Highly Efficient Differentiation and Enrichment of Spinal Motor Neurons Derived from Human and Monkey Embryonic Stem Cells

Abstract

BackgroundThere are no cures or efficacious treatments for severe motor neuron diseases. It is extremely difficult to obtain naïve spinal motor neurons (sMNs) from human tissues for research due to both technical and ethical reasons. Human embryonic stem cells (hESCs) are alternative sources. Several methods for MN differentiation have been reported. However, efficient production of naïve sMNs and culture cost were not taken into consideration in most of the methods.Methods/Principal FindingsWe aimed to establish protocols for efficient production and enrichment of sMNs derived from pluripotent stem cells. Nestin+ neural stem cell (NSC) clusters were induced by Noggin or a small molecule inhibitor of BMP signaling. After dissociation of NSC clusters, neurospheres were formed in a floating culture containing FGF2. The number of NSCs in neurospheres could be expanded more than 30-fold via several passages. More than 33% of HB9+ sMN progenitor cells were observed after differentiation of dissociated neurospheres by all-trans retinoic acid (ATRA) and a Shh agonist for another week on monolayer culture. HB9+ sMN progenitor cells were enriched by gradient centrifugation up to 80% purity. These HB9+ cells differentiated into electrophysiologically functional cells and formed synapses with myotubes during a few weeks after ATRA/SAG treatment.Conclusions and SignificanceThe series of procedures we established here, namely neural induction, NSC expansion, sMN differentiation and sMN purification, can provide large quantities of naïve sMNs derived from human and monkey pluripotent stem cells. Using small molecule reagents, reduction of culture cost could be achieved.

Advances in stem cell therapy for spinal cord injury

“…Transplanted ES cells will form teratomas, and thus, ES cells must be predifferentiated prior to grafting. Protocols have been developed to differentiate ES cells into neural precursors (27)(28)(29)(30) and specific neuronal (30)(31)(32) and glial lineages (33,34). Predifferentiated mouse ES cells transplanted into the injured rat spinal cord differentiated into neurons and glia and showed partial functional recovery (35).…”

Potential for pharmacological manipulation of human embryonic stem cells

“…WNT, TGFb, and FGF/ERK Mature neurons (Sasaki et al, 2010) Cobalt chloride HIF-1a stabilization Cardiomyocyte differentiation to functionally mature cardiomyocytes (Ng et al, 2011) Corticosteroids Trophoblast and mesodermal (Barbaric et al, 2010) CSA Calcineurin NFAT Cardiomyocyte differentiation from ESC-derived mesodermal cells in visceral endodermal stromal cell co-culture (Mummery et al, 2003) Decreased hypertrophy of ESC-derived cardiomyocytes (Lim et al, 2000) Cyclopamine SMO Hh Astrocytes Cymarin RARs/RXRs Mesodermal/endodermal (Desbordes et al, 2008) Dorsomorphin ALK2, 3 and 6 TGFb Neurogenesis Wada et al, 2009;Zhou et al, 2010) …”

“…Of further interest was their finding that dorsomorphin was ineffective at inducing neural conversion in mESCs, demonstrating that small molecules may have species-specific effects . Additionally, it was demonstrated that dorsomorphin is important in the initial differentiation of NSCs/NPCs for the induction of spinal motor neuron differentiation from hESCs (Wada et al, 2009).…”

Modelling of Neurological Diseases Using Induced Pluripotent Stem Cells

“…Because neuroectodermal cells differentiated from hESCs generally have a rostral character, motoneuron differentiation is then achieved by the caudalizing factor retinoic acid followed by the ventralizing factor Shh (Li et al, 2005). Small molecules that can activate the Shh signaling, purmorphamine and SAG (a chlorobenzothiophene-containing Hh pathway agonist), have been shown to be effective as substitutes of Shh in motoneuron differentiation , Wada et al, 2009). The use of Hb9 (homeobox gene selectively expressed in motoneurons) promoter driven GFP hESCs allows isolation of a fairly pure population of motoneurons (Singh Roy et al, 2005).…”

“…The use of Hb9 (homeobox gene selectively expressed in motoneurons) promoter driven GFP hESCs allows isolation of a fairly pure population of motoneurons (Singh Roy et al, 2005). Enrichment of derived motoneurons, up to 80%, was also shown to be achieved by gradient centrifugation in Biocoll (Wada et al, 2009) …”