Comparison of the neuropoietic activity of gene-modified versus parental mesenchymal stromal cells and the identification of soluble and extracellular matrix-related neuropoietic mediators

Abstract

Introduction: Transplanting mesenchymal stromal cells (MSCs) or their derivatives into a neurodegenerative environment is believed to be beneficial because of the trophic support, migratory guidance, immunosuppression, and neurogenic stimuli they provide. SB623, a cell therapy for the treatment of chronic stroke, currently in a clinical trial, is derived from bone marrow MSCs by using transient transfection with a vector encoding the human Notch1 intracellular domain. This creates a new phenotype, which is effective in experimental stroke, exhibits immunosuppressive and angiogenic activity equal or superior to parental MSCs in vitro, and produces extracellular matrix (ECM) that is exceptionally supportive for neural cell growth. The neuropoietic activity of SB623 and parental MSCs has not been compared, and the SB623-derived neuropoietic mediators have not been identified.

Methods: SB623 or parental MSCs were cocultured with rat embryonic brain cortex cells on cell-derived ECM in a previously characterized quantitative neuropoiesis assay. Changes in expression of rat neural differentiation markers were quantified by using rat-specific qRT-PCR. Human mediators were identified by using expression profiling, an enzymatic crosslinking activity, and functional interference studies by means of blocking antibodies, biologic inhibitors, and siRNA. Cocultures were immunolabeled for presynaptic vesicular transporters to assess neuronal specialization.

Results: Among six MSC/SB623 pairs, SB623 induced expression of rat neural precursor, oligodendrocyte, and astrocyte markers on average 2.6 to 3 times stronger than did their parental MSCs. SB623 expressed significantly higher FGF2, FGF1, and BMP4, and lower FGFR1 and FGFR2 levels; and human FGF1, FGF2, BMPs, and HGF were implicated as neuropoietic mediators. Neural precursors grew faster on SB623- than on MSC-derived ECM. SB623 exhibited higher expression levels and crosslinking activity of tissue transglutaminase (TGM2). TGM2 silencing reduced neural precursor growth on SB623-ECM. SB623 also promoted the induction of GABA-ergic, but not glutamatergic, neurons more effectively than did MSCs.

Conclusions: These data demonstrate that SB623 cells tend to support neural cell growth more effectively than their parental MSCs and identify both soluble and insoluble mediators responsible, at least in part, for enhanced neuropoietic potency of SB623. The neuropoiesis assay is a useful tool for identifying beneficial factors produced by MSCs and their derivatives.

Figure 1

Comparison of neuropoietic activity of SB623 and MSCs in cocultures with rat embryonic neural cells. (A) Rat neural cells were grown in the presence or absence of MSCs or SB623 (rat/human cell ratio was 10:1) and immunostained for rat nestin (upper panel) and GFAP (middle panel) on day 5, or for CNP on day 12. Nuclei were stained with DAPI (B) An example of microplate neuropoiesis assay data: a comparison of rat neural differentiation marker induction in cocultures of rat cells with MSCs and SB623 from Donor A. Rat neural cells (5,000/well) were cocultured with 500, 250, and 125 cells/well of either MSCs or SB623; and expression of rat-specific nestin, GFAP, and CNP, and human-specific GAP was quantified by using qRT-PCR. Stimulation with 10% MSC-CM and no stimulation (“No add”) were used as positive control and background, respectively. Relative units correspond to standard samples used in qPCR run. Error bars represent SD of biologic duplicates. (C) Neuropoietic coefficients of MSCs and SB623 from Donor A were calculated based on data presented in (B) by first subtracting the background expression of a corresponding neural marker and then normalizing the expression of the neural marker to the human GAP for each number of human cells per well, followed by averaging normalized values. Error bars represent SD from three normalized values.

Figure 2

Role of human growth factors in induction of rat neural markers in various culture settings; qRT-PCR. (A) Effect of FGF2 inhibition on rat nestin. Rat neural cells were stimulated with SB623 cell-derived conditioned medium (25%), which increased the nestin expression. The presence of FGF2-neutralizing antibody (bFM1), but not FGF2-specific nonneutralizing antibody (bMF2), concentration-dependently inhibited the nestin increase. (B) Effect of human FGF1 inhibition on rat nestin. Rat neural cells were stimulated by MSCs (60 or 200 cells/well), MSC-CM (5%), or recombinant FGF1, or FGF2 (both at 5 ng/ml), which led to the induction of rat nestin. The application of the anti-human FGF1 neutralizing antibody decreased nestin mRNA levels induced by all stimuli but the recombinant FGF2. (C) Effect of BMP inhibition on rat GFAP induction. Rat GFAP was induced by coculturing rat neural cells with SB623 (200 cells/well). A BMP inhibitor noggin concentration-dependently decreased the GFAP induction. (D) Effect of HGF silencing in MSC on rat CNP induction. Rat CNP expression was stimulated by coculturing rat neural cells with MSC transfected with either HGF siRNA (siHGF) or control siRNA (siControl). A lower CNP induction was observed in cocultures with siHGF-transfectants.

Figure 3

Comparison of presynaptic puncta formation in cocultures of rat embryonic neural cells with MSCs or SB623; immunostaining. (A) Immunostaining for VGLUT (day 7) and VGAT (day 11); rat/human cell ratio, 50:1. Bar, 50 μm. (B) Quantification of VGAT-immunoreactive puncta per neurite length (averaged from 10 microscopic fields, one to four neurites/field), day 11. (C) Typical distribution and size of VGAT-positive puncta in axonal processes in cocultures with MSCs (upper) or SB623 (lower), day 13. Neurites are outlined manually. Bar, 50 um.

Figure 4

Comparison of SB623- and MSC-derived ECM in supporting nestin-positive cell growth. (A) Rat neural cells were grown for 5 days on MSC- or SB623-derived ECM and then immunostained for nestin (upper panel) and counterstained for nuclei (lower panel); magnification 100×. (B) Nestin-positive cells grown and stained as described in (A) were counted, and their numbers expressed as percentage of total nuclei. (C) Rat nestin expression in cells growing on MSC- or SB623-ECM was quantified by using qRT-PCR and normalized by the LDH activity released from either MSC- or SB623-ECM-producing cells during ECM preparation, correspondingly, to account for possible differences in cell numbers.

Figure 5

Comparison of expression and activity of TGM2 in SB623 and MSC; its functional analysis in ECM using siRNA. (A) Expression levels of TGM2 normalized to GAP were determined by using qRT-PCR in SB623/MSC pairs from several donors. Levels in SB623 cells were expressed relative to levels in parental MSCs, which were set on 1. (B) TGM2-crosslinking activity was measured by amounts of biotinylated cadaverine incorporated into PLL in the presence of SB623 or MSC cell lysates. The activity was then normalized to the total protein, and expressed compared with the parental MSCs, where the values were set on 1. (C) TGM2 was detected in ECM of MSCs and SB623 by using immunoblotting. The TGM2 signal was quantified densitometrically and normalized to the total ECM protein per lane. The total ECM protein was assessed by using duplicated gel: the gel was stained for protein; photographed; and the density of corresponding lane minus background determined. (The whole blot and gel are shown in Additional file 6: Figure S5). (D) Nestin expression was quantified by using qRT-PCR in rat neural cells grown on ECM produced by SB623 transfected with either siTGM2 or siControl. Nestin levels on siControl-ECM were set on 1. The graph represents means from three experiments; error bar represents standard error of mean; *P < 0.05. (E) Rat nestin mRNA expression in cocultures with either siTGM2 or siControl transfectants. The graph represents mean from two experiments.