Innervated organoid compositions and methods of making same
Disclosed are in vitro methods for the differentiation of precursor cells into a neural crest cell (NCC) primed to a neurogenic lineage. The methods may include, for example, the steps of activating a Hedgehog signaling pathway (“HH signaling pathway”) in a precursor cell, wherein the precursor cell may be contacted with a neural crest cell induction medium for differentiation of the precursor cell into a neural crest cell. Compositions for carrying out the disclosed methods are also disclosed.
1 . A method of making an innervated colonic organoid model, comprising the step of
a. activating a Hedgehog signaling pathway (“HH signaling pathway”) in a precursor cell, wherein said precursor cell is contacted with a neural crest cell (“NCC”) induction medium until said precursor cell differentiates into an enteric neural crest cell (ENCC) primed to a neurogenic lineage, and wherein said precursor cell is a pluripotent stem cell;
b. contacting the ENCC primed to a neurogenic lineage with a hindgut spheroid; and
c. transplanting said hindgut spheroid and said ENCCs primed to a neurogenic lineage into a kidney capsule of an immunodeficient host until an innervated colonic organoid forms; wherein said hindgut spheroid is obtained from definitive endoderm.
2 . The method of claim 1 , wherein said innervated colonic organoid comprises NC cells differentiated into neurons, wherein said innervated colonic organoid is characterized by the formation of crypts and colonic epithelium and nerve cells, wherein said nerve cells are selected from TUJ1+neurons, S100b+ glia, and combinations thereof.
3 . The method of claim 1 , wherein said innervated colonic organoid is characterized by expression of one or markers selected from gut epithelium (CDH1+), colon (SATB2+), goblet (MUC2+), endocrine (GLP1+)-like cells, or a combination thereof.
4 . The method of claim 1 , wherein said neural crest cell induction medium comprises
a) knockout Dulbecco's Modified Eagle Medium (“DMEM”) and Knockout Serum Replacement (“KSR);
b) L-glutamine;
c) β-mercaptoethanol; and
d) LDN193189.
5 . The method of claim 4 , wherein said neural crest cell induction medium comprises CHIR99021 and/or retinoic acid.
6 . The method of claim 1 ,
wherein said ENCC have elevated expression of neuronal progenitor markers, loss of SRYbox transcription factor 10 (SOX10) expression, and co-expression of an enteric HOX code;
wherein said ENCC cells are sox10−, PHOX2B+, TUBB3high;
wherein said ENCC cells are TUBB3high, ELAVL4+; and/or
wherein said ENCC cells have reduced expression of NC markers transcription factor AP2-alpha (TFAP2A), snail family transcriptional repressor (SNAIL), and SRY-box transcription factor 10 (SOX10) and elevated expression of neurogenic markers tubulin beta-3 (TUBB3), ELAV Like RNA Binding Protein 4 (ELAV4), and paired-like homeobox 2B (PHOX2B).
7 . The method of claim 1 , wherein said precursor cell is an induced pluripotent stem cell.
8 . The method of claim 1 , wherein said HH signaling pathway activation is from about day 0 to about day 4 of said contacting step, or from about day 0 to about day 10 of said contacting step.
9 . The method of claim 1 , wherein activation of said HH signaling pathway comprises contacting said precursor cell with one or both of an HH agonist and an HH antagonist that acts as a partial agonist.
10 . The method of claim 9 , wherein said HH agonist, said HH antagonist that acts as a partial agonist, or combination thereof, is contacted with said precursor cell from day 0 to day 10 of said contacting step, or from day O to about day 4, or about day 4 to about day 10.
11 . The method of claim 9 , wherein said HH agonist is selected from a SMOOTHENED agonist (“SAG”), recombinant Sonic Hedgehog (Shh), Purmorphamine, and combinations thereof; and/or wherein said HH antagonist that acts as a partial agonist is selected from Cyclopamine, Vismodegib (GDC-0449), and combinations thereof.
12 . The method of claim 1 , wherein said ENCC is derived in the absence of SMOOTHENED agonist (SAG) and wherein said ENCC is characterized by elevated expression of p75 neurotrophin receptor (p75NTR), human natural killer-1 (HNK1), rearranged during transfection (RET), transcription factor activating protein 2 beta (TFAP2B), snail family transcriptional repressor 1 (SNAI1), snail family transcriptional repressor 2 (SNAI2), and/or SRYbox transcription factor 10 (SOX10) expression, and/or expression of an enteric HOX code, wherein expression of an enteric HOX code is selected from expression of one or more of homeobox B3 (HOXB3), homeobox B3 (HOXB4), and homeobox B3 (HOXB5).
13 . The method of claim 1 , wherein said precursor cell is a population of precursor cells, wherein said method provides a substantially homogenous population of ENCCs primed to a neurogenic lineage.
14 . The method of claim 1 , wherein said ENCC is capable of differentiation into other non-neurogenic lineages.
15 . The method of claim 1 , wherein said precursor cell is contacted with an HH activator for a period of time sufficient to increase the number of SOX2+NANOG− cells, having expression of sex-determining region Y-box 2 (SOX2) and lacking expression of nanog homeobox (NANOG).
16 . The method of claim 6 , wherein the neuronal progenitor markers comprise tubulin beta-3 (TUBB3), and co-expression of an enteric HOX code comprises expression of homeobox B3 (HOXB3), homeobox B4 (HOXB4), and homeobox B5 (HOXB5).
17 . The method of claim 1 , wherein the activation of a Hedgehog signaling pathway (“HH signaling pathway”) is from day 0 to day 10.
18 . The method of claim 17 , wherein the activation of a Hedgehog signaling pathway (“HH signaling pathway”) is from day 0 to day 4.
19 . The method of claim 1 , wherein the innervated colonic organoid expresses Protein Gene Product 9.5 (PGP9.5), neurofilament (NF), and TUJ1.
20 . The method of claim 1 , wherein greater than about 50% of neurons are mature enteric neurons expressing Calretinin, Protein Gene Product 9.5 (PGP9.5+) and neurofilament (NFL+).
21 . The method of claim 1 , wherein the innervated colonic organoid comprises increased numbers of mature neurons (PGP9.5+, NFL+) as compared to an innervated colonic organoid that has not been treated with an HH agonist.