Controlled induction of human pancreatic progenitors produces functional beta-like cells
Inventors
Hebrok, Matthias • Russ, Holger Andreas
Assignees
University of California San Diego UCSD • University of California Irvine
Publication Number
US-10975355-B2
Publication Date
2021-04-13
Expiration Date
2036-04-22
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Abstract
Methods are provided for the simple, fast, effective and safe directed differentiation of embryonic stem cells into pancreatic beta-like cells secreting insulin in response to glucose levels. The cells are useful transplant therapeutics for diabetic individuals.
Core Innovation
The disclosed invention provides a simplified, rapid, and safe method for the directed differentiation of embryonic stem cells into functional pancreatic beta-like cells that secrete insulin in response to glucose. The process utilizes a suspension-culture-based differentiation protocol, which enables the correct temporal specification of pancreatic and endocrine progenitors, resulting in glucose-responsive beta-like cells generated in vitro. A key aspect of the method is the exclusion of commonly used bone morphogenetic protein (BMP) inhibitors during the initial stages of differentiation to prevent precocious induction of endocrine fate.
Sequential induction of pancreatic lineage is achieved by first exposing foregut cells derived from embryonic stem cells to retinoic acid, followed by treatment with a combination of epidermal growth factor (EGF) and keratinocyte growth factor (KGF), yielding highly pure PDX1+ and PDX1+/NKX6.1+ progenitor populations. Endocrine differentiation is then precisely timed in these PDX1+/NKX6.1+ progenitors via induction of NEUROG3 expression, facilitating the formation of functional, glucose-responsive beta-like cells that express insulin.
The primary problem addressed by this invention is the inefficiency and lack of specificity in existing differentiation protocols, which often result in polyhormonal cells that do not secrete insulin in response to glucose and lack appropriate beta cell transcription factors, such as NKX6.1. Additionally, the severe shortage of donor material and variability in islet preparations limits the scalability and reproducibility of cadaveric islet transplantation for diabetes treatment. The simplified, scalable protocol disclosed allows for fast, reproducible production of functional human beta-like cells and supports detailed research into pancreas development and beta cell biology.
Claims Coverage
The patent includes two independent claims focused on the controlled differentiation of embryonic stem cells into pancreatic progenitor and beta-like cells, with specific inventive features relating to the sequence and combination of signaling factors and the prevention of premature endocrine induction.
Generation of PDX1+/NKX6.1+ progenitor cells by temporal exposure to specific factors and omission of BMP inhibitor prior to NKX6.1 expression
A method of generating a PDX1+/NKX6.1+ progenitor cell comprises: - Contacting an embryonic stem cell with an effective amount of a retinoic acid compound to induce a PDX1+ progenitor cell. - Subsequently, contacting the PDX1+ progenitor cell with effective amounts of epidermal growth factor and keratinocyte growth factor. - Importantly, the PDX1+ progenitor cell is not contacted with a bone morphogenic protein (BMP) inhibitor prior to expression of NKX6.1 by the cell. - This sequence induces formation of a PDX1+/NKX6.1+ progenitor cell.
Production of functional INS+/NKX6.1+ beta-like cells by precise induction of NEUROG3 expression in PDX1+/NKX6.1+ progenitors
A method for further differentiating the PDX1+/NKX6.1+ progenitor cell includes: - Inducing expression of NEUROG3 in the PDX1+/NKX6.1+ progenitor cell by contacting it with an effective amount of an inhibitor of bone morphogenetic protein, an inhibitor of TGFβ/ALK, or an inhibitor of sonic hedgehog. - This leads to the production of an INS+/NKX6.1+ cell, which is a beta-like cell responsive to glucose levels.
Use of the derived INS+/NKX6.1+ beta-like cell for transplantation
A method comprising transplanting the INS+/NKX6.1+ cell produced by the above differentiation protocol into a human, including a diabetic human.
In summary, the inventive features cover controlled temporal induction of pancreatic progenitors and beta-like cells from embryonic stem cells through a specific sequence and combination of factors, deliberate omission of BMP inhibitors during early specification, and application of the resulting beta-like cells for transplantation therapies.
Stated Advantages
Provides a fast and reproducible supply of functional human beta-like cells.
The resulting beta-like cells are glucose responsive and exhibit key features of bona fide human beta cells.
Beta-like cells remain functional after short-term transplantation and reduce blood glucose levels in diabetic mice.
The protocol enables accurate recapitulation of key steps of human pancreas development.
The method reduces the formation of unwanted polyhormonal endocrine cells by preventing precocious endocrine induction.
The system is scalable and facilitates detailed investigations into human pancreas development and beta cell function.
Eliminates dependence on variable cadaveric islet preparations and overcomes donor shortage issues.
Documented Applications
Transplant therapeutics for diabetic individuals, using human beta-like cells produced by the disclosed methods.
Tool for accelerating understanding of the biology of human beta cells, including large scale drug screens and gene function studies.
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