated animals compared to PBS treatment (Fig 17 A-C).

Studies have shown that glial scarring impedes axonal regeneration, and that transected axons do not project beyond the borders of glial scar barriers.

Therefore, reduced glial scar formation is thought to have a stimulatory effect on axonal regeneration and functional recovery. At 8 weeks post-transplantation, GFAP immunoreactivity signal- a surrogate marker of astrocyte reactivity was significantly reduced in the peri-infarct region of MSC/Ngn1+HGF treated chronic stroke rat brain (Fig 18 A-C)) compared to control groups. To assess the state of ongoing inflammation after stem cell treatment we analyzed the IBA1 intensity in the ischemic brain. Stem cell treatment greatly reduced IBA1 fluorescence intensity but there was no significant difference between cell types suggesting all cells exerted similar immunosuppressive effects in chronic stroke (Fig 19 A-C). As there was a similar level of immune-suppressive effect among

MSC, MSC/Ngn1, MSC+HGF, and MSC/Ngn1+HGF cells, we analyzed the expression of transforming growth factor-β, TGF- β which is a key player of immune suppression after Mesenchymal stem cell transplantation. qRT-PCR analysis showed that the expression of TGF- β was similar between all the cells (Fig 20).

Fig. 17. Enhanced angiogenesis by MSCs and MSC/Ngn1 cells overexpressing HGF. A. Representative photographs of Lectin immunostaining 8 weeks after cell transplantation B. Low magnification lectin immunofluorescence image showing cortical and striatal ischemic penumbra region used for quantification of lectin +ve vascular structures. Quantification of vascular density is illustrated in C. Results from four animals per group are presented as mean± S.E. Statistically significant differences between the groups were determined by ANOVA.

Fig 18. Reduced GFAP immunoreactivity in chronic stroke brain by MSC/Ngn1 cells overexpressing HGF. A. Representative photographs of GFAP immunostaining 8 weeks after cell transplantation B. Low magnification lectin immunofluorescence image showing cortical and striatal ischemic penumbra region used for quantification of GFAP immunofluorescence intensity.

Quantification of GFAP Fluorescence intensity is illustrated in C. Results from four animals per group are presented as mean± S.E. Statistically significant differences between the groups were determined by ANOVA.

Fig 19. Reduced IBA1 immunoreactivity in chronic stroke brain by Mesenchymal stem cells. A. Representative photographs of IBA1 and ED1 immunostaining 8 weeks after cell transplantation. Quantification of IBA1 and ED1 immunoreactivity from the ischemic penumbra region is illustrated in B and C respectively. Results from four animals per group are presented as mean±

S.E. Statistically significant differences between the groups were determined by ANOVA.

Fig 20. Expression of TGF-β, a key player of immune suppression by HGF overexpressing MSC and MSC/Ngn1 cells. A. Expression of TGF-β in MSC, MSC/Ngn1, MSC+HGF, and MSC/Ngn1+HGF cells was verified by real-time PCR analysis. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control. B. Quantitative assessment of TGF-β mRNA expression in naïve and gene-modified MSCs.

3. Neuronal differentiation of MSC/Ngn1+HGF cells in chronic stroke.

We previously showed that the functional recovery in acute stroke model animals with MSC/Ngn1 cells was due, at least in part, to neuronal differentiation of the transplanted cells. Transplanted MSC/Ngn1 cells were differentiated into neuronal cells and were functionally connected to host neural networks. In this study, we assessed whether HGF overexpression alters the neuronal differentiation ability of MSC/Ngn1 cells. Human mitochondrial antigen (hMT) was used to detect transplanted human cells. We found the highest number of hMT positive cells in the peri-infarct striatum of MSC/Ngn1+HGF treated chronic stroke animals compared to other groups (Fig 21 A). Further to analyze the differentiation of these MSC/Ngn1+HGF cells, double immunofluorescence analysis against hMT and neuronal marker NeuN or hMT and astrocytic marker, GFAP was done (Fig 21 B). These hMT positive MSC/Ngn1+HGF cells mostly expressed neuronal marker NeuN. These cells never expressed astrocytic marker, GFAP suggesting that Ngn1 promotes MSC to differentiate into neuronal cells in vivo but not astroglial cells.

Fig 21.Trans-differentiation of grafted MSC/Ngn1+HGF into neuronal cells.

A. Human mitochondrial antigen (hMt) is utilized to detect bone marrow-derived MSCs. B) Representative histological images. Among grafted cells, MSC/Ngn1+HGF cells remain at the highest level in the peri-infarct area, where they differentiate into NeuN+ cells (filled arrowheads). These cells never express GFAP (an astrocyte marker), suggesting that Ngn1 promotes MSC to differentiate neuronal cells in vivo (trans-differentiation) but not astroglia cells.

Note that hMT-positive cells are only detected in the transplanted side (ipsilateral) but not in the contralateral side of the brain, suggesting the specificity of the assay.

Part D. Chronic stroke model of Nestin-GFP transgenic mice as a tool for studying non-canonical neurogenesis.

1. Characterization of Nestin-GFP cells in adult mouse brain.

In order to study the effects of MSC/Ngn1+HGF cell transplantation in post-stroke neurogenesis in chronic stroke, we took advantage of Nestin-GFP transgenic mice which express Green fluorescence protein (GFP) under the control of the second intron of the nestin promoter. This construct limits GFP expression to neural precursors of the mice although nestin protein is also expressed in muscle precursors. As shown in Figure 22 A, GFP fluorescence from a 2 mm thick coronal section of the freshly dissected brain of P90 Nestin-GFP mice is mostly localized to the sub-ventricular region (SVZ). However, weak GFP fluorescence can be observed in all other parts of the brain including cortex and striatum. The immunohistochemical analysis shows that Nestin-GFP+ cells in both cortex and striatum were different from Nestin-Nestin-GFP+ in SVZ and subgranular zone of the dentate gyrus in terms of their molecular expression.

Nestin-GFP+ cells in SVZ and subgranular zone were positively stained with anti-GFAP antibody representing them as classical type B NSCs (Fig. 22 D, E).

In contrast, Nestin-GFP+ cells in cortex and striatum were negatively stained with anti-GFAP antibody. However, these parenchymal Nestin-GFP+ cells were exclusively positive for CSPG4 (NG2) proteoglycan and Olig2 which is the marker for Oligodendrocyte progenitor cells (Fig 22 B, C). We also observed

that these cortical and striatal Nestin-GFP/NG2 cells express proliferation marker BrdU suggesting their progenitor character (Fig 22 B,C). Results from 9, 5um coronal brain sections from 3 adult Nestin GFP+ mouse strongly indicate that the majority of Nestin-GFP+ cells in neocortex and striatum are a subpopulation of NG2+ progenitors. Based on these data, we can conclude that the Nestin-GFP transgene reveals the majority of NG2+ progenitors in the non-classical NSC niche in cortex and striatum.

Fig 22. Nestin-GFP transgene reveals NG2+ oligodendrocyte progenitor cells (OPCs) in non-Canonical NSC niche of adult mouse brain. (A) 2 mm coronal brain section showing GFP expression in SVZ (yellow inset) and Striatum & Neo-cortex (red inset) of P90 Nestin-GFP mice. Immuno-histochemical analysis showing that Nestin-GFP+ cells in Non-Canonical NSC

niche; Neo-cortex(B) and Striatum (C) express OPC marker NG2 and Olig2 while Nestin-GFP+ cells in Canonical NSC niche; Subgranular zone of dentate gyrus(E) and Sub-ventricular zone of lateral ventricle (D) express classical Type B quiescent neural stem cell marker, GFAP. Nestin-GFP+ OPCs in Neo-cortex and striatum cells are labeled with proliferation marker BrdU suggesting of their progenitor characteristics.

2. Characterization of Cortical Nestin-GFP cells in-vitro.

We then assessed the in-vitro neurosphere forming capability of these cortex derived Nestin-GFP+ cells. FACS sorted cortical Nestin-GFP+ cells gave rise to the formation of GFP+ neurospheres within 10 days in neural stem cell culture medium (Fig 23 A-C). These GFP+ neurospheres were exclusively positive for NG2 and Nestin protein assessed by immunocytochemistry which indicates that these cells continue to express NG2 and Nestin even in in-vitro neurosphere culture (Fig 23 D).

Fig. 23. Adult Non-SVZ derived NestGFP cells form neurospheres in-vitro. (A) Adult (P90) Nestin-GFP mice were sacrificed and Neo-cortex was carefully isolated. Neo-cortex was dissociated with Milteyni Biotech’s Adult brain dissociation kit to obtain Neo-cortical cells. B. Nestin-GFP+ cells were sorted by flow cytometry. A total of 2.7% Nestin-GFP cells were isolated. C.

FACS sorted Nestin-GFP+ cells were cultured in the presence of FGF-2 and EGF over the period of 2 weeks in non-coated dish. Neo-cortex derived Nestin-GFP+ cells formed neurospheres in vitro. D. Immuno-cytochemical analysis showed that these GFP+ neurospheres consistently expressed Nestin and NG2.

3. Enhanced proliferation and neuronal differentiation of adult brain parenchymal Nestin-GFP progenitors by MSC/Ngn1+HGF cells in chronic 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

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