Novel Lumbosacral Neural Crest Progenitor Differentiation for In Vitro Female Reproductive Tract Pain Modeling

Introduction: Chronic pelvic pain affects more than one fifth of the global female population, but a targeted pain management remedy remains elusive. Despite the complex nature of pain biology, conventional rodent models, often employed due to ethical concerns, translate poorly into the clinic. An alternative would be developing a representative human in vitro model containing stem-cell-derived sensory neurons (SNs) that innervate the female reproductive tract (FRT), which could be used as a patient-specific drug-screening platform once the model is established. However, prevailing protocols often ignore the regional specificity of these neurons along the rostral-caudal (head to tail) axis, resulting in a skewed representation that fails to reflect the properties of FRT-related sensory neurons close to the lumbosacral (closer to the caudal or tail) end of the body axis. Addressing this gap, we aim to develop protocols for the differentiation of lumbosacral neural crest progenitors (NCPs) that have the capacity to become peripheral nervous system (PNS) SNs. Here we demonstrate a novel method to efficiently generate NCPs with lumbar identity that can mature into pain-sensing nociceptors. This is a first step towards an in vitro model for FRT pain to develop personalized pain management strategies.

Materials and

Methods: Following the methodology outlined in Iyer et al. (2022), H9 hESCs were first differentiated into NMPs with discrete HOX profiles for 72-216 hours using FGF8, CHIR, GDF11, and LDN, validated using immunocytochemistry (ICC) and qRT-PCR, and cryopreserved for batch-to-batch consistency. To optimize region-specific NCC differentiation, thawed NMPs with specific HOX profiles were differentiated in media containing CHIR, SB, and BMP4.These NCCs were differentiated into SNs using DAPT and cAMP, then matured and maintained in media with NT-3, NGF, BDNF, and GDNF. Region-specific NCPs and SNs were characterized using ICC.

Results, Conclusions, and Discussions: Cryopreserved NMPs used for optimization experiments were SOX2+/Brachyury+/CDX2+ and expressed cervical (HOXC6) or lumbar (HOXD10) HOX genes, indicative of their bipotent (neural, mesodermal) differentiation potential and caudal axial positions. Within 8 days of NCP induction, optimized cultures were up to 95% SOX10+ while remaining negative for spinal (PAX6) or mesodermal (Brachyury) markers (Figure 1B). These yields were higher than comparable protocols (Fan et. al. 2022) with modest (~60-75%) differentiation efficiency and/or rely on FACS for purification. We also observed that monolayer cultures with high SOX10 expression spontaneously formed aggregated, three-dimensional structures characteristic of the migratory properties of NCPs. Finally, our region-specific NCPs were able to mature into ISL1+/BRN3a+/TUJ+/PRPH+ SNs with around 14.05% efficiency (Figure 1C). These express early nociceptor markers (TRPV1, CGRP) after one week of maturation (Figure 1D) and functional markers (NaV1.8) after 3 weeks. We also found expression of estrogen receptors (ER, GPER) on SNs (Figure 1E), demonstrating their receptiveness to estrogen in FRT-specific models of pain. As we continue to optimize the differentiation of NCPs in lumbosacral regions, we will validate the electrophysiological properties of nociceptors using calcium imaging and derive additional SN subtypes (mechanoreceptors, proprioceptors) and glia (Schwann cells) that populate FRT circuits. Ultimately, we anticipate developing a modular toolbox that can be used in diverse tissue-engineered models corresponding to different regions along the body axis.

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