Neural Stem Cells & Small Molecules: Mini-review
The prevalence of neurodegenerative disorders throughout the world, which are caused by damage to the Central Nervous System (CNS) and Peripheral Nervous System (PNS), is estimated to be 1 billion people . With the average life expectancy increasing, it is likely that this figure is going to escalate, unless a way in which these disorders can be directly and efficiently targeted to alleviate suffering or reverse their progress altogether, can be found.
Whilst there are a range of pharmaceuticals available which are used in an attempt to reduce symptoms or slow the progress of these disorders , much research is being undertaken to find novel ways to replace the damaged neurons and therefore target the disorder directly. One promising way is the use of stem cells. These could be either: Embryonic Stem Cells (ESCs), induced Pluripotent Stem Cells (iPSCs), Neural Stem Cells (NSCs), neural progenitor stem cells (NPCs) or mesenchymal stem cells (MSCs)
- ESCs are pluripotent cells derived from the inner cell mass of preimplantation embryos and which have the ability to give rise to all the cell types of the embryo, though not the extraembryonic tissues .
- iPSCs are generated by reprogramming somatic cells .
- NSCs and NPCs were first detected in the late 1980s . NSCs are multipotent cells that generate neurons, astrocytes and oligodendrocytes , whereas the NPCs can be unipotent, bipotent or multipotent and will not self-renew indefinitely. The NPCs are the non-stem cell progeny of the NSCs.
- MSCs are multipotent adult stem cells which have the ability to self-renew and differentiate in to functional cell types, according to the tissue in which they are found .
Each of these cell lines could have a huge positive impact on research of neurodegenerative disorders, however, they have proven difficult to grow and maintain in vitro. Long term, successful culture of these cells is variable and may be due to the differences in serum-free media and growth factors used . In addition, feeder cells may also be required to maintain the stem cells in their undifferentiated state . Further, current protocols used to push these cells towards differentiating into cells of a neural lineage are lengthy (30-50 days) and have demonstrated only limited success [10, 11].
The use of small molecules within the sphere of neural stem cells is one which has shown great promise. Several groups have been able to direct differentiation of iPSCs, ESCs and NSCs using small molecules, dramatically reducing the time needed to produce cells which show functional neuronal phenotypes. For advantages of using small molecules in stem cell biology, please refer to the HelloBio minireview: Role of Small Molecules in Stem Cell Biology. Small molecules have been used which target several different pathways:
Inhibition of SMAD signaling
Work by Chambers et al (2009) found that the inhibition of SMAD signalling using the small molecules Noggin and SB 431542, hESCs undertook complete neural conversion within approximately 19 days .
Building on this work, the same group then improved their strategy by replacing Noggin with a BMP inhibitor (LDN193189) and including three small molecules. These molecules were a potent inhibitor of VEGF/FGF/PDGF tyrosine kinase signalling (SU5402; Cat. No. HB3133), a WNT agonist that selectively inhibits GSK-3β (CHIR99021) and a Notch inhibitor that acts through γ-secretase (DAPT; Cat. No. HB3345). This strategy greatly improved the results from the 2009 paper: their data showed that by day 8 there had been induction of neural crest cells. Further, by day 15, cells were expressing markers for sensory neurons .
The most recent work from this group has, again, shown changes to the initial protocol but with even more success at activating neurogenesis. In their 2017 paper , Qi et al used a combination of small molecules which have dual SMAD inhibition properties (SU5402, PD0325901 and DAPT, see Diagram 1). By day 13, 70% of cells expressed TUJ1, which is a marker of neuronal fate. They were also able to produce neurons, following this protocol, but under Good Manufacturing Practice (GMP) compatible culturing conditions. This resulted in neurons capable of firing action potentials and forming excitatory synapses.
WNT signalling has been found to have a significant role in neurogenesis. Not only does the presence of WNT in cell culture increase the population of neural stem cells through self-renewing division, it has also been shown that WNT3a promotes differentiation into the neural and astrocyte lineage by inhibiting neural stem cell maintenance  and WNT7a signalling induces differentiation in neural stem cells of the neocortex . In addition, it has been found that activating WNT signalling under dual SMAD inhibition conditions yielded 75% post-mitotic neurons in 11 days of differentiation .
Retinoids have been demonstrated to be useful in promoting neurogenesis in vitro and have been used as supplements in culture media. However, these molecules, such as all-trans-retinoic acid (ATRA) have had a number of limitations, such as light sensitivity, and therefore results have been variable. In order to overcome this, Christie et al, (2010) produced a small molecule, EC 23 (Cat. No. HB2592), a synthetic retinoid derivative . As well as being light stable, it has a similar biological activity to ATRA and was found to significantly enhance neurogenesis in NPCs and ESCs. It was found to be most effective at lower nanomolar concentrations.
ISX compounds promote neurogenesis in NSCs through the upregulation of genes associated with neurogenesis and cell cycle progression in primary mouse dentate gyrus cells in vitro. In addition, it was determined that the compound was able to pass through the blood-brain barrier and promote hippocampal neurogenesis, following an intraperitoneal injection .
The small molecule A83-01 (Cat. No. HB3218) is a TGF-β inhibitor which can be used to maintain pluripotency of iPSCs . However, it has also been found to be essential in direct-to-iNSC reprogramming of adult human dermal fibroblasts (AHDF), in combination with other small molecules. In a study by Zhu et al, (2014) the addition of A83-10 with CHIR99021, after treatment with OCT-4 plus SOX2 confirmed the ability of this small molecule to reprogram AHDF cells to iNSCs, with PAX-6 expression . Further expansion of these cells showed that cells were able to maintain PAX-6, PLZF and Otx2 expression, supporting hNSC identity. The cells were shown to maintain this stable expression throughout expansion over 5 months. In addition, the growth rate of these cells was comparable to stem cell-derived embryonic hNSCs.
Several groups have therefore used IBMX to determine if the application of this small molecule to undifferentiated cells would induce cells toward a neuronal phenotype.
Tio et al, (2010) added IBMX to culture with MSCs derived from human umbilical cord blood, along with retinoic acid (RA) . This combination induced NF-L expression, which is a component of the cytoskeleton of neurons (Wang et al, 2012) .
IBMX has also been tested for effects on neuronal differentiation by Lepski et al, (2013) on NPCs . Their results showed that Forskolin or IBMX significantly enhanced neuronal function maturation. Further, within 1 week of treatment with IBMX, the expression of NA+ and K+ channels increased, along with an increased number of neurons which were positive for microtubule-associated protein-2 (MAP-2), a marker for mature neurons.
Mu et al, (2015) found similar results in MSCs from bone marrow: MSCs expressed the highest mRNA levels of nestin, MAP-2, Nse and Gfap, markers of mature neurons with IBMX / indomethacin . Protein levels of MAP-2 and GFAP were also higher in MSCs cultured in the presence of IBMX / indomethacin than those cultured with other small molecules. In addition, the authors looked at the cytotoxicity of the small molecules they used and determined that IBMX did not significantly affect proliferation and viability of MSCs.
Small molecules for neural stem cells from Hello Bio
- A83-01 Selective TGF-βRI (ALK5), ALK4 and ALK7 inhibitor. Maintains human induced pluripotent stem cell (hiPSC) self-renewal.
- DAPT γ-secretase inhibitor. Also induces neuronal differentiation.
- DBZ γ-secretase and NOTCH inhibitor.
- DMH-1 Selective ALK-2 inhibitor. Promotes iPSC neurogenesis.
- E C23 Synthetic retinoid. Induces neural differentiation of hESCs.
- Forskolin Cell permeable, reversible adenylyl cyclase activator. Neural differentiation inducer.
- IBMX Non-selective, competitive cAMP and cGMP PDE inhibitor. Facilitates neural progenitor cell differentiation
- ISX9 Neurogenic agent. Induces SVZ progenitor neuronal differentiation and cardiomyogenic differentiation
- Metformin hydrochloride LKBI/AMPK activator. Also promotes neurogenesis.
- Neuropathiazol Selective neuronal differentiation. Inducer in hippocampal neural progenitors.
- Rosiglitazone Potent and selective PPARγ agonist. Promotes adipocyte differentiation and enhances NPC proliferation.
- SAG Cell permeable sonic hedgehog (Shh) agonist and SMO agonist. Enhances neural differentiation
- SB 203580 Potent selective p38 MAPK inhibitor. Stimulates neural stem cell proliferation.
- SU 5402 Potent FGFR and VEGFR inhibitor. Attenuates integrin β4-induced neural stem cell differentiation.
- WHI-P 154 Non-selective JAK3 inhibitor and potent EGFR inhibitor. Induces neural progenitor cell differentiation
The studies presented here have shown that the addition of small molecules to stem cell cultures promote neurogenesis in vitro. Further, there is evidence that cells from these studies can subsequently be implanted in vivo, where they will become functional and establish long distance projections . Whilst this, in itself is exciting, there are additional benefits to using small molecules to promote neurogenesis in stem cells: culture of stem cells with small molecules significantly reduces the time to create differentiated cell lines (19 days for complete neural conversion versus 30-50 days using alternative methods). The use of the small molecules also reduces the need for expensive growth factors and serum, which also removes the issue of batch to batch variation. Finally, it has been shown that these cells can be grown under GMP conditions.
It is expected that the use of small molecules will quickly enable researchers to develop cell based strategies for disease modelling in vitro, as well as the very real possibility of developing cell therapy solutions for CNS disorders, thereby addressing a global need.
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