A human iPSC-derived midbrain neural stem cell model of prenatal opioid exposure and withdrawal: A proof of concept
Rhea Sullivan1, Quinn Ahrens1, Sara L Mills-Huffnagle2
1Department of Pediatrics, Penn State College of Medicine, Hershey, Pennsylvania, United States of America.
Insights
Prenatal opioid exposure impacts infant brain development. This study created an in vitro model using human stem cells to show how morphine affects neural progenitor cells and their differentiation.
Area of Science:
- Neuroscience
- Developmental Biology
- Stem Cell Biology
Background:
- Clinical studies link prenatal opioid exposure to neurodevelopmental delays.
- The precise mechanisms by which opioids affect the developing brain are not fully understood.
Purpose of the Study:
- To develop and optimize an in vitro model of prenatal morphine exposure and withdrawal.
- To investigate the impact of morphine on human midbrain neural progenitor cells.
Main Methods:
- Utilized human induced pluripotent stem cell (iPSC)-derived midbrain neural progenitors in a monolayer culture.
- Optimized neural induction protocols to enhance opioid receptor expression (OPRM1, OPRK1).
- Assessed toll-like receptor 4 (TLR4) expression and cyclic adenosine monophosphate (cAMP) levels during withdrawal.
Main Results:
- A longer neural induction period increased mu and kappa opioid receptor expression.
- Morphine withdrawal led to cAMP overshoot.
- Morphine exposure and withdrawal altered progenitor cell fate, increasing nestin-positive progenitors and decreasing NEUN-positive neurons, while withdrawal reversed the latter.
Conclusions:
- The developed in vitro model effectively mimics aspects of prenatal opioid exposure and withdrawal.
- Morphine impacts neural progenitor cell differentiation and receptor expression.
- This model provides a platform for mechanistic studies on neurodevelopment under opioid influence.
Abstract:
A growing body of clinical literature has described neurodevelopmental delays in infants with chronic prenatal opioid exposure and withdrawal. Despite this, the mechanism of how opioids impact the developing brain remains unknown. Here, we developed an in vitro model of prenatal morphine exposure and withdrawal using healthy human induced pluripotent stem cell (iPSC)-derived midbrain neural progenitors in monolayer. To optimize our model, we identified that a longer neural induction and regional patterning period increases expression of canonical opioid receptors mu and kappa in midbrain neural progenitors compared to a shorter protocol (OPRM1, two-tailed t-test, p = 0.004; OPRK1, p = 0.0003). Next, we showed that the midbrain neural progenitors derived from a longer iPSC neural induction also have scant toll-like receptor 4 (TLR4) expression, a key player in neonatal opioid withdrawal syndrome pathophysiology. During morphine withdrawal, differentiating neural progenitors experience cyclic adenosine monophosphate overshoot compared to cell exposed to vehicle (p = 0.0496) and morphine exposure conditions (p, = 0.0136, 1-way ANOVA). Finally, we showed that morphine exposure and withdrawal alters proportions of differentiated progenitor cell fates (2-way ANOVA, F = 16.05, p < 0.0001). Chronic morphine exposure increased proportions of nestin positive progenitors (p = 0.0094), and decreased proportions of neuronal nuclear antigen positive neurons (NEUN) (p = 0.0047) compared to those exposed to vehicle. Morphine withdrawal decreased proportions of glial fibrillary acidic protein positive cells of astrocytic lineage (p = 0.044), and increased proportions of NEUN-positive neurons (p < 0.0001) compared to those exposed to morphine only. Applications of this paradigm include mechanistic studies underscoring neural progenitor cell fate commitments in early neurodevelopment during morphine exposure and withdrawal.
