Peihua Jiang Monell Chemical Senses Center Topics
Overview of the sense of taste Identification of taste stem/progenitor cells Development of taste organoid culture system Use of taste organoid culture system to study taste tissue homeostasis Senses: creating an internal representation of the external world
Chemosensory The Sense of taste
Sweet Umami Bitter
Sour Salty
• The sense of taste influences our dietary intakes Taste buds in fungiform, foliate, and circumvallate papillae
Yarmolinsky et al., 2009 Cell 139: 234-244 Taste Receptors
Yarmolinsky et al., Cell 139, 234-244 (2009) Taste transduction elements
Taste transduction elements play key functions in T1R2+3 Taste cells
Gustducin Plc2
IP3 DAG Ca++
IP R TRPM5 3 3 Chemosensory cells in Na+ extra-oral tissues (e.g., Intestinal tuft cells)
Calhm1/Calhm3
ATP
Howitt et al., Science 351:1329-33 (2016) Inactivated Tas1r2 receptor gene explains why cats cannot taste sweet
Cats are indifferent to sweet-tasting foods Cats are obligate carnivores Cats lack a functional T1R2, therefore, a functional sweet taste receptor Taste receptors are shaped by and reflect a species' food choices
Beauchamp et al., Journal of Comparative and Physiological Psychology, 91(5): 1118-1127, 1977 Li et al., PLoS Genet. 1(1): e3 (2005) Tas1r genes are pseudogenized in dolphin and sea lion
* denotes an open reading frame-disrupting mutation Jiang et al., PNAS, 109:4956-61 (2012) Taste renewal: Tracking adult taste stem/progenitor cells
Taste cells continuously turn over throughout life, and adult taste stem/progenitor cells generate new taste cells to maintain taste tissue homeostasis. The average lifespan of taste bud cells has been estimated to be 8- 12 days.
Loss of taste in humans (e.g., cancer patients receiving chemotherapy or radiotherapy, elderly) can have a significant impact on quality of life, often lead to lost appetite and malnutrition Implications: Cell-based therapy for those suffering from taste loss Flavor discovery Adult taste stem/progenitor cells
What are adult stem/progenitor cells: Unique cells that are capable of self-renewal Have the ability to differentiate through a committed lineage and can differentiate to yield the major specialized cell types of the tissue or organ for tissue renewal and maintenance They are multipotent Green: Adult taste stem cells Blue: precursor cells Red: terminally differentiated cells (type I, II and III)
Type I: supporting cells Type II: sweet, bitter or umami taste receptor cells Type III: sour or salty cells How to identify adult taste stem cells?
Lineage tracing - labeling the cells in taste tissue with molecular markers and then determining the specialized cell types they can generate
Cell culture - isolating the cells, growing them in cell culture, and manipulating them, by adding growth factors, to determine what types they can generate
Transplanting Lgr5-GFP expressing cells are taste stem cells
GFP DAPI
Circumvallate Papilla • Lgr5-GFP+ cells are found in the posterior portion of the tongue (circumvallate and foliate papillae) GFP Yee et al., Stem Cells, 32: 991-1000 (2013) Lineage tracing to determine whether Lgr5- expressing cells generate taste cells
Tamoxifen inducible Cre Line x Reporter Line
tdTo mato Lgr5/ tdTo tdTo mato mato tdTo mato Time Lgr5+ cells give rise to taste bud cells and are self-renewing
1 TM injection 1 day Lgr5-GFP-IRES- creER+/-; Rosa26- tdTomato+/- mice • Green (Lgr5+ stem/progenitor cells) • Red (tdTomato, marked stem/progenitor cells and daughter cells)
1 month
Yee et al., Stem Cells, 32: 991-1000 (2013) Lgr5+ cells produce all three subtypes of taste cells in the circumvallate papilla
Type I: NTPDase2
Type II: Trpm5
Type III: 5-HT
Yee et al., Stem Cells, 32: 991-1000 (2013) Conclusion
Lgr5 marks taste stem/progenitor cells in the posterior tongue Question: Whether single Lgr5+ cells are multipotent or lineage- specific (e.g., sweet progenitor cells produce sweet taste cells) Growing taste bud-like structures (“organoids”) from Lgr5+ taste stem/progenitor cells
Dissociation Sorting
E(gf)N(oggin)R(spondin) medium
Ren et al., PNAS, 111:10401-6 (2014) Organoids derived from single Lgr5+ cells contain actively cycling cells
Ren et al., PNAS, 111:10401-6 (2014) Mature taste-like cells are generated in cultured organoids derived from single Lgr5+ cells
Green: anti-gustducin (type II)
Red: anti-Car4 (type III)
Taste organoid Ren et al., PNAS, 111:10401-6 (2014) Taste bud Mature Taste Cells Derived from Lgr5+ Cells Respond to Tastants
Sweeteners: Acesulfame-K Sucralose
Bitter compounds: Denatonium
Plc2 blocker: U73122
Ren et al., PNAS, 111:10401-6 (2014) Growing taste organoids from Trpm5-GFP trangenic mice
When taste cells start to differentiate?
Day 6 Day 7 Day 8 Day 9 Day 10
Ren et al., Sci Rep 7(1), 4004 (2017) A close-up view of organoid K-means clustering analysis of the RNA-seq data
Cluster 1 (559 genes) Cluster 1 (521 genes) Cluster 2 (1998 genes) D = Day Cluster 2 (1784 genes) Cluster 3 (9658 genes) Cluster 3 (10893 genes) Cluster 4 (1909 genes) Cluster 4 (926 genes) 3.5 Dataset 1 3.5 Dataset 2 3.0 3.0 2.5 2.5 2.0 2.0 1.5 1.5 1.0 1.0 0.5 0.5 0.0 0.0
Center Normalized Log2(RPKM)CenterNormalized D2 D4 D6 D8 D2 D4 D6 D8 D10 D12 D14 D10 D12 D14
Taste receptor genes and taste signaling component genes are found in subcluster 1
Ren et al., Sci Rep 7(1), 4004 (2017) Temporal expression of taste receptor genes and signaling components in organoids
100 80 60 40 Tas2r105 20 10 Tas2r135 Tas2r126 8 Tas2r113 6 Tas2r137 Tas2r108 4 Tas2r118 2.02 Tas1r3 Tas1r2 Tas1r1 1.5 Gnat3 (gustducin) Trpm5 Gng13 (G13) 1.0
0.5
0.0
RPKM (Reads Per Kilobase of transcript per Million mapped reads) mapped transcript of per Million Per (Reads Kilobase RPKM
D2 D4 D6 D8 D10 D12 D14 Days in culture
Ren et al., Sci Rep 7(1), 4004 (2017) Shh 516 Manoranjan signaling cell signaling generation in taste - 22 ( 2012 et et al., Pediatric Research ) Ren et al., Sci Rep 7 ( 1 71 ), 4004 : ( 2017
) GANT61 is an inhibitor of GLI1 and GLI2 + GANT61 Day +GANT61 0 41014
D4-D14 D0-D10 D0-D4 Control D 0 Brightfield - D 4 D 0 - D 10 Day - 14 Car Organoids D 4 4 - D 14 Merged Conclusion
Single Lgr5+ cells can give rise to all types of differentiated taste cells Lgr5+ cells are multipotent stem/progenitor cells Taste organoid culture system recapitulates the process of native taste cell generation Adult taste stem cell-derived organoids can be used to study taste tissue homeostasis Next steps
Taste stem cells can grow and generate mature taste cells when cultured in dish (taste organoids) in the absence of neuronal innervation.
Yet, taste buds degenerate when gustatory nerves are transected.
What is the niche factor(s) derived from gustatory nerves that regulate taste tissue homeostasis Taste tissue homeostasis in the absence of innervation (glossopharyngeal cut)
Control d6 GP nerve transection d6 GP nerve transection d14
Car4 (marks type III taste cells) staining of the circumvallate papilla
Future direction: What is the niche factor produced by neurons that innervate taste tissue to regulate taste tissue homeostasis?
Lin et al., unpublished data Acknowledgements
Monell University of Cinninatti My laboratory Eitaro Aihara Wenwen Ren Weiwei Lei Jamie Watson Tokyo University of Agriculture Amanda Soohoo Ken Iwatsuki Nishi Gheewala Minliang Zhou Xiaoli Lin USDA Collaborators Joe Urban Bob Margolskee Ichiro Matsumoto Wuhan University Huabin Zhao
Funding: NIDCD, PA health fund, Gates Foundation