Modulation of the Mesenchymal Stem Cell Secretome Using Computer-Controlled Bioreactors: Impact on Neuronal Cell Proliferation, Survival and Differentiation

Abstract

In recent years it has been shown that the therapeutic benefits of human mesenchymal stem/stromal cells (hMSCs) in the Central Nervous System (CNS) are mainly attributed to their secretome. The implementation of computer-controlled suspension bioreactors has shown to be a viable route for the expansion of these cells to large numbers. As hMSCs actively respond to their culture environment, there is the hypothesis that one can modulate its secretome through their use. Herein, we present data indicating that the use of computer-controlled suspension bioreactors enhanced the neuroregulatory profile of hMSCs secretome. Indeed, higher levels of in vitro neuronal differentiation and NOTCH1 expression in human neural progenitor cells (hNPCs) were observed when these cells were incubated with the secretome of dynamically cultured hMSCs. A similar trend was also observed in the hippocampal dentate gyrus (DG) of rat brains where, upon injection, an enhanced neuronal and astrocytic survival and differentiation, was observed. Proteomic analysis also revealed that the dynamic culturing of hMSCs increased the secretion of several neuroregulatory molecules and miRNAs present in hMSCs secretome. In summary, the appropriate use of dynamic culture conditions can represent an important asset for the development of future neuro-regenerative strategies involving the use of hMSCs secretome.

Figure 1. Expansion and characterization of hBM-MSCs in static culture and 500 mL computer-controlled bioreactors.

hBM-MSCs adhered to both the tissue culture flasks and the microcarriers in the suspension bioreactors on day 1, and proliferated up to day 4 (A). FACS analysis for hMSC markers, CD13, CD73, CD90, CD105 were >99.9% while non-hMSC markers were expressed <2.0%. Mean fluorescence intensity is also displayed (B). Additionally, key process parameters (i.e. dissolved oxygen, pH and temperature) in our computer-controlled bioreactor system were well maintained at pre-determined setpoints for the culture period (C). Scale bars represent 100 μm.

Figure 2. In vitro differentiation of hNPCs.

Morphology and immunofluorescence staining of undifferentiated hNPCs ((A–C): a neuroshpere) into PPRF-h2 showed that the majority of the cells express mainly (A) Nestin⁺ cells, which evidences their progenitor profile. hBM-MSCs CM collected from static and dynamic culture conditions was able to significantly increase the survival and differentiation of hNPCs into (E,F) immature (DCX⁺ cells) and (I,J,M,N) mature (Map-2⁺/NeuN⁺ cells) neurons when compared to the (D,H,L) control group ((G,K,O); mean ± SEM., n = 3, p < 0.001). At the same time, the (F,J,N) hBM-MSCs CM collected from the dynamic culture conditions also showed increased levels of DCX⁺ (p < 0.05) and MAP-2⁺ (p < 0.05) cells, and NeuN⁺ (p = 0.077) cells compared to the (E,I,M) static hBM-MSCs CM in the induction hNPC differentiation ((G,K,O); mean ± SEM., n = 3). qRT-PCR analysis to NOTCH1 on hNPCs-differentiated cells showed a higher expression (p < 0.05; mean ± SEM; n = 3) in the (P) hBM-MSCs dynamic secretome group when compared to the control group. CT: Control (Neurobasal-A media), CMs: hBM-MSCs static conditioned media. CMd: hBM-MSCs dynamic conditioned media (Scale bar: 50 μm). Data are expressed as mean ± SEM. *p<0.05; **p<0.01; ***p<0.001.

Figure 3. In vivo injection of the hMSC Secretome (i.e. CM) increases proliferation and neuronal differentiation.

Injection of the hBM-MSC CM from static and dynamic conditions into the DG of adult rat hippocampus. After 7 days post-injection, both CM (static and dynamic) were able to increase the (B,C) levels of endogenous proliferating cells (Ki-67⁺ cells) in the DG when compared to the (A) sham group ((J), mean ± SEM., n = 5, p < 0.05). Moreover, although both CM were able to increase the number of newborn neurons ((E,F); DCX⁺ cells) compared to the (D) sham group, the dynamic CM showed higher numbers of the newborn (neurons) cell densities ((K), mean ± SEM, n = 5, p < 0.05) compared to the static CM. For the induction of neuronal differentiation, both CM (from the static and dynamic conditions) were able to increase significantly the number of Ki-67⁺/DCX⁺ cells when compared to the control group ((L), mean ± SEM., n = 5, p < 0.05), with this phenomena being more evident for the group cultured with the dynamic CM. hBM-MSCs CM (from static and dynamic conditions) was also able to increase astrocytic cell densities in the DG of hippocampus. Immunohistochemical analysis of GFAP (Astrocytes; (M–O)) revealed increased numbers promoted by the injection of hBM-MSCs CM (P, statistically significant to the Sham group, mean ± SEM, n = 5, p < 0.05) 7 days post-injection. SH: Sham (animals injected with Neurobasal-A media) CMs: hBM-MSCs static conditioned media. CMd: hBM-MSCs dynamic conditioned media (Scale bar: 100 μm). Data are expressed as mean ± SEM. *p<0.05; **p<0.01.

Figure 4. Graphical representation of the hBM-MSCs Secretome (i.e. CM) proteomic analysis by mass spectrometry.

CM analysis shows that the pattern of protein expression is modulated when we change from (A) static to (B) dynamic culture condition. Indeed, the (C) Venn diagram indicates more proteins were identified in the dynamic CM (130 proteins) when compared do the static CM (120 proteins). Specific neuroregulatory molecules such as (D) cystatin C (Cys C), (E) glia-derived nexin (GDN), (F) galectin-1 (Gal-1) and (G) pigment epithelium-derived factor (PEDF) were found to be upregulated in the dynamic hBM-MSCs secretome (data are expressed as mean ± SEM, n=3). CMs: hBM-MSCs static conditioned media. CMd: hBM-MSCs dynamic conditioned media.

Figure 5. Graphical representation for the hBM-MSCs Secretome (i.e. CM) classical trophic factors by Bioplex analysis.

Comparative analysis showed an upregulation in the concentration levels of (A) brain-derived neurotrophic factor (BDNF), (B) vascular endothelial growth factor (VEGF), (C) nerve growth factor (NGF), (D) insulin growth factor 1 (IGF-1) and (E) miR-16 in the hMSC dynamic CM when compared to the static condition. Trophic factor values were normalized to cell density (i.e. pg (of each factor)) per 10,000 cells in each condition, respectively. CMs: hBM-MSCs static conditioned media. CMd: hBM-MSCs dynamic conditioned media. Values are shown as mean ± SEM, n = 3; ***p < 0.001.