Professor Northeastern University Boston, Massachusetts, United States
Introduction:: Neural progenitor cells (NPCs) are capable of self-renewal and neurogenesis, making them ideal candidates for neurodegenerative disease treatments[1]. However, NPCs exhibit limited stemness and differentiation potential in 2D culture. In the adult mammalian brain, NPCs are found in the neurovascular niche (NVN) where they reside close to brain microvascular networks (BMVNs) comprised of brain endothelial cells (BECs), pericytes, and astrocytes[1]. It is well-known that these cells, specifically BECs, govern NPC behavior through various niche factors[1]. The goal of this work is to recapitulate the human NVN in vitro to further elucidate the influences of neurovascular niche on NPC fate. Towards this, we developed a perfused BMVNs within a microfluidic device (MFD) to support NPC neuronal differentiation. We hypothesize that NPCs cultured with BMVNs will exhibit enhanced neurogenesis compared to NPCs alone. This would demonstrate the supportive role of BMVNs and validate our model for future NPC studies.
Materials and Methods:: NPCs (Stemcell Technologies) were aggregated into neurospheres, resuspended in fibrin gels alone (N condition) or with primary BECs expressing tdTomato (tdT), pericytes, and astrocytes (NEPA condition), and then injected into MFDs (AIM Biotech). All samples were incubated (37°C and 5% CO2) in neuron differentiation medium. To promote microvessel perfusion, interstitial flow of 1ul/s was applied across the fibrin gel in all samples, which enhances the vascular lumen connectivity, pericytes/astrocytes association and permeability similar to blood brain barrier (data not shown). On Day 10, differentiated neurons were labeled with either Tuj1 antibodies to highlight neurites or with Fluo-8 to observe calcium (Ca2+) oscillations. To confirm brain MVN anastomosis, microspheres were perfused through microvessels in NEPA samples. Fluorescence images were acquired using an Eclipse Ti2 microscope (Nikon). To quantify neurogenesis, the total neurite outgrowth and number of neurons exhibiting Ca2+ oscillations was measured for neurospheres in N and NEPA conditions. Significance was determined using Welch’s t-test.
Results, Conclusions, and Discussions:: Results and Discussion: On Day 10, neurites extending from neurospheres cultured alone appeared speckled (Fig. 1Ai), possibly indicating neurite regression. In contrast, neurites extending from neurospheres cultured with BMVNs appeared continuous and were observed interacting with microvessels (Fig. 1Aii), confirming proper neuritogenesis. For these reasons, total neurite outgrowth was found to be significantly enhanced for neurospheres cultured in NEPA conditions compared to N conditions (Fig. 1B). Similarly, significantly more neurons exhibiting Ca2+ oscillations were measured in neurospheres cultured with BMVNs than those cultured alone (Fig. 1C). These data confirmed NPC differentiation into functional neurons, specifically in NEPA conditions. In addition, microspheres were observed flowing through BMVNs (Fig. 1D), confirming luminal flow similar to blood flow found in brain microvessels[1]. Collectively, these data infer that our bioengineered 3D BMVNs support NPC neurogenesis. This was probably due to microvessels in MFDs adopting the neurotrophic role of blood vessels found in the NVN[1]. These results validate the use of our model for the study of the interaction between NPCs and BMVNs.
Conclusions: We have developed the first in vitro model of the human NVN with perfused BMVNs capable of supporting NPC neurogenesis. This platform will allow us to study NPC fate and the therapeutic potential of NPCs for neurodegenerative diseases. The presence of perfused microvessels will also facilitate pharmacological studies that require vascular drug delivery and changes of BBB in various neurodegenerative diseases.
Acknowledgements (Optional): : This work was supported by NIH R01NS107462, R21NS121736, NSF CBET-1350240.
