reprogramming-induced-pluripotent-stem-cell-lines-from-frozen-buffy

Reprogramming Induced Pluripotent Stem Cell Lines from Frozen Buffy Coat Samples

Human pluripotent stem cells (hPSCs) are a valuable tool for disease modeling. Further stem cells can be reprogrammed from adult somatic cells, called ...

University of Georgia

Sensory neurons (SNs) detect a wide range of information from the body and the environment that is critical for homeostasis. There are three main subtypes of SNs: nociceptors, mechanoreceptors, and proprioceptors, which express different membrane proteins, such as TRKA, TRKB, or TRKC, respectively. Human pluripotent stem cell technology provides an ideal platform to study development and diseases of SNs, however there is not a viable method to isolate individual SN subtype for downstream analysis available. Here, we employ the method immunopanning to isolate each SN subtype. This method is very gentle and allows proper survival after the isolation. We use antibodies against TRKA, TRKB, and TRKC to isolate nociceptors, mechanoreceptors, and proprioceptors, respectively. We show that our cultures are enriched for each subtype and express their respective subtype markers. Furthermore, we show that the immunopanned SNs are electrically active and respond to specific stimuli. Thus, our method can be used to purify viable neuronal subtypes using respective membrane proteins for downstream studies.

Isolation of human pluripotent stem cell-derived sensory neuron subtypes by immunopanning.

The technology of human pluripotent stem cell (hPSC)-based 3D organoid/assembloid cultures has become a powerful tool for the study of human embryonic development, disease modeling and drug discovery in recent years. The autonomic sympathetic nervous system innervates and regulates almost all organs in the body, including the heart. Yet, most reported organoids to date are not innervated, thus lacking proper neural regulation, and hindering reciprocal tissue maturation. Here, we developed a simple and versatile sympathetic neuron (symN)-innervated cardiac assembloid without the need for bioengineering. Our human sympathetic cardiac assembloids (hSCAs) showed mature muscle structures, atrial to ventricular patterning, and spontaneous beating. hSCA-innervating symNs displayed neurotransmitter synthesis and functional regulation of the cardiac beating rate, which could be manipulated pharmacologically or optogenetically. We modeled symN-mediated cardiac development and myocardial infarction. This hSCAs provides a tool for future neurocardiotoxicity screening approaches and is highly versatile and modular, where the types of neuron (symN or parasympathetic or sensory neuron) and organoid (heart, lung, kidney) to be innervated may be interchanged.

A modular platform to generate functional sympathetic neuron-innervated heart assembloids.

The extracellular matrix (ECM) is a mixture of glycoproteins and fibrous proteins that provide the biophysical properties necessary to maintain cellular homeostasis. ECM integrity is of particular importance during development, where it allows proper migration and cellular differentiation. Laminins are ECM heterotrimeric proteins consisting of α, β, and γ chains. There are five known α chains, four β chains, and three γ chains. Thus, there are 60 potential combinations for laminin trimers, however only 16 laminin trimers have been identified to date. Furthermore, none of them contain laminin β4 and its function is unknown. Here, we sought to characterize the role of  (the gene encoding laminin β4) during human embryonic development of the peripheral sensory nervous system. Using human pluripotent stem cells (hPSCs), we found that  is expressed in the ectoderm in the early stages of sensory neuron (SN) specification. SNs, part of the peripheral nervous system, are specialized neurons that detect pain, temperature, and touch. Surprisingly, more than 20 million people in the US have some form of peripheral nerve damage (including SNs), however there are very few treatment options available. Learning about the biology of peripheral neurons will uncover potential new therapeutic targets, thus we focused on understanding the effects of  in SNs. First, we knocked out  in hPSCs, using CRISPR/Cas9, and found that loss of  impairs the migration of the SN progenitors neural crest cells (NCCs) and harms SN development and survival. To assess if  has clinical relevance, we studied the genetic disorder Familial Dysautonomia (FD), which specifically affects the peripheral nervous system. FD is caused by a mutation in  (a component of the Elongator complex) leading to developmental and degenerative defects in SNs. A previous report showed that patients with severe FD harbor additional single nucleotide variants in . We found that these variants sharply downregulate the expression of  and laminin β4 levels in SNs differentiated from induced pluripotent stem cells (iPSCs) reprogramed from patients with severe FD. Moreover, a healthy ECM is sufficient to rescue the developmental phenotypes of FD, further confirming that ECM defects contribute significantly to the etiology of FD. Finally, we found that /laminin β4 is necessary for actin filament accumulation and it interacts with laminin α4 and laminin γ3, forming the laminin-443, a previously unreported laminin trimer. Together, these results show that  is a critical, but largely unknown gene required for SN development and survival.

Laminin β4 is required for the development of human peripheral sensory neurons.

Accessing untransformed human primary parasympathetic neurons is extremely challenging due to their inaccessibility in patients. Therefore, a human pluripotent stem cell-based approach to generate pure parasympathetic neurons is valuable for studying their development, maturation, and pathology. Here, we present a protocol to derive parasympathetic neurons from human pluripotent stem cells. We describe steps for human pluripotent stem cell replating, neural crest replating, cryobanking, and parasympathetic neuron differentiation. We then detail procedures for functional assays via multielectrode array (MEA) analysis. For complete details on the use and execution of this protocol, please refer to Wu et al..

Article	Year
Isolation of human pluripotent stem cell-derived sensory neuron subtypes by immunopanning. Frontiers in cell and developmental biology\| PubMed ID: 37206924	2023
A modular platform to generate functional sympathetic neuron-innervated heart assembloids. Research square\| PubMed ID: 38562819	2024
Laminin β4 is required for the development of human peripheral sensory neurons. bioRxiv : the preprint server for biology\| PubMed ID: 39605577	2024
Protocol to derive postganglionic parasympathetic neurons using human pluripotent stem cells for electrophysiological and functional assessment. STAR protocols\| PubMed ID: 40748761	2025

Christina James

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