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Differentiation of human induced pluripotent stem cells into dorsal and ventral spinal neural 
progenitor cells for application in spinal cord therapeutics
Anne Huntemer-Silveira1, Dane Malone2, Patrick Walsh3, Anna Frie1, & Ann Parr2,3
1Department of Neuroscience, 2Department of Neurosurgery, 3Stem Cell Institute, University of Minnesota, MN USA
BACKGROUND
Regional Identity in the Spinal Cord
RESULTS
Protocol and all characterization has been replicated in 4 cell lines. 
RT-qPCR: Actin used as a reference gene. Analyzed with ddCT method. Significance 
evaluated one way ANOVA. 
Immunocytochemistry: Cells were formalin fixed and stained using established 
primary and secondary antibodies. Images acquired at 20X. 
Establishment of Spinal Neural Progenitor Cell (sNPC) Lines
Figure 2. 
Directed 
differentiation 
of dorsal and 
ventral spinal 
neurons from a 
shared lineage2. 
Protocol and 
timeline for the 
differentiation of 
dorsal and 
ventral spinal 
neurons. 
Figure 1. Regional progenitor domains in the developing 
spinal cord (left) and the mature cell populations they 
give rise to (right). In SCI, damage to the dorsal cord 
(top/blue) is most often associated with sensory 
impairment, while damage to the ventral cord 
(bottom/orange) is associated with motor impairment. 
➢ SCI is associated with profound sensory and motor deficits that arise due to 
damage in the dorsal and ventral spinal cord, respectively.
Bench to Bedside to Bench: Improving Clinical Outcomes
➢ Cell replacement therapy (CRT) utilizes stem cell derived neural progenitor cells 
to restore lost connections and promote sensorimotor recovery following SCI, 
though clinical application has had mixed results.
➢ Evidence suggests that regional specification of transplants to recapitulate 
specific phenotypic regions may improve the integration of transplanted cells1.
Project Overview
We report here our work generating human induced pluripotent stem cell 
(hiPSC)-derived, region specific dorsal and ventral spinal neural progenitor cells 
from a shared lineage. Utilizing established markers to characterize spinal 
neuron development, we show that these populations are produced with high 
consistency and can be utilized in both in vitro and in vivo models of SCI.
➢ Dorsal horn sensory circuitry remains understudied despite the majority of SCI 
patients experiencing sensory dysfunction.
METHODS
FUTURE DIRECTIONS
➢ To enhance the translation of CRT from 
bench to bedside, we propose a 
combinatorial approach that employs 
regionally specified transplants paired 
with the use of 3D printed bio-scaffolds 
to improve cell integration.
➢ Thus far we successfully 3D-printed our 
ventral spinal NPCs onto a bioscaffold 
for implantation in both acute and 
chronic SCI rat models.
➢ Future research will also explore in vitro 
modeling of hiPSC-derived populations 
to study synaptic mechanisms of pain 
and injury. 
Figure 3. Development and functional characterization of regionally specific spinal cord 
bioscaffolds for use in SCI. Top: Dorsal and ventral spinal cord neural progenitor cells are 
generated from a shared lineage of induced pluripotent stem cells (iPSCs) and are optimized 
for 3D-printing into scaffolds. Bottom: Transplanted scaffold with bioprinted sNPCs twelve 
weeks post-transplantation. Transplanted human sNPCs (SC121) express NF200, a mature 
neuronal marker and integrate with host tissue along the edges of the scaffolds. 
References
1. Dell'Anno, M.T., et al. (2018).  Human 
neuroepithelial stem cell regional specificity enables 
spinal cord repair through a relay circuit. 
2. Walsh, P., et al. (2018). Defined Culture Conditions 
Accelerate Small-molecule-assisted Neural Induction 
for the Production of Neural Progenitors from 
Human-induced Pluripotent Stem Cells. 
3. Gupta, S., Sivalingam, D., Hain, S., Makkar, C., Sosa, 
E., Clark, A., & Butler, S. J. (2018). Deriving Dorsal 
Spinal Sensory Interneurons from Human Pluripotent 
Stem Cells.
DAPI PAX3 OLIG2  PAX6 DAPI NKX6.2 PAX7  MSX1
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Funding
Dorsal-Ventral 
Specification: At day 6, cells 
exhibit gene expression 
consistent with their 
respective phenotypes. 
Dorsal sNPCs primarily 
express Pax3, Pax7, and 
MSX1. Ventral sNPCs 
primarily express NKX6.2 
and Olig2. 
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Rostral-Caudal 
Specification: At day 20, 
dorsal (top) and ventral 
(bottom) spinal neurons 
exhibit Hox gene 
expression consistent 
with a rostral spinal 
identity, spanning 
hindbrain, cervical, and 
thoracic levels.
Mature Dorsal and Ventral 
Spinal Neurons Express Post-
Mitotic Neural M rkers: 
Expression of neural markers 
NeuN (neurons), S100B 
(astrocytes), and PDGFRA 
(oligodendrocytes) in dorsal 
(bottom left) and ventral 
(bottom right) spinal neurons 
at Day 20. NeuN expressing 
dorsal and ventral spinal 
neurons co-express TUBB3. 
Application, Calcium Imaging in Co-Culture: Calcium 
imaging of co-cultures at one month containing hiPSC-
derived nociceptors and RFP+ dorsal neurons (TUBA1B). 
Activation of nociceptors with capsaicin yields a fluorescent 
respond in dorsal neurons that do not otherwise respond 
to capsaicin, suggesting the formation of functional 
connections following 30 days in culture.