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Differentiation of human induced pluripotent stem cells (hiPSCs) into functional neurons is an exciting area of research, with considerable therapeutic potential in neurodegenerative diseases. In the past, conversion of hiPSCs into neural lineages was a time-consuming process hampered by the poor yield of selective survival protocols and the heterogenous nature of embryoid body formation. Employing small molecules that target the signaling pathways underlying neural cell differentiation has the potential to overcome these problems.
In their paper from 2009, Lorenz Studer’s lab outlined a novel method of neural differentiation, inhibiting SMAD signaling with the noggin protein and the small molecule inhibitor SB 431542 (Cat.No 1614). This method induced rapid and consistent conversion of hiPSCs, yielding a homogeneous population of PAX6+ neuroectoderm cells by day 11 of differentiation. From these, they could derive neural crest cells, rosette neural stem cells, dopaminergic neurons and motor neurons (Chambers et al, 2009).
Subsequently, the same group (Chambers et al, 2012) improved on this method, replacing Noggin with the BMP inhibitor LDN 193189 (Cat.No. 6053) and adding an additional three small molecules, SU 5402 (Cat.No. 3300), CHIR 99021 (Cat.No. 4423), and DAPT (Cat.No 2634), to accelerate the production of post-mitotic neurons from hiPSCs. These compounds inhibit VEGF/FGF/PDGF signaling, GSK-3β and Notch signaling, respectively. Using this protocol, named LSB3i for its components, they showed induction of neural crest cells by day 8. By day 15, further differentiation resulted in cells expressing markers for sensory neurons, which were determined to be primarily functional nociceptors owing to their expression of vanilloid receptors and their electrophysiological profile (Chambers et al, 2012).
Further research revealed that the WNT signaling pathway plays a critical role in determining CNS versus neural crest cell fates. The LSB3i protocol relies on activation of the WNT pathway by CHIR 99021 in combination with dual SMAD inhibition to induce a neural crest lineage and peripheral neurons. Subsequent adjustments to this protocol (Qi et al, 2017) replaced CHIR 99021 with the WNT signaling inhibitor XAV 939 (Cat.No. 3748), giving rise to an almost pure (>98%) population of PAX6+ cells from day 6 of differentiation. The efficiency of this conversion was further improved by a combination of dual SMAD inhibition with PD 0325901 (Cat.No. 4192), SU 5402 and DAPT, resulting in 70% TUJ1 (a marker of neuronal fate) positive neurons by day 13 of differentiation (Fig.1). Qi et al, also applied the protocol in good manufacturing practice (GMP) compatible culture conditions. In longer term culture, following this protocol yielded neurons capable of firing action potentials and forming excitatory synapses. Of relevance to the clinical applications of hiPSC derived cells, in vivo transplantation of these cortical neurons showed formation of long distance projections in the mouse cortex (Qi et al, 2017).
Figure 1: Derivation of functional cortical neurons from hiPSCs using small molecules
The protocols outlined above, and other recent publications, show that hiPSCs can be robustly differentiated into neurons suitable for GMP regulated implantation into humans. Clinicaltrials.gov indicates that there are approximately 10 clinical trials, either completed or recruiting patients, to investigate the transplantation of stem cell-derived neural precursors or neurons in Parkinson’s disease, amylotrophic lateral sclerosis, multiple sclerosis and traumatic spinal cord injury. The use of small molecules to drive neural differentiation presents a significant improvement on previous protocols, in terms of time required and cell yield, and paves the way for the use of the resulting cells as therapies in neurodegenerative diseases. Keep a look out for the new and exclusive range of GMP stem cell compounds coming soon from Tocris!
Chambers et al (2009) Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD. Nat Biotechnol. 27, 275. PMID: 19252484
Chambers et al (2012) Combined small molecule inhibition accelerates developmental timing and converts human pluripotent stem cells into nociceptors. Nat Biotechnol. 30, 715. PMID: 22750882
Qi et al (2017) Combined small-molecule inhibition accelerates the derivation of functional, early-born, cortical neurons from human pluripotent stem cells. Nat Biotechnol. 35, 154. PMID: 28112759