Methods and compositions for producing pancreatic beta cells
Inventors
Hebrok, Matthias • Russ, Holger Andreas • Ding, Sheng • Zhu, Saiyong
Assignees
J David Gladstone Institutes • University of California San Diego UCSD
Publication Number
US-11332716-B2
Publication Date
2022-05-17
Expiration Date
2036-07-26
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Abstract
Compositions and methods of producing mammalian cell populations that include a high proportion of pancreatic beta cells are described herein. Such cell populations are useful for treatment of diabetes. Also provided are materials and methods for the direct differentiation of stem cells, such as embryonic stem cells, into functional pancreatic beta cells. The disclosure provides the benefit of direct differentiation, which results in the production of functional pancreatic beta cells efficiently and at low cost.
Core Innovation
The invention relates to compositions and methods for producing mammalian cell populations that include a high proportion of pancreatic beta-like cells and/or mature pancreatic cells. Such populations are particularly useful for the treatment of diabetes. The methods described allow for the direct differentiation of stem cells, such as human embryonic stem cells, into functional pancreatic beta cells efficiently and at low cost.
The disclosed technology solves the problem of scarcity and inefficiency in producing functional pancreatic beta-like cells by providing a simplified suspension-culture-based differentiation protocol. This protocol allows for the correct temporal specification of pancreatic and endocrine progenitors into glucose-responsive pancreatic beta cells in vitro, preventing the formation of nonfunctional polyhormonal endocrine cells. The method involves the exclusion of commonly used BMP inhibitors during early differentiation stages resulting in the prevention of premature endocrine induction, and sequential exposure to retinoic acid followed by combined EGF/KGF treatment which establishes highly pure progenitor populations expressing PDX1 and NKX6.1 transcription factors.
Upon precise temporal induction of the endocrine differentiation factor NEUROG3 in PDX1+/NKX6.1+ progenitors, functional pancreatic beta cells are generated that exhibit key features of bona fide human pancreatic beta cells. These cells remain functional after short-term transplantation and effectively reduce blood glucose levels in diabetic mice. The disclosed methods also involve exposure to vitamin C and BayK-8644, compounds that improve the generation of functional pancreatic beta cells, enhancing insulin production and glucose-stimulated insulin secretion.
Claims Coverage
The patent contains one independent claim that broadly covers a method of generating functional pancreatic beta-like cells from pancreatic endodermal progenitor cells and administering these cells for treatment of diabetes.
Method for generating pancreatic beta-like cells from pancreatic endodermal progenitor cells
A method comprising: (a) contacting pancreatic endodermal progenitor cells with a first composition consisting essentially of vitamin C, BayK-8644, heparin, zinc sulfate, Alk5 inhibitor II, LDN-193189, T3 thyroid hormone, and Compound E for three to fifteen days to generate a first cell population; (b) contacting the first cell population with a second composition consisting essentially of vitamin C, BayK-8644, heparin, zinc sulfate, Alk5 inhibitor II, T3 thyroid hormone, cysteine, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid, and R8428 for three to fifteen days to generate a second cell population; and (c) culturing the second cell population as 3D aggregates in low-attachment plates for three to fifteen days to thereby generate a third cell population comprising functional pancreatic beta-like cells.
Treatment of diabetes by administration of functional pancreatic beta-like cells
Administering the functional pancreatic beta-like cells generated by the method to a mammal in need thereof, whereby the mammal has type I, type II, or type 1.5 diabetes.
The claims cover a method for generating functional pancreatic beta-like cells by sequential treatment with specific compositions containing vitamin C, BayK-8644, growth factors, signaling inhibitors, and antioxidants, followed by 3D culturing. The claimed method includes administration of the produced cells for diabetes treatment.
Stated Advantages
Provides efficient and direct differentiation of stem cells into functional pancreatic beta cells without generating nonfunctional polyhormonal cells.
Yields a fast and reproducible supply of glucose-responsive pancreatic beta cells.
Avoids precocious endocrine induction by omitting BMP inhibitors during early differentiation stages.
Improves pancreatic beta cell survival and function upon transplantation, allowing immediate functionality post-transplant.
Utilizes vitamin C and BayK-8644 to enhance insulin production and glucose responsiveness of generated pancreatic beta cells.
Documented Applications
Use of generated pancreatic beta-like cells for the treatment of diabetes mellitus type 1, type 2, or type 1.5 in mammals including humans.
Cell therapy applications involving transplantation of functional pancreatic beta cells to restore insulin production and glucose regulation.
Basic research tool to study pancreatic development and beta cell biology.
Tools for drug discovery, toxicology screening, and identification of therapeutic targets.
Use in bioengineering devices and development of pancreatic islets or organs on chips.
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