Method of making in vivo human small intestine organoids from pluripotent stem cells
Disclosed are methods for making a vascularized hollow organ derived from human intestinal organoid (HIOs). The HIOs may be obtained from human embryonic stem cells (ESC's) and/or induced pluripotent stem cells (iPSCs), such that the HIO forms mature intestinal tissue. Also disclosed are methods for making a human intestinal tissue containing a functional enteric nervous system (ENS).
1. A method of making an intestinal organoid having enteric neuronal and glial cell types, comprising the steps of:
a) providing a mid/hindgut spheroid;
b) providing vagal neural crest cells;
c) combining and mechanically aggregating the mid/hindgut spheroid together with the vagal neural crest cells to form a mid/hindgut spheroid-neural crest cell aggregate; and
d) maintaining said mid/hindgut spheroid-neural crest cell aggregate in a growth media to form an intestinal organoid, wherein said intestinal organoid comprises neuronal cell types and glial cell types;
wherein the neuronal cell types express tyrosine hydroxylase (TH), calbindin, calretinin, and serotonin (5-HT),
wherein the mid/hindgut spheroid is derived from definitive endoderm;
wherein the vagal neural crest cells express Hoxb3, Hoxb5, or Hoxb7 and are derived from a neurosphere, wherein said neurosphere is derived from a human precursor cell selected from one or both of an embryonic stem cell (ESC) or an induced Pluripotent Stem Cell (IPSC); and
wherein the enteric neuronal cell types exhibit neuronal activity as measured by rhythmic waves of calcium transients.
2. The method of claim 1 wherein said neuronal cell types comprise βIII-tubulin.
3. The method of claim 1 , further comprising the step of transplanting said intestinal organoid in vivo into an intestinal vasculature of a mammal for a period of time sufficient to mature into an intestinal organoid comprising nNOS+ inhibitory neurons.
4. The method of claim 3 wherein said intestinal organoid comprises a functional enteric nervous system (ENS) and is capable of contractile activity.
5. The method of claim 1 , further comprising the step of transplanting said intestinal organoid in vivo into an intestinal vasculature of a mammal for a period of time sufficient to mature into an intestinal organoid comprising neurons, wherein said neurons are embedded within desmin+ smooth muscle layers.
6. The method of claim 1 , further comprising the step of transplanting said intestinal organoid in vivo into an intestinal vasculature of a mammal for a period of time sufficient to mature into an intestinal organoid comprising glial cells embedded within a mesenchymal layer of said intestinal organoids.
7. The method of claim 1 , further comprising the step of transplanting said intestinal organoid in vivo into an intestinal vasculature of a mammal for a period of time sufficient to mature into an intestinal organoid comprising dopaminergic neurons, interneurons, sensory neurons, excitatory neurons, and inhibitory neurons.
8. The method of claim 1 , wherein the mid/hindgut spheroid and vagal neural crest cells are mechanically aggregated by low speed centrifugation.
9. The method of claim 1 , wherein the enteric neuronal cell types are positive for the pan-neuronal marker PGP9.5 and βIII-tubulin, and comprise: dopaminergic neurons expressing TH, interneurons expressing 5-HT and ChAT, sensory neurons expressing calbindin, and/or excitatory neurons expressing calretinin; wherein the glial cell types are positive for the glial marker S100; and wherein the enteric neuronal and glial cell types are embedded in the mesenchyme of the intestinal organoid.