Neural differentiation medium for human pluripotent stem cells to model physiological glucose levels in human brain

Highlights

• Neural differentiation media that models physiological glucose levels.

• Neural media supports human pluripotent stem cell neuronal differentiation.

• Media is needed for studying impact of diabetic conditions on human neurons.

Abstract

Cortical neurospheres (NSPs) derived from human pluripotent stem cells (hPSC), have proven to be a successful platform to investigate human brain development and neuro-related diseases. Currently, many of the standard hPSC neural differentiation media, use concentrations of glucose (approximately 17.5–25 mM) and insulin (approximately 3.2 μM) that are much greater than the physiological concentrations found in the human brain. These culture conditions make it difficult to analyse perturbations of glucose or insulin on neuronal development and differentiation. We established a new hPSC neural differentiation medium that incorporated physiological brain concentrations of glucose (2.5 mM) and significantly reduced insulin levels (0.86 μM). This medium supported hPSC neural induction and formation of cortical NSPs. The revised hPSC neural differentiation medium, may provide an improved platform to model brain development and to investigate neural differentiation signalling pathways impacted by abnormal glucose and insulin levels.

Introduction

Human pluripotent stem cells (hPSC) are increasingly being used as a unique research tool to study the human nervous system, supporting fundamental and translational research (Lee et al., 2020). The 3-dimensional structured cortical NSPs derived from hPSC, provide a more equivalent platform for brain neurogenesis and brain disorder modelling in comparison to 2D structured cultures, as they support spatial organization of cells, which is crucial in regulating stem cell/progenitor differentiation (Hattori, 2014; Koo et al., 2019). This platform offers new hope for improving our understanding of early human brain developmental in health and in disease. To bring this potential forward it is important that cells are cultured in conditions appropriate for the intended research\therapeutic aims. To date, there are standard versions of neural media used to culture hPSC and, more specifically, neuronal progenitors. While many of the current standard media contain most of the components found in natural physiological conditions (Denham and Dottori, 2011; Bagley et al., 2017; Mansour et al., 2018), they are not necessarily at physiological concentrations. Bardy et el (Bardy et al., 2015) show, that while commonly used media such as DMEM/F12 and neurobasal promote neuronal differentiation and survival, they actually impair neurophysiological functions, such as calcium activity, electrical activity, synaptic activity and action potential firing, compared to neurons maintained in perfusate from mouse or artificial cerebrospinal fluid (CSF).

Glucose and insulin are two neural media components that are commonly used in non-physiologically relevant concentrations. DMEM/F12 and neurobasal contain highly elevated levels of glucose (17.5–25 mM) and supplemented insulin (3.2 μM) compared to glucose levels found in the brain extracellular fluid (ECF) (0.67–2.2 mM) (Zilberter et al., 2010; Gallagher et al., 2009; Cavus et al., 2005) and insulin concentrations in the CSF (8−7 pmol/l) (Born et al., 2002). Recent studies show that human-derived neural stem cells are highly sensitive to insulin, and that the optimal insulin concentration for cell viability was no more than 0.86 μM (Rhee et al., 2013). Insulin has been shown to act as a neuro-protector (Mielke et al., 2006), increase neuronal activity and has a role in synaptic maintenance (Passafaro et al., 2001). Similarly, the insulin receptor has proven to play a key role in the dynamics of dendrite formation, spine density and neurite growth (Chiu and Cline, 2010; Choi et al., 2005). Furthermore, the recommended glucose level for culturing human neurons is approximately 2.5 mM, similar to that found in the healthy human brain (Bardy et al., 2015). Other studies showed that neural stem cells cultured in 25 mM glucose concentrations presented oxidative stress (Yu et al., 2016; Chen et al., 2018) and suppressed neural stem cell differentiation (Yu et al., 2016).

The impact of dysglycaemia and abnormal insulin concentrations on neuronal development in vivo has also been well documented. Disruption of glucose homeostasis may affect brain physiology and lead to brain disorders (Zilberter et al., 2010; Mergenthaler et al., 2013). Type 1 diabetes (T1D) individuals, suffering from chronic hyperglycaemia, show cortical atrophy and structural brain abnormalities, contributing to neurocognitive impairment (Kodl et al., 2008). There have also been reports of decreased mean grey and white matter volume in the brain (Cameron et al., 2014; Rodriguez, 2012), as well as lower measures of intelligence, attention, processing speed, long-term memory, and executive skills, in children with T1D (Northam et al., 2001). Both in vitro and in vivo studies investigating maternal diabetes induced hyperglycaemia, revealed suppressed autophagy, delayed neurogenesis, neural tube defects (Li et al., 2013; Yang et al., 2013), and enhanced neural progenitor apoptosis (Salbaum and Kappen, 2010; Adastra et al., 2011), all contributing to neurodevelopmental defects in the embryo.

The demand for a medium that mimics more closely the physiological concentrations of glucose in the human CNS, is of great importance in investigating the impact of different conditions and their aetiology on the early developing cerebral cortex of the human brain. Here we assess the generation and maintenance of hPSC-derived cortical NSPs, in neural medium containing glucose levels similar to that found in the human brain (2.5 mM) and lower insulin levels as recommended for hPSC-derived neural stem cells (0.86 μM), using analyses of mRNA and protein expression. As the physiological concentrations are considerably lower than those found in the standard traditional cell culture medium protocols, we investigated whether the redefined medium impacted hPSC neuronal differentiation, cell survival and NSP formation.