Neurochemical and Morphological Comparisons of Motor Nerve Organoids and Spinal-Cord Explants

Abstract

Organoids are multicellular structures formed in vitro from populations of individual cells allowing modeling of structural and functional aspects of organs and tissues in normal and diseased states. They offer unique opportunities to model and treat disease. Using a mouse embryonic stem cell line, we have cultured organoids that express markers of spinal cord motor neurons as well as motor neurons found within the peripheral nervous system. The morphology and select neurotransmitter content of the organoids and spinal cord explants were compared at different developmental time points. We found indications of maturation in the organoids over time, mirrored by similar trends in the spinal cord explants. Although the organoids contained the same neurotransmitters as the spinal cord explants, the developmental changes of these neurotransmitter levels were less marked in organoids. Given these differences, further work is required to optimize organoid growth conditions to better reproduce in vivo models when using organoids to study development.

Keywords: capillary electrophoresis-mass spectrometry; development; morphology; neurotransmitter; organoid; spinal cord explants.

Figure 1.

Schematic of organoid culture. Mouse embryonic stem cells (mESCs) were cultured on feeder layers of rat embryonic fibroblasts in growth media for 9 days. The resulting neurospheres were then transferred to Matrigel droplets and cultured in differentiation media containing retinoic acid and purmorphamine.

Figure 2.

Schematic correlating spinal cord extract and motor organoid developmental time points. Developmental time points for the spinal cord explants and motor organoids were correlated using the onset of neurogenesis (gestational day 11 [GD11] in the spinal cord explants and DIV 6 in the organoids). Therefore, P0 in the spinal cord explants correlates to DIV 16 in the organoids and P7 correlates to DIV 23. Time points at P14 and P21 for the spinal cord explants and at DIV 55 for the organoids were also analyzed due to the possibility of delayed maturation in the organoids.

Figure 3.

Neurons and astrocytes comprise motor organoids. A-E: Green: Beta-III Tubulin, expressed in neurons. Red: Aldehyde Dehydrogenase 1 Family Member 1 (ALDH1L1), expressed in astrocytes. Blue: 4′,6-diamidino-2-phenylindole (DAPI), a chromatin marker. Scalebars 10 μm. A-B: Spinal cord explants at Postnatal days 0, 7, 14, and 21 (P0, P7, P14, and P21). C-E: Organoids at 16 Days In Vitro (DIV), 23 DIV, and 55 DIV.

Figure 4.

Motor neuron gene Hb9 is expressed in motor organoids and spinal cord explants. A-E: Green: Beta-III tubulin, expressed in neurons. Red: Homeobox-9 (Hb9), expressed in nuclei of motor neurons. Blue: 4′,6-diamidino-2-phenylindole (DAPI), a chromatin marker. Scalebars 10 μm. A-B: Spinal cord explants at Postnatal days 0, 7, 14, and 21 (P0, P7, P14, and P21). C-E: Motor organoids at Day In Vitro (DIV) 16, DIV 23, and DIV 55. F: Hb9 expression decreased significantly over time for spinal cords (p=0.002) and organoids (p=0.003), one-way ANOVA. * Represents p<0.05; ** represents p<0.01; *** represents p<0.001.

Figure 5.

Myelin Protein Zero is present in organoids and spinal cord explants. A-E: Green: Beta-III Tubulin, expressed in neurons. Red: Myelin Protein Zero, expressed in motor neurons. Blue: 4′,6-diamidino-2-phenylindole (DAPI), a chromatin marker. Scalebars 10 μm. A-B: Spinal cord explants at Postnatal days 0, 7, 14, and 21 (P0, P7, P14, and P21). C-E: Organoids at 16 Days In Vitro (DIV), 23 DIV, and 55 DIV.

Figure 6.

Schematic of Capillary Electrophoresis-Mass Spectrometry. Schematic of Capillary Electrophoresis-Mass Spectrometry (CE-MS) instrument used in this work. The sample of interest is deposited in the sample well. Compounds in the sample are separated in the capillary separation channel in a conductive background electrolyte (BGE) solution based upon their size, charge, and shape. The compounds are then ionized via electrospray ionization (ESI) and undergo mass spectrometry (time of flight [ToF] mass analyzer), which allows for identification by their mass to charge ratios.

Figure 7.

ACh, GABA, Asp, and Glu levels in spinal cord explants and organoids. A: From left to right, levels of ACh, GABA, Glu, and Asp in mouse spinal cord explants at P0 (N=6), P7 (N =8), P14 (N=4), and P21 (N=4), measured by CE-MS. B: From left to right, levels of ACh, GABA, Glu, and Asp in pooled organoid samples, measured by CE-MS. Each data point represents 4 pooled organoids. N = 1 for ACh DIV 16 and DIV 23. N = 2 for GABA DIV 16. N = 3 for Glu DIV 16, Asp DIV 16, and Asp DIV 23. N = 4 for all other time points. ACh was only detected in 1 of the 4 pooled samples at both DIV 16 and DIV 23 time points, and no GABA was detected in all 4 pooled samples at the DIV 23 time point. Red data points represent outliers with Grubb’s test with α=0.05. Mann-Whitney U test was used with a two-tailed hypothesis and assuming 0.05 significance level. * represents p<0.05; ** represents p<0.01; *** represents p<0.001. For the spinal cord samples, the Ns are 6 for P0, 8 for P7, 4 for P14, 4 for P21. For the organoids, the Ns are vary for the different transmitters and are 1,1, 4 for ACh, 2,4,4 for GABA, 4,3,4 for Gly, and 3,3,4 for Asp where each measurement represents four pooled organoids and the three numbers represent the 16, 23, and 55 DIV timepoints.