RESUMEN
Neural crest stem cells (NCPCs) have been shown to differentiate into various cell types and tissues during embryonic development, including sensory neurons. The few studies addressing the generation of NCPCs and peripheral sensory neurons (PSNs) from human induced pluripotent stem cells (hiPSCs), generated sensory cells without displaying robust activity. Here, we describe an efficient strategy for hiPSCs differentiation into NCPCs and functional PSNs using chemically defined media and factors to achieve efficient differentiation, confirmed by the expression of specific markers. After 10 days hiPSCs differentiated into NCPCs, cells were then maintained in neural induction medium containing defined growth factors for PSNs differentiation, followed by 10 days in neonatal human epidermal keratinocytes- (HEKn-) conditioned medium (CM). We observed a further increase in PSN markers expression and neurites length after CM treatment. The resulting neurons elicited action potentials after current injection and released substance P (SP) in response to nociceptive agents such as anandamide and resiniferatoxin. Anandamide induced substance P release via activation of TRPV1 and not CB1. Transcriptomic analysis of the PSNs revealed the main dorsal root ganglia neuronal markers and a transcriptional profile compatible with C fiber-low threshold mechanoreceptors. TRPV1 was detected by immunofluorescence and RNA-Seq in multiple experiments. In conclusion, the developed strategy generated PSNs useful for drug screening that could be applied to patient-derived hiPSCs, consisting in a powerful tool to model human diseases in vitro.
RESUMEN
BACKGROUND: Sugarcane breeding has significantly progressed in the last 30 years, but achieving additional yield gains has been difficult because of the constraints imposed by the complex ploidy of this crop. Sugarcane cultivars are interspecific hybrids between Saccharum officinarum and Saccharum spontaneum. S. officinarum is an octoploid with 2n = 80 chromosomes while S. spontaneum has 2n = 40 to 128 chromosomes and ploidy varying from 5 to 16. The hybrid genome is composed of 70-80% S. officinaram and 5-20% S. spontaneum chromosomes and a small proportion of recombinants. Sequencing the genome of this complex crop may help identify useful genes, either per se or through comparative genomics using closely related grasses. The construction and sequencing of a bacterial artificial chromosome (BAC) library of an elite commercial variety of sugarcane could help assembly the sugarcane genome. RESULTS: A BAC library designated SS_SBa was constructed with DNA isolated from the commercial sugarcane variety SP80-3280. The library contains 36,864 clones with an average insert size of 125 Kb, 88% of which has inserts larger than 90 Kb. Based on the estimated genome size of 760-930 Mb, the library exhibits 5-6 times coverage the monoploid sugarcane genome. Bidirectional BAC end sequencing (BESs) from a random sample of 192 BAC clones sampled genes and repetitive elements of the sugarcane genome. Forty-five per cent of the total BES nucleotides represents repetitive elements, 83% of which belonging to LTR retrotransposons. Alignment of BESs corresponding to 42 BACs to the genome sequence of the 10 sorghum chromosomes revealed regions of microsynteny, with expansions and contractions of sorghum genome regions relative to the sugarcane BAC clones. In general, the sampled sorghum genome regions presented an average 29% expansion in relation to the sugarcane syntenic BACs. CONCLUSION: The SS_SBa BAC library represents a new resource for sugarcane genome sequencing. An analysis of insert size, genome coverage and orthologous alignment with the sorghum genome revealed that the library presents whole genome coverage. The comparison of syntenic regions of the sorghum genome to 42 SS_SBa BES pairs revealed that the sorghum genome is expanded in relation to the sugarcane genome.
