Induction of hepatocytes by stem cell differentiation using RNA

Abstract

To provide a novel method for inducing or generating hepatocytes from human induced pluripotent stem cells with unprecedented efficiency and functionality. [Solution] The core of the present invention is the use of mRNA discovered through experiments at numerous decisive differentiation points in a previously unknown manner along the pathway from pluripotency to mesoderm to endoderm to hepatocyte pathway. This disclosure provides a method for inducing stem cell differentiation by modulating cell growth dynamics and related parameters (thereby using specific combinations of cell density, reagent concentration, and mRNA combinations to control the direction of differentiation / induction).

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 62 / 423,113, filed on November 16, 2016, which is hereby incorporated by reference herein in its entirety.

[0002] Field of the Invention This disclosure relates to directing the induction of hepatocytes from pluripotent stem cells through a dynamically controlled cell growth process that utilizes specific combinations and ranges of cell density, reagent concentration, and specific combinations of mRNA.

Background Art

[0003] Background of the Invention Recent efforts in the generation and resultant differentiation of human stem cells have changed the paradigm regarding cell - fate plasticity, models of human disease, and clinically relevant therapeutic agents. Both embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) generated from somatic cells can be differentiated into an ever - growing list of specific cell types indistinguishable from their corresponding primary cells. As a result, stem cells are extremely promising for the development of novel human cell therapies. iPSCs exhibit particular potential in the field of personalized medicine due to their unlimited cell availability, the non - invasive procedure for obtaining such cells, and the potential ability to tailor each treatment to an individual patient's immune system by liberating them from immunosuppressive drugs.

[0004] Significant research funding is being spent on developing cell replacement therapies to treat or prevent various human diseases. For example, liver diseases (e.g., hepatic fibrosis and cirrhosis, which can often lead to late-stage liver failure) can be treated by transplantation of donated human liver organs or organ-derived hepatocytes. However, there are still considerable hurdles to overcome in finding a reliable supply of donor livers. Now, many academic and industrial groups have developed methods to orient ESCs or iPSCs into hepatocytes using numerous recombinant growth factors in the form of recombinant proteins, but these factors are expensive and difficult to control at effective doses.

[0005] To mitigate the cost burden and inconsistencies, some researchers have attempted to identify small molecules that could influence signaling pathways as agonists or antagonists of growth factor receptors. While typically less expensive than growth factors, one major drawback of small molecules is the nonspecific effects they can exert on unintended targets (e.g., cell membrane-bound receptors, intracellular organelles, or genomic components).

[0006] Another key component of typical differentiation protocols is the culture medium for culturing cells, which can consist of nutrients (lipids, amino acids, carbohydrates, vitamins, etc.), appropriate concentrations of salt, pH buffers, critical elements, and common protein factors (e.g., insulin or serum albumin). Different cell types have different nutrient requirements and medium components, further complicated by cell type-specific growth factors and small molecules for signaling.

[0007] In the clinical application of stem cell-derived tissue cells, most components of established differentiation media require individual assurance under the latest cGMP (Conditional Good Manufacturing Practice) standards; for example, growth factors must be manufactured using special procedures and require individual assurance. [Overview of the project] [Means for solving the problem]

[0008] Summary of the Invention This disclosure provides a method for inducing stem cell differentiation by modulating cell growth dynamics and related parameters (thereby using specific combinations of cell density, reagent concentration, and mRNA to control the orientation of differentiation / induction).

[0009] To achieve the objectives and in accordance with the objectives of the present invention, as embodied and broadly described herein, one aspect of the present invention relates to a method for inducing the differentiation of stem cells into hepatocytes, the method comprising: (a) culturing induced pluripotent stem cells as starting cells under conditions for differentiation; (b) inducing the starting cells to move from a pluripotent state toward a mesoendodermal lineage; (c) orienting the differentiating cells toward endodermal cells through transfection of the cultured cells with a first combination of mRNA at an effective dose and within a specific time range; (d) further orienting the endodermal cells toward hepatic progenitor cells through transfection with a second combination of mRNA; (e) further maturing the hepatic progenitor cells toward hepatocytes with a third combination of mRNA; and (f) obtaining hepatocytes by passage the progenitor cell clusters into a monolayer or by collecting clusters formed from the hepatic progenitor cells and re-platering them into a monolayer.

[0010] In one embodiment, the present invention relates to a method for inducing the differentiation of stem cells into hepatocytes, the method comprising: (a) culturing induced pluripotent stem cells as starting cells under conditions for differentiation; (b) inducing the starting cells to move from a pluripotent state toward a mesoendodermal lineage; (c) orienting the differentiating cells toward endodermal cells through transfection of cultured cells with a first combination of mRNA at an effective dose and within a specific time range; (d) further orienting the endodermal cells toward hepatic progenitor cells through transfection with a second combination of mRNA; (e) further maturing the hepatic progenitor cells toward hepatocytes with a third combination of mRNA; and (f) obtaining hepatocytes by passage the progenitor cell clusters into a monolayer or by collecting clusters formed from the hepatic progenitor cells and re-platening them into a monolayer, wherein the first combination of mRNA includes FoxA2 mRNA.

[0011] In another embodiment, the present invention relates to a method for inducing the differentiation of stem cells into hepatocytes, the method comprising: (a) culturing induced pluripotent stem cells as starting cells under conditions for differentiation; (b) inducing the starting cells to move from a pluripotent state toward a mesoendodermal lineage; (c) orienting the differentiating cells toward endodermal cells through transfection of the cultured cells with a first combination of mRNA at an effective dose and within a specific time range; (d) further orienting the endodermal cells toward hepatic progenitor cells through transfection with a second combination of mRNA; (e) further maturing the hepatic progenitor cells toward hepatocytes with a third combination of mRNA; and (f) obtaining hepatocytes by passage the progenitor cell clusters into a monolayer or by collecting clusters formed from the hepatic progenitor cells and re-platening them into a monolayer, wherein the first combination of mRNA comprises Sox17 mRNA.

[0012] In another embodiment, the present invention relates to a method for inducing the differentiation of stem cells into hepatocytes, the method comprising: (a) culturing induced pluripotent stem cells as starting cells under conditions for differentiation; (b) inducing the starting cells to move from a pluripotent state toward a mesoendodermal lineage; (c) orienting the differentiating cells toward endodermal cells through transfection of the cultured cells with a first combination of mRNA at an effective dose and within a specific time range; (d) further orienting the endodermal cells toward hepatic progenitor cells through transfection with a second combination of mRNA; (e) further maturing the hepatic progenitor cells toward hepatocytes with a third combination of mRNA; and (f) obtaining hepatocytes by passage the progenitor cell clusters into a monolayer or by collecting clusters formed from the hepatic progenitor cells and re-platening them into a monolayer, wherein the first combination of mRNA comprises FoxA2 and Sox17 mRNA.

[0013] In another embodiment, the present invention relates to a method for inducing the differentiation of stem cells into hepatocytes, the method comprising: (a) culturing induced pluripotent stem cells as starting cells under conditions for differentiation; (b) inducing the starting cells to move from a pluripotent state toward a mesoendodermal lineage; (c) orienting the differentiating cells toward endodermal cells through transfection of the cultured cells with a first combination of mRNA at an effective dose and within a specific time range; (d) further orienting the endodermal cells toward hepatic progenitor cells through transfection with a second combination of mRNA; (e) further maturing the hepatic progenitor cells toward hepatocytes with a third combination of mRNA; and (f) obtaining hepatocytes by passage the progenitor cell clusters into a monolayer or by collecting clusters formed from the hepatic progenitor cells and re-platening them into a monolayer, wherein the first combination of mRNA comprises FoxA2, ​​Sox17, GATA4, and GATA6 mRNA.

[0014] In another embodiment, the present invention relates to a method for inducing the differentiation of stem cells into hepatocytes, the method comprising: (a) culturing induced pluripotent stem cells as starting cells under conditions for differentiation; (b) inducing the starting cells to move from a pluripotent state toward a mesoendodermal lineage; (c) orienting the differentiating cells toward endodermal cells through transfection of the cultured cells with a first combination of mRNA at an effective dose and within a specific time range; (d) further orienting the endodermal cells toward hepatic progenitor cells through transfection with a second combination of mRNA; (e) further maturing the hepatic progenitor cells toward hepatocytes with a third combination of mRNA; and (f) obtaining hepatocytes by passage the progenitor cell clusters into a monolayer or by collecting clusters formed from the hepatic progenitor cells and re-platening them into a monolayer, wherein the second combination of mRNA includes Hex mRNA.

[0015] In another embodiment, the present invention relates to a method for inducing the differentiation of stem cells into hepatocytes, the method comprising: (a) culturing induced pluripotent stem cells as starting cells under conditions for differentiation; (b) inducing the starting cells to move from a pluripotent state toward a mesoendodermal lineage; (c) orienting the differentiating cells toward endodermal cells through transfection of the cultured cells with a first combination of mRNA at an effective dose and within a specific time range; (d) further orienting the endodermal cells toward hepatic progenitor cells through transfection with a second combination of mRNA; (e) further maturing the hepatic progenitor cells toward hepatocytes with a third combination of mRNA; and (f) obtaining hepatocytes by passage the progenitor cell clusters into a monolayer or by collecting clusters formed from the hepatic progenitor cells and re-platening them into a monolayer, wherein the second combination of mRNA includes Tbx3 mRNA.

[0016] In another embodiment, the present invention relates to a method for inducing the differentiation of stem cells into hepatocytes, the method comprising: (a) culturing induced pluripotent stem cells as starting cells under conditions for differentiation; (b) inducing the starting cells to move from a pluripotent state toward a mesoendodermal lineage; (c) orienting the differentiating cells toward endodermal cells through transfection of the cultured cells with a first combination of mRNA at an effective dose and within a specific time range; (d) further orienting the endodermal cells toward hepatic progenitor cells through transfection with a second combination of mRNA; (e) further maturing the hepatic progenitor cells toward hepatocytes with a third combination of mRNA; and (f) obtaining hepatocytes by passage the progenitor cell clusters into a monolayer or by collecting clusters formed from the hepatic progenitor cells and re-platening them into a monolayer, wherein the second combination of mRNA comprises Tbx3 and Hex mRNA.

[0017] In another embodiment, the present invention relates to a method for inducing the differentiation of stem cells into hepatocytes, the method comprising: (a) culturing induced pluripotent stem cells as starting cells under conditions for differentiation; (b) inducing the starting cells to move from a pluripotent state toward a mesoendodermal lineage; (c) orienting the differentiating cells toward endodermal cells through transfection of the cultured cells with a first combination of mRNA at an effective dose and within a specific time range; (d) further orienting the endodermal cells toward hepatic progenitor cells through transfection with a second combination of mRNA; (e) further maturing the hepatic progenitor cells toward hepatocytes with a third combination of mRNA; and (f) obtaining hepatocytes by passage the progenitor cell clusters into a monolayer or by collecting clusters formed from the hepatic progenitor cells and re-platening them into a monolayer, wherein the second combination of mRNA comprises Tbx3, GATA4, GATA6, and Hex mRNA.

[0018] In another embodiment, the present invention relates to a method for inducing the differentiation of stem cells into hepatocytes, the method comprising: (a) culturing induced pluripotent stem cells as starting cells under conditions for differentiation; (b) inducing the starting cells to move from a pluripotent state toward a mesoendodermal lineage; (c) orienting the differentiating cells toward endodermal cells through transfection of the cultured cells with a first combination of mRNA at an effective dose and within a specific time range; (d) further orienting the endodermal cells toward hepatic progenitor cells through transfection with a second combination of mRNA; (e) further maturing the hepatic progenitor cells toward hepatocytes with a third combination of mRNA; and (f) obtaining hepatocytes by passage the progenitor cell clusters into a monolayer or by collecting clusters formed from the hepatic progenitor cells and re-platening them into a monolayer, wherein the third combination of mRNA includes HNF1a mRNA.

[0019] In another embodiment, the present invention relates to a method for inducing the differentiation of stem cells into hepatocytes, the method comprising: (a) culturing induced pluripotent stem cells as starting cells under conditions for differentiation; (b) inducing the starting cells to move from a pluripotent state toward a mesoendodermal lineage; (c) orienting the differentiating cells toward endodermal cells through transfection of the cultured cells with a first combination of mRNA at an effective dose and within a specific time range; (d) further orienting the endodermal cells toward hepatic progenitor cells through transfection with a second combination of mRNA; (e) further maturing the hepatic progenitor cells toward hepatocytes with a third combination of mRNA; and (f) obtaining hepatocytes by passage the progenitor cell clusters into a monolayer or by collecting clusters formed from the hepatic progenitor cells and re-platening them into a monolayer, wherein the third combination of mRNA includes HNF4a mRNA.

[0020] In another embodiment, the present invention relates to a method for inducing the differentiation of stem cells into hepatocytes, the method comprising: (a) culturing induced pluripotent stem cells as starting cells under conditions for differentiation; (b) inducing the starting cells to move from a pluripotent state toward a mesoendodermal lineage; (c) orienting the differentiating cells toward endodermal cells through transfection of cultured cells with a first combination of mRNA at an effective dose and within a specific time range; (d) further orienting the endodermal cells toward hepatic progenitor cells through transfection with a second combination of mRNA; (e) further maturing the hepatic progenitor cells toward hepatocytes with a third combination of mRNA; and (f) obtaining hepatocytes by passage the progenitor cell clusters into a monolayer or by collecting clusters formed from the hepatic progenitor cells and re-platening them into a monolayer, wherein the third combination of mRNA includes HNF4a, HNF1a, HNF6, CEB / Pa, and CEB / Pb mRNA.

[0021] In another embodiment, the present invention relates to a method for inducing the differentiation of stem cells into hepatocytes, the method comprising: (a) culturing induced pluripotent stem cells as starting cells under conditions for differentiation; (b) inducing the starting cells to move from a pluripotent state toward a mesoendodermal lineage; (c) orienting the differentiating cells toward endodermal cells through transfection of the cultured cells with a first combination of mRNA at an effective dose and within a specific time range; (d) further orienting the endodermal cells toward hepatic progenitor cells through transfection with a second combination of mRNA; (e) further maturing the hepatic progenitor cells toward hepatocytes with a third combination of mRNA; and (f) obtaining hepatocytes by passage the progenitor cell clusters into a monolayer or by collecting clusters formed from the hepatic progenitor cells and re-platening them into a monolayer, wherein the starting cells are collected from body fluid or tissue.

[0022] One aspect of the present invention relates to cells obtained by a method for inducing the differentiation of stem cells into hepatocytes, the method comprising: (a) culturing induced pluripotent stem cells as starting cells under conditions for differentiation; (b) inducing the starting cells to exit the pluripotent state towards the mesendoderm lineage; (c) directing the differentiating cells towards endoderm cells through transfection of the cultured cells with a first combination of mRNAs at an effective dose and within a specific time period; (d) further directing the endoderm cells towards hepatocyte progenitor cells through transfection with a second combination of mRNAs; (e) further maturing the hepatocyte progenitor cells into hepatocytes with a third combination of mRNAs; and (f) obtaining the hepatocytes by passaging the progenitor cell clusters into a monolayer or collecting the clusters formed from the hepatocyte progenitor cells and replating them into a monolayer.

[0023] One aspect of the present invention relates to a composition for treating a disease, disorder, or dysplasia, comprising cells obtained by a method for inducing the differentiation of stem cells into hepatocytes, the method comprising: (a) culturing induced pluripotent stem cells as starting cells under conditions for differentiation; (b) inducing the starting cells to exit the pluripotent state towards the mesendoderm lineage; (c) directing the differentiating cells towards endoderm cells through transfection of the cultured cells with a first combination of mRNAs at an effective dose and within a specific time period; (d) further directing the endoderm cells towards hepatocyte progenitor cells through transfection with a second combination of mRNAs; (e) further maturing the hepatocyte progenitor cells into hepatocytes with a third combination of mRNAs; and (f) obtaining the hepatocytes by passaging the progenitor cell clusters into a monolayer or collecting the clusters formed from the hepatocyte progenitor cells and replating them into a monolayer.

[0024] One aspect of the present invention is a method for treating a disease, disorder, or malformation, the method comprising the steps of administering at least one of the following to a subject in need: cells obtained by a method for inducing differentiation of stem cells into hepatocytes, the method comprising: (a) culturing induced pluripotent stem cells as starting cells under conditions for differentiation; (b) inducing the starting cells to move from a pluripotent state toward a mesoendodermal lineage; (c) orienting the differentiating cells toward endodermal cells through transfection of cultured cells with a first combination of mRNA at an effective dose and within a specific time range; (d) further orienting the endodermal cells toward hepatic progenitor cells through transfection with a second combination of mRNA; (e) further maturing the hepatic progenitor cells toward hepatocytes with a third combination of mRNA; and (f) passage the progenitor cell clusters into a monolayer, or collecting clusters formed from the hepatic progenitor cells and re-plate them into a monolayer. Therefore, the present invention relates to a method encompassing the step of obtaining the above-mentioned hepatocytes, cells, and a composition for treating a disease or malformation comprising cells obtained by a method for inducing the differentiation of stem cells into hepatocytes, wherein the method comprises the steps of (a) culturing induced pluripotent stem cells as starting cells under conditions for differentiation; (b) inducing the starting cells to move from a pluripotent state toward a mesoendodermal lineage; (c) orienting the differentiated cells toward endodermal cells through transfection of cultured cells with a first combination of mRNA at an effective dose and within a specific time range; (d) further orienting the endodermal cells toward hepatic progenitor cells through transfection with a second combination of mRNA; (e) further maturing the hepatic progenitor cells into hepatocytes with a third combination of mRNA; and (f) obtaining the above-mentioned hepatocytes by passage the progenitor cell clusters into a monolayer or by collecting clusters formed from the hepatic progenitor cells and re-platening them into a monolayer.

[0025] In another embodiment, the present invention relates to a method for treating a disease, disorder, or malformation, the method comprising the steps of administering at least one of the following to a subject in need thereof, wherein the cells are obtained from a recipient subject: cells obtained by a method for inducing differentiation of stem cells into hepatocytes, the method comprising: (a) culturing induced pluripotent stem cells as starting cells under conditions for differentiation; (b) inducing the starting cells to move from a pluripotent state toward a mesoendodermal lineage; (c) orienting the differentiating cells toward endodermal cells through transfection of cultured cells with a first combination of mRNA at an effective dose and within a specific time range; (d) further orienting the endodermal cells toward hepatic progenitor cells through transfection with a second combination of mRNA; (e) further maturing the hepatic progenitor cells toward hepatocytes with a third combination of mRNA; and (f) passage the progenitor cell clusters into a monolayer or collect clusters formed from the hepatic progenitor cells. The present invention relates to a method comprising the steps of obtaining the above-mentioned hepatocytes by re-plateping them into a monolayer, a composition for treating a disease or malformation comprising cells and cells obtained by a method for inducing the differentiation of stem cells into hepatocytes, wherein the method comprises the steps of (a) culturing induced pluripotent stem cells as starting cells under conditions for differentiation; (b) inducing the starting cells to move from a pluripotent state toward a mesoendodermal lineage; (c) orienting the differentiated cells toward endodermal cells through transfection of cultured cells with a first combination of mRNA at an effective dose and within a specific time range; (d) further orienting the endodermal cells toward hepatic progenitor cells through transfection with a second combination of mRNA; (e) further maturing the hepatic progenitor cells toward hepatocytes with a third combination of mRNA; and (f) obtaining the above-mentioned hepatocytes by passage the progenitor cell clusters into a monolayer or by collecting clusters formed from the hepatic progenitor cells and re-plate them into a monolayer.

[0026] In another embodiment, the present invention is a method for treating a disease, disorder, or dysplasia, the method comprising administering to a subject in need thereof at least one of the following, wherein the starting cells are obtained from a recipient: cells obtained by a method for inducing the differentiation of stem cells into hepatocytes, the method comprising: (a) culturing induced pluripotent stem cells as starting cells under conditions for differentiation; (b) inducing the starting cells to exit the pluripotent state and move towards the mesendoderm lineage; (c) directing the differentiating cells towards endodermal cells through transfection of the cultured cells with a first combination of mRNAs at an effective dose and within a specific time period; (d) further directing the endodermal cells towards hepatocyte progenitor cells through transfection with a second combination of mRNAs; (e) further maturing the hepatocyte progenitor cells into hepatocytes with a third combination of mRNAs; and (f) obtaining the hepatocytes by passaging the progenitor cell clusters into a monolayer or collecting the clusters formed from the hepatocyte progenitor cells and replating them into a monolayer, a cell, and a composition for treating a disease or dysplasia comprising cells obtained by a method for inducing the differentiation of stem cells into hepatocytes, the method comprising: (a) culturing induced pluripotent stem cells as starting cells under conditions for differentiation; (b) inducing the starting cells to exit the pluripotent state and move towards the mesendoderm lineage; (c) directing the differentiating cells towards endodermal cells through transfection of the cultured cells with a first combination of mRNAs at an effective dose and within a specific time period; (d) further directing the endodermal cells towards hepatocyte progenitor cells through transfection with a second combination of mRNAs; (e) further maturing the hepatocyte progenitor cells into hepatocytes with a third combination of mRNAs; and (f) obtaining the hepatocytes by passaging the progenitor cell clusters into a monolayer or collecting the clusters formed from the hepatocyte progenitor cells and replating them into a monolayer, relating to a composition.

[0027] One aspect of the present invention relates to a method for generating differentiated hepatocytes from induced pluripotent stem cells, the method comprising: (a) culturing the induced pluripotent stem cells as starting cells under conditions for differentiation; (b) inducing the starting cells to move from a pluripotent state toward a mesoendodermal lineage; (c) orienting the differentiated cells toward endodermal cells through transfection of the cultured cells with a first combination of mRNA at an effective dose and within a specific time range; (d) further orienting the endodermal cells toward hepatic progenitor cells through transfection with a second combination of mRNA; (e) further maturing the hepatic progenitor cells toward hepatocytes with a third combination of mRNA; and (f) obtaining hepatocytes by passage the progenitor cell clusters into a monolayer or by collecting clusters formed from the hepatic progenitor cells and re-platening them into a monolayer.

[0028] One aspect of the present invention relates to a method for generating endodermal cells from induced pluripotent stem cells, the method comprising: (a) culturing the induced pluripotent stem cells as starting cells under conditions for differentiation; (b) inducing the starting cells to move from a pluripotent state toward a mesoendodermal lineage; and (c) orienting the differentiated cells toward the endoderm through transfection of the cultured cells with endoderm-specific mRNA at an effective dose and within a specific time range.

[0029] In one aspect, the Disclosure provides novel and implementable processes for controlling cell density and division rate to achieve desired differentiation outcomes. In several aspects, the processes include, for example, optimization of timing, order of addition, RNA dose and ratio of various RNAs during RNA transfection, and their duration or number of iterations. In several aspects, the Disclosure further relates to the selection of culture vessel surface and environmental conditions (e.g., oxygen concentration). The Disclosure further provides processes and methods for selecting desired cells or increasing their percentage in the overall population, as well as methods for cryopreservation and re-culturing differentiated cells. The methods of the Disclosure encompass simplified protocols and efficient methods for maturing hepatocytes through a three-dimensional stage. In several aspects, the mature and differentiated hepatocytes generated from stem cell manipulation secrete glycogen. In one aspect, the Disclosure provides a newly developed protocol for generating more functional and more mature hepatocytes that function in vivo. In several aspects, the mature hepatocytes of the Disclosure are useful for treating various liver diseases, conditions, and injuries.

[0030] In certain embodiments, exemplary methods for generating mature and functional hepatocytes through stem cell induction may be represented by regimens and steps as described and shown in the examples herein. By contacting mRNA with cells at critical fate change points under suitable dose and delivery conditions, very high efficiency was achieved at low cost without the use of large amounts of expensive growth factors. Since mRNA is more specific in directing cell and differentiation events through the functional proteins it encodes, the disclosed method is far more powerful than any known method for generating human hepatocytes and opens the way to human medicine in the treatment of liver diseases, conditions, and injuries.

[0031] In some respects, this disclosure also provides novel methods for achieving cell fate determination without using, or with reduced amounts of, small molecules that influence signaling pathways as agonists or antagonists of growth factor receptors (which are often variable in purity, stability, and toxicity).

[0032] In another aspect of this disclosure, the above method offers significant advantages in the simplicity of establishing a differentiation medium through the use of appropriately supplied mRNA of differentiation-directing genes. This is in contrast to previous approaches of laborious testing of “differentiation medium” by removing or adding one component at a time. In one aspect, the optimal combination of mRNAs and appropriate medium, as well as other parameters disclosed herein, can simplify the process for generating differentiated functional hepatocytes and are an integral part of the present invention.

[0033] Furthermore, other methods also rely on animal products (e.g., serum or Matrigel that must undergo centrifugation and / or be produced using GMP practices). Another aspect of the present invention is to create novel methods that are primarily based on a single type of molecule suitable for uniform assurance and quality control processes.

[0034] This disclosure provides a differentiation method that utilizes the highly efficient and well-regulated expression of master regulatory genes or key transcription factors in tissue-specific differentiation. More specifically, these factors are introduced into pluripotent stem cells in the form of appropriately modified and purified mRNA molecules, as shown through the provided model.

[0035] In one aspect, the present disclosure provides a method for inducing cell differentiation, the method comprising: utilizing key cell fate factors and fusions between conventional transcription factors (TFs) having transactivation domains optimized for orienting stem cells toward different cell types; introducing these factors as synthetic messenger RNA (mRNA) into pluripotent stem cells cultured at a preferred density in a manner that produces an appropriate level of transgene expression; and maintaining the cells under conditions optimized to produce highly efficient specific differentiation, thereby inducing the pluripotent or progenitor state of stem cells or progenitor cells toward a specific lineage or tissue cell type.

[0036] In another aspect, the Disclosure provides a method for altering the pluripotency or progenitor state of stem cells or progenitor cells toward a specific lineage or tissue cell type, the method comprising at least one of the following steps: generating stem cells (collectively referred to as stem cells) expressing deterministic cell fate genes, including key cell fate factors and fusions among conventional transcription factors (TFs) having transactivation domains optimized for orienting stem cells toward different cell types; introducing these factors as synthetic messenger RNA (mRNA) into pluripotent stem cells cultured at a preferred density in a manner that produces an appropriate level of transgene expression; and maintaining the cells under conditions optimized to produce highly efficient specific differentiation.

[0037] In one aspect, the present disclosure provides a method for generating differentiated hepatocytes from iPSCs, the method comprising: a) preparing the iPSCs as starting cells by culturing them under validated conditions as disclosed herein to prepare them as starting cells for differentiation; b) inducing the starting cells to move from a pluripotent state toward a mesoendodermal lineage; c) orienting the differentiated cells toward the endoderm by using mRNA of an endoderm-specifying gene through transfection of cultured cells at disclosed doses and within a specific time range; d) further orienting the endodermal cells toward hepatic progenitor cells by using further mRNA molecules of genes or combinations of mRNA molecules of genes through transfection; e) further maturing the hepatic progenitor cells toward hepatocytes with further mRNA molecules of genes or combinations of mRNA molecules of genes; and f) obtaining hepatocytes by passage the progenitor cell clusters into a monolayer or by collecting clusters formed from hepatic progenitor cells and re-platening them into a monolayer.

[0038] In one aspect, the present disclosure provides a method for generating endodermal cells from iPSCs, the method comprising: a) culturing iPSCs as starting cells under validated conditions as disclosed herein to prepare the cells as starting cells for differentiation; b) inducing the starting cells to move from a pluripotent state toward a mesoendodermal lineage; and c) orienting the differentiating cells toward endoderm by using mRNA of endoderm-specific genes through cultured cell transfection at disclosed doses and within a specific time range. The present invention provides, for example, the following items: (Item 1) A method for inducing the differentiation of stem cells into hepatocytes, wherein the method is (a) A step of culturing induced pluripotent stem cells as starting cells under conditions for differentiation; (b) A step of inducing the starting cells to evolve from a pluripotent state towards a mesoendoderm lineage; (c) A step of orienting the differentiating cells toward endodermal cells through transfection of cultured cells with a first combination of mRNA at an effective dose and within a specific time range; (d) A step of further orienting the endodermal cells toward hepatic progenitor cells through transfection with a second combination of mRNA; (e) A step of further maturing the hepatic progenitor cells into hepatocytes with a third combination of mRNA; and (f) A step of obtaining hepatocytes by subculturing the progenitor cell cluster into a monolayer, or by collecting the cluster formed from the hepatic progenitor cells and re-platening it into a monolayer. A method of including. (Item 2) The first combination of mRNAs is the method described in item 1, which includes FoxA2 mRNA. (Item 3) The first combination of mRNAs is the method described in item 1, which includes Sox17 mRNA. (Item 4) The first combination of mRNAs is the method described in item 1, comprising FoxA2 and Sox17 mRNA. (Item 5) The method according to item 1, wherein the first combination of mRNAs includes FoxA2, ​​Sox17, GATA4, and GATA6 mRNA. (Item 6) The second combination of mRNA is the method described in item 1, which includes Hex mRNA. (Item 7) The second combination of mRNA is the method described in item 1, which includes Tbx3 mRNA. (Item 8) The second combination of mRNAs is the method described in item 1, comprising Tbx3 and Hex mRNA. (Item 9) The second combination of mRNA is Tbx3, GATA4, GATA6, and Hex The method described in item 1, including mRNA. (Item 10) The third combination of mRNA is the method described in item 1, which includes HNF1a mRNA. (Item 11) The third combination of mRNA is the method described in item 1, which includes HNF4a mRNA. (Item 12) The method according to item 1, wherein the third combination of mRNAs includes HNF4a, HNF1a, HNF6, CEB / Pa, and CEB / Pb mRNA. (Item 13) The starting cells are collected from the body fluids or tissues of the subject, as described in item 1. (Item 14) Cells obtained by the method described in item 1. (Item 15) A composition for treating a disease, disorder, or malformation, comprising the cells described in item 14. (Item 16) A method for treating a disease, disorder, or malformation, the method comprising the step of administering at least one of the cells described in item 14 and the compositions described in item 15 to a subject in need thereof. (Item 17) The cells are derived from the recipient subject, as described in item 16. (Item 18) The starting cells are obtained from the recipient, as described in item 16. (Item 19) A method for generating differentiated hepatocytes from induced pluripotent stem cells, wherein the method is: (a) A step of culturing the induced pluripotent stem cells as starting cells under conditions for differentiation; (b) A step of inducing the starting cells to evolve from a pluripotent state toward a mesoendoderm lineage; (c) A step of orienting the differentiating cells toward endodermal cells through transfection of cultured cells with a first combination of mRNA at an effective dose and within a specific time range; (d) A step of further orienting the endodermal cells toward hepatic progenitor cells through transfection with a second combination of mRNA; (e) A step of further maturing the hepatic progenitor cells into hepatocytes with a third combination of mRNA; and (f) A step of obtaining hepatocytes by subculturing the progenitor cell cluster into a monolayer, or by collecting the cluster formed from the hepatic progenitor cells and re-platening it into a monolayer. A method of including. (Item 20) A method for generating endodermal cells from induced pluripotent stem cells, wherein the method is: (a) A step of culturing the induced pluripotent stem cells as starting cells under conditions for differentiation; (b) the step of inducing the starting cells to move from a pluripotent state toward a mesoendoderm lineage; and (c) A step of orienting differentiated cells toward the endoderm by transfection of cultured cells with endoderm-specific mRNA at an effective dose and within a specific time range, A method of including.

[0039] Further objects and advantages of the present invention are partially shown in the following description, are partially obvious from that description, or can be learned through the practice of the present invention. The objects and advantages of the present invention are realized and achieved by the elements and combinations particularly pointed out in the appended claims.

[0040] It should be understood that both the general description above and the detailed description below are merely illustrative and descriptive of the present invention and not limitations as described in the claims.

[0041] The accompanying drawings are incorporated herein and constitute part of this specification, illustrating several embodiments of the invention and, together with the description, serve to illustrate the principles of the invention. [Brief explanation of the drawing]

[0042] A patent or application file shall include at least one color-finished drawing. A copy of the published patent or patent application with the color drawing shall be provided by the Patent Office upon request and payment of the required fees.

[0043] The aforementioned aspects and advantages of the present invention may become apparent from the following detailed description with reference to the accompanying drawings.

[0044] [Figure 1A] Figure 1A shows endodermal cells from various starting densities and illustrates an exemplary embodiment of endoderm induction.

[0045] [Figure 1B] Figure 1B illustrates an exemplary embodiment of endoderm induction, showing endodermal cells induced from iPSCs using Sox17 mRNA at various densities (e.g., from low to high densities as described in the examples).

[0046] [Figure 2] Figure 2 illustrates exemplary embodiments of hepatic progenitor induction, showing hepatic progenitor cells forming clusters starting from various endodermal cell densities. Figure 2A shows exemplary cell densities / clusters associated with the induction. Figure 2B shows exemplary cell densities / clusters associated with the induction. Figure 2C shows exemplary cell densities / clusters associated with the induction.

[0047] [Figure 3] Figure 3 shows hepatocytes directly derived from hepatic progenitor cells in a monolayer culture, illustrating an exemplary embodiment of hepatocyte induction.

[0048] [Figure 4] Figure 4 shows hepatocyte progenitor cells that have grown as three-dimensional (3D) spheres (which then mature into hepatocytes), illustrating an exemplary embodiment of hepatocyte maturation in 3D spheres.

[0049] [Figure 5]Figure 5 shows that cells in hepatocyte spheres (H&E on the left) are glycogen-positive (PAS on the right), illustrating an exemplary embodiment of hepatocytes functioning in glycogen secretion in 3D. Figure 5A shows an exemplary diagram of hepatocytes / glycogen. Figure 5B shows an exemplary diagram of hepatocytes / glycogen.

[0050] [Figure 6] Figure 6A shows cells in hepatocyte spheres exhibiting hepatocyte markers, illustrating exemplary embodiments of endoderm and hepatocytes derived from human iPSCs that specifically exhibit cell markers. Figure 6A shows AFP staining, and Figure 6B shows A1 antitrypsin.

[0051] [Figure 7] Figure 7 shows hepatocytes directly derived from 3D hepatic progenitor cell spheres that have been re-plated as a monolayer culture, illustrating an exemplary embodiment in which human hepatocytes produced through 3D spheres can be re-plated into a monolayer and exhibit mature hepatocyte morphology.

[0052] [Figure 8] Figure 8 shows that 2 million iPSCs from the starting population were transfected using MaxCyte STX placed in an Optimization 2, OC-100 processing assembly. Photographs were taken 24 hours after transfection at 10× using an EVOS imaging system. Figure 8A shows an exemplary transfection and associated iPSC. Figure 8B shows an exemplary transfection and associated iPSC. Figure 8C shows an exemplary transfection and associated iPSC. Figure 8D shows an exemplary transfection and associated iPSC. Figure 8E shows an exemplary transfection and associated iPSC. Figure 8F shows an exemplary transfection and associated iPSC. [Modes for carrying out the invention]

[0053] Detailed description of the invention In describing the present invention, all terms not defined herein have the general meanings recognized in the art. The following descriptions are illustrative and not limiting to the extent that they relate to specific embodiments or particular uses of the present invention. The following descriptions are intended to encompass all options, modifications, and equivalents that fall within the spirit and scope of the present invention.

[0054] The concept of a "master control" gene—that is, a single key gene (typically a transcription factor gene, sometimes a small number of genes working together) that can determine the fate of cells and tissues, and ultimately the formation of entire organs during development—has been generally accepted, based on research in muscle (MyoD), eye (Pax6), and other fields of developmental biology. Shinya Yamanaka's discovery that differentiated cells can be restored to a pluripotent state by the expression of a selected group of transcription factors expressed in stem cells demonstrated the power of a few key transcription factors in driving cells through long, multi-stage fate changes. Other group studies on iPSC generation have broadened the selection of reprogramming factors, showing that several variations may be acceptable in transcription factor selection for the purpose of reprogramming. In Yamanaka's original study, the expression of reprogramming factors was achieved through the application of viral vectors integrated into the genome, as the long-term expression of these factors is required to bring about cell transformation. While the accompanying genomic modifications symbolize a significant hurdle for the therapeutic application of iPSCs, the possibility of reactivated expression from embedded viral cassettes is also a concern, even for in vitro studies. Applying mRNA transfection to reprogramming, as recently disclosed by the inventors' group, is particularly intriguing because this system allows the expression of the reprogramming cocktail and even individual component factors to be regulated within a short timeframe simply by changing which transcripts are added to the cell culture medium. Once the transfection of a particular factor is complete, ectopic expression within the target cell terminates rapidly due to the rapid decay of mRNA in the cytoplasm. Even if the mRNA does not remain in the target cell without disappearing, its ability to be directly translated in the cytoplasm without the need for nuclear translocation, as in the case of transfected DNA and embedded viral vectors, more than compensates for the short half-life of mRNA, allowing for highly efficient expression within a short timeframe (which is critical for cell fate determination).

[0055] Long-lasting DNA vectors (e.g., episomal plasmids), when used to alter the quality of cell fate, require weaning to reduce any risk of random genomic integration. RNA viruses or viral derivatives (e.g., Sendai virus or Venezuelan encephalitis (VEE) virus), even after being stripped to be modified non-infectious RNA replicons, still possess viral elements that are susceptible to recombination with the host genome. It is always difficult to be completely certain that cells are free from viral vectors without the monotonous and tedious discovery of proof in the form of negative data. This invention discloses a number of progressive steps focused on applying the advantages of mRNA-based cell fate determination to directed differentiation. In summary, this disclosure teaches single or multiple rounds of ectopic transcription factor expression in a simplified manner for directing cell differentiation.

[0056] Nevertheless, technical barriers exist to mRNA-based stem cell differentiation. Not all stem cell types and culture media contribute equally to efficient mRNA delivery, which is currently an obstacle to mRNA-based differentiation. It is also generally known that stem cells, particularly most human stem cell lines, are quite difficult to culture without forming a compartment of transfection resistance. Part of the teachings of this invention is that pluripotent stem cells can be grown under conditions in which the majority of their cells can be transfected with modified mRNA. In another embodiment, the doses of RNA and transfection reagents (both related to toxicity) should be provided to the cells at levels that can exert master regulatory gene effects while supporting the viability of target cells in the face of pro-apoptotic and anti-apoptotic forces generated by the cell fate alteration process.

[0057] Therefore, in view of previously known stem cell differentiation procedures and associated challenges, the novel methods, materials, and protocols described herein generate different cell types from iPSCs or ESCs with improved process efficiency and cell quality. The present invention achieves significant improvements through the enhancement of TF mRNA delivered to target stem cells. The present invention also provides a novel protocol that supports the generation of tissue cells from human stem cells without leaving a footprint, without the use of feeder cells or any other potentially foreign-contaminated reagents. The novel protocol expands the benefits of its modified mRNA and helps to clear away remaining obstacles to the therapeutic application of stem cell-derived technologies.

[0058] Considering that differentiation from a pluripotent state to a final differentiated state often involves numerous steps and takes several weeks to even several months, growth factor-based stepwise strategies are inherently inefficient, monotonous, and tedious. Therefore, embodiments of the present invention fundamentally eliminate the need for growth factors in guiding hepatocyte generation.

[0059] More specifically, the present invention relates to altering the pluripotency or progenitor state of stem cells or progenitor cells (collectively referred to as stem cells) toward a specific lineage or tissue cell type by expressing decisive cell fate genes, including key cell fate factors and fusions among conventional transcription factors (TFs) having transactivation domains optimized for directing stem cells toward different cell types; introducing these factors as synthetic messenger RNA (mRNA) into pluripotent stem cells cultured at a preferred density in a manner that produces an appropriate level of transgene expression; and maintaining the cells under conditions optimized to produce an efficiency of specific differentiation that was previously unattainable. Factors expressed through mRNA introduction may also include growth factors, cytokines, hormones, signal peptides, and other secretory factors or modified enzymes that influence cell fate. Using similar procedures, microRNAs (miRNAs) or other non-protein-coding RNAs may be introduced into cells in cell state transition to direct differentiation. Compared to other methods known in the art, the present invention dramatically reduces the time, cost, and effort involved in stem cell differentiation into hepatocytes.

[0060] The present invention describes a method for altering the pluripotency or progenitor state of stem cells or progenitor cells toward a specific lineage or tissue cell type, the method comprising at least one of the following: expressing deterministic cell fate genes, which include key cell fate factors and are optimized to orient stem cells toward different cell types; introducing these factors as synthetic messenger RNA (mRNA) into pluripotent stem cells cultured at a preferred density in a manner that produces an appropriate level of transgene expression; and maintaining the cells under conditions optimized to produce highly efficient specific differentiation.

[0061] In certain embodiments, fully stabilized and enlarged iPSCs are provided.

[0062] In certain embodiments, it is not necessary to clear episomes or RNA viruses (e.g., Sendai), and the iPSCs can be traced through 10+ passages after isolation.

[0063] In certain embodiments, the process does not have a feeder.

[0064] In certain embodiments, the process comprises all synthetic or human reagents, contains no non-human animal-derived components, and is free of foreign matter.

[0065] In certain embodiments, the process leaves no trace of existence: there is no random integration of DNA into the genome (as often occurs in episomes).

[0066] In certain embodiments, the process generates a completely specially created genetic background via patient-specific starting tissue and / or genome editing.

[0067] In another experiment, as an alternative to the process outlined in Table 1, iPSC cells grown as spheres in a suspension were directly transfected using electroporation (e.g., using a MaxCyte STX electroporator) without plated onto a plate surface. In one embodiment, 2 million starting iPSCs in spheres were transfected in suspension with various mRNAs (e.g., Sox17 or Pax6) or mock-transfected. The amount of mRNA tested in Figure 8 was 2500 ng. The cells were then grown in NBM in the case of Sox17 transfection, or in MEMalpha with KSR in the case of Pax6 transfection. Transfection could be repeated one, two, three, four, five, or even more times if the transition took a longer period. As a result, after the first transfection with Sox17 mRNA, the cell clusters became noticeably smaller and the spheres became less dense, losing their distinct "edge" or external boundary. In contrast, mock-transfected spheres were well-preserved and showed clearly visible outer "margins" in 2D images. The smaller spheres of untransfected or mock-transfected iPSCs had a transparent appearance, while the larger spheres did not appear as transparent due to their thicker cell layers. For comparison, iPSC spheres transfected with Pax6 (neuronal differentiation TF) mRNA progressed towards the ectoderm, i.e., neural progenitor cells, and these spheres had darker and less defined "margins" than the mock-transfected ones, but were larger in size and had more defined boundaries than those transfected with Sox17.

[0068] By the same principles and similar methods, germ-layer-specific intermediate cells (e.g., endodermal cells) and further downstream intermediate cells (e.g., hepatocyte progenitor cells, pancreatic progenitor cells, etc.) can also be transfected with additional TF mRNA in spheres. Cells thus transfected are resistant to toxicity derived from small molecules, growth factors, or other elements in the cell culture and should generally be more efficiently differentiated than 2D transfection using chemical reagents. This observation has not yet been found in scientific publications and was made incidentally during testing of an electroporation apparatus, and has served as a feasible method as part of this disclosure.

[0069] definition To facilitate understanding of the present invention, many terms are defined below. Terms as defined herein have the meanings generally understood by those skilled in the art in the field directly related to the present invention. Terms such as “a,” “an,” and “the” are not intended to refer only to singular entities, but include a comprehensive class of which specific examples may be used for illustrative purposes. The terminology used herein is used to describe specific embodiments of the present invention, but their use, except as outlined in the claims, does not define the scope of the present invention.

[0070] The term "hepatocyte-like cell" is intended to mean a cell that shares characteristics with hepatocytes. Hepatocyte-like cells are further defined by morphological and specific marker characteristics. Since induced pluripotent stem cell-derived hepatocyte-like cells share similar characteristics with hepatocytes (including marker and hormonal characteristics), they can be used interchangeably with induced pluripotent stem cell-derived liver cells or hepatocytes.

[0071] An "embryoid body" is a cell aggregate derived from pluripotent cells, where cell aggregation may be initiated by any method that prevents cells from adhering to a surface and forming representative colony growth. As used herein, an "embryoid body" is a three-dimensional spheroid aggregate of pluripotent stem cells (including, but not limited to, embryonic stem cells derived from the blastocyst stage of an embryo of a mammalian source). Embryoid bodies may be formed from embryonic stem cells derived through any technique generally known in the art (including, but not limited to, somatic cell nuclear transfer or somatic cell reprogramming to produce induced pluripotent stem cells).

[0072] As used herein, the term “induced pluripotent stem cell” means a pluripotent stem cell derived from a somatic cell (e.g., an adult somatic cell). Induced pluripotent stem cells are similar to embryonic stem cells in their ability to differentiate into any adult cell type, but they are not derived from an embryo.

[0073] As used herein, “cell,” “cell line,” and “cell culture” include offspring. It is also understood that not all offspring are exactly identical in DNA content due to intentional or accidental mutations. Variant offspring having the same function or biological characteristics are included, as screened in the originally transformed cells.

[0074] As used herein, “composition” refers to a combination of an active agent and at least one other compound or molecule, either inactive (e.g., a detectable agent or label) or active (e.g., an adjuvant).

[0075] As used herein, “culturing” means maintaining cells under conditions that allow them to grow as a group and avoid aging. “Culturing” may also include conditions under which the cells can differentiate into similar or alternative forms.

[0076] As used herein, “differentially expressed” refers to the differential production of RNA (including, but not limited to, mRNA, tRNA, miRNA, siRNA, snRNA, and piRNA) transcribed from a gene or regulatory region of the genome, or a protein product encoded by a gene, compared to the level of RNA production from the same gene or regulatory region in normal or control cells. In other contexts, “differentially expressed” also refers to nucleotide sequences or proteins in cells or tissues that have varying temporal and / or spatial expression profiles compared to normal or control cells.

[0077] As used herein, “overexpressed” or “overexpression” means an increased level of expression of an RNA or protein product encoded by a gene compared to the level of expression of that RNA or protein product in normal or control cells.

[0078] As used herein, “underexpressed” or “underexpression” means a reduced level of expression of the RNA or protein product encoded by that RNA or gene, compared to the level of expression of that RNA or protein product in normal or control cells.

[0079] As used herein, “differentiate” or “differentiation” refers to the process of differentiating a precursor or progenitor cell (i.e., a hepatic progenitor cell) into a specific cell type (e.g., a hepatocyte).

[0080] As used herein, “effective amount” means a quantity sufficient to produce a beneficial or desired biological, emotional, medical, or clinical response in a cell, tissue, system, animal, or human. An effective amount may be administered in one or more doses, applications, or dosages. The term also includes, within its range, quantities effective to enhance normal physiological function.

[0081] As used herein, “expansion” or “expanded” in the context of cells refers to an increase in the number of characteristic cell types from an initial population of cells (which may or may not be identical). The initial cells used for expansion do not need to be the same cells that will be produced from the expansion. For example, the expanded cells may be produced by the ex vivo or in vitro growth and differentiation of the initial population of cells.

[0082] As used herein, “expression” refers to the process by which polynucleotides are transcribed into RNA transcripts. In the context of mRNA and other translated RNA species, “expression” also refers to the process by which the transcribed RNA is subsequently translated into peptides, polypeptides, or proteins.

[0083] As used herein, “induced pluripotent stem cells” or “iPS cells” or “iPSCs” refer to cells that are artificially derived (not naturally derived) from non-pluripotent cells and capable of differentiating into multiple cell types.

[0084] As used herein, "integration-free iPS cell" means an iPS cell that does not contain foreign transgenes that are integrated into the genome of non-pluripotent cells.

[0085] As used herein, “isolated” means that a polynucleotide, peptide, polypeptide, protein, antibody, or fragment thereof has been separated from the components (cells and others) to which it is normally associated in nature. Polynucleotides, peptides, polypeptides, proteins, antibodies, or fragments thereof that do not exist in nature do not require “isolation” to distinguish them from their naturally occurring counterparts.

[0086] As used herein, “concentrated” refers to a molecule (including, but not limited to, polynucleotides, peptides, polypeptides, proteins, antibodies, or their fragments) that is distinguishable from its naturally occurring counterpart by having a greater concentration or number of molecules per unit volume than that of its naturally occurring counterpart.

[0087] As used herein, “diluted” refers to a molecule (including, but not limited to, polynucleotides, peptides, polypeptides, proteins, antibodies, or their fragments) that is distinguishable from its naturally occurring counterpart by having a lower concentration or number of molecules per unit volume than that of its naturally occurring counterpart.

[0088] As used herein, “separated” means physically separated from the original source or population, so that the separated compound, drug, particle, or molecule can no longer be considered part of the original source or population.

[0089] As used herein, for the purposes of treatment, “mammal” means any animal classified as a mammal (including humans), domesticated and farm animals, non-human primates, and zoo, sport, or pet animals (including, but not limited to, dogs, horses, cats, and cows).

[0090] As used herein, “stem cell” refers to any self-replicating, totipotent, pluripotent, or multipotent, or progenitor or precursor cell that is capable of differentiating into a number of cell types.

[0091] As used herein, “totipotent” refers to all cell types in an organism, plus any cell that can differentiate into and give rise to extraembryonic or placental cells.

[0092] As used herein, “pluripotent” refers to a cell that can differentiate into and give rise to all cell types that make up an organism, including any fetal or adult cell type, with the exception of extraembryonic or placental cells.

[0093] As used herein, “multipotent” refers to cells that can develop into more than one cell type, but are more limited in the cell types into which they can develop than pluripotent cells.

[0094] Where used interchangeably in this specification, “subject,” “individual,” or “patient” refers to a vertebrate organism.

[0095] As used herein, "substantially pure population of cells" "Pure cell population" refers to a population of cells that possess specific cellular marker characteristics and potential differentiation ability, comprising about 50%, preferably about 75-80%, more preferably about 85-90%, and most preferably at least about 95% of the cells constituting the total cell population. Accordingly, "substantially pure cell population" means a population of cells that contains less than about 50%, preferably less than about 20-25%, more preferably less than about 10-15%, and most preferably less than about 5%, of cells that do not exhibit specific cellular marker characteristics and potential differentiation ability under the specified assay conditions.

[0096] As used herein, “pre-differentiation” refers to the process by which a precursor or progenitor cell (e.g., pluripotent stem cell) differentiates into an intermediate cell type (e.g., hepatic progenitor cell) that has the potential to further differentiate into the final effector cell (e.g., hepatocyte).

[0097] As used herein, “therapeutic” refers to the treatment, cure, and / or improvement of a disease, disorder, condition, or adverse event, or a reduction in the rate of progression of a disease, disorder, condition, or adverse event. The term also includes palliative treatment and partial improvement of a disease, disorder, condition, or adverse event, to the extent that it enhances normal physiological function.

[0098] The terms “treating” and “treatment,” as used herein, generally refer to obtaining a desired pharmacological and / or physiological effect. This effect may be preventative, in that it prevents or partially prevents a disease, its symptoms, or condition, and / or therapeutic, in that it partially or completely cures a disease, condition, symptoms, or adverse effects caused by the disease. The term “treatment,” as used herein, encompasses any treatment in mammals, particularly humans, and includes: (a) preventing the occurrence of the disease in a subject who may be predisposed to the disease but has not yet been diagnosed with the disease; (b) inhibiting the disease, i.e., preventing its occurrence; or (c) alleviating the disease, i.e., reducing or improving the disease and / or its symptoms or condition. The term “treatment,” as used herein, refers to therapeutic treatments and preventative or protective measures. Those in need of treatment include those who already have the disorder and those whose disorder should be prevented.

[0099] As used herein, “preventive” refers to preventing or stopping a disease or condition before it occurs, even if it has not yet been diagnosed, or while the disease or condition is still in an asymptomatic stage.

[0100] As used herein, “active agent” refers to a substance, compound, or molecule that is biologically active or otherwise induces a biological or physiological effect in a subject to which it is administered.

[0101] As used herein, “pharmaceutically acceptable carrier” means a diluent, adjuvant, excipient, or vehicle that, together with the active agent, the chondrocytes of the Disclosure, or a composition comprising the chondrocytes of the Disclosure, is approved by a federal or state regulatory authority or is listed in the United States Pharmacopeia or other generally recognized pharmacopoeia for use in animals and / or humans.

[0102] Unless otherwise defined herein, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art.

[0103] cell type Examples of cell types include, for instance, endodermal cells, hepatic progenitor cells, and hepatocytes.

[0104] Examples of suitable surfaces for culture vessels include, but are not limited to, vitronectin, E-cadherin, Corning® Synthemax® II, or Matrigel for iPSCs; Matrigel for endoderms; and Matrigel or collagen for liver progenitor cells.

[0105] In one aspect, exemplary methods for dedifferentiating or reprogramming somatic cells may involve the use of one or more synthetic mRNA reprogramming factors selected from Oct4, Sox2, Klf4, cMyc, Nanog, and Lin28, as well as transactivation domains, thereby reprogramming or dedifferentiating the somatic cells. Methods and compositions for IPSC regulation are described in USSN 13 / 893,16 and USSN 14 / 292,317 (these contents are incorporated herein by reference).

[0106] In certain embodiments, there are protocols for the use of suspended cell cultures, and low-cell-adhesion culture plates and containers may be used for such suspended cultures.

[0107] In certain embodiments, environmental conditions (e.g., oxygen concentration) can be adjusted for optimal induction conditions.

[0108] In certain embodiments, processes and methods are provided for selecting desired cells from an entire cell culture population or for increasing their percentage confluence or cell density.

[0109] In certain embodiments, a method for cryopreserving hepatocyte-like cells is provided. In one embodiment, some of the differentiated cells are cryopreserved for optimal cell viability during preservation. In some embodiments, HSA and DMSO may be added to the culture medium to increase cell viability during preservation. In some embodiments, for example, 2.5% HSA containing 10% DMSO in the culture medium may be used. The cell number may be optimized using this application for further improvement of viability during preservation.

[0110] Methods for re-culturing differentiated cells are also provided. Cells can be re-culturing in most commercially available culture vessels: for example, T75 flasks, T25 flasks, 6-well plates, and 96-well plates. Cells can be re-culturing at various cell densities for various applications.

[0111] In certain embodiments, the disclosure also provides methods for managing physical stress on cells and thereby improving their viability during handling throughout the differentiation process. Certain types of cells during differentiation, such as iPSCs, are very small. These small cells are very sensitive to centrifugal force. iPSCs are very sensitive to excessive centrifugal force. Some types of cells during differentiation, such as iPSCs and endodermal stage cells, are very sticky. These cells are very sensitive to shear force. When handling these cells, we avoided using any small tips with a 10 mL pipette and also avoided repeatedly moving the cells up and down with the pipette. For maintenance, these cells may be cultured as colonies and then dissociated as clusters instead of single cells. If single cells are needed for differentiation, dissociation may be terminated before cell detachment, the dissociation solution may be removed, and the cells may be further dissociated with the remaining dissociation solution. This protocol is commonly used in cell cultures.

[0112] This specification is best understood in light of the teachings of the references cited herein. The embodiments herein provide illustrations of embodiments of the invention and should not be construed as limiting the scope of the invention. Those skilled in the art will readily recognize that many other embodiments are encompassed by the invention. All publications and patents cited herein are incorporated herein by reference in whole. This specification supersedes any such materials to the extent that the incorporated materials are inconsistent with or contradict this specification. Any reference herein is not an endorsement that such references are prior art to the invention.

[0113] Unless otherwise indicated, all figures used herein, including in the claims, representing amounts of components, reaction conditions, etc., should be understood in all cases to be modified by the term “about.” Thus, unless otherwise indicated, numerical parameters are approximations and may vary depending on the desired properties sought to be obtained by the invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should be interpreted in light of significant figures and common rounding approaches.

[0114] The use of the phrases “a” or “an” may mean “one” when used with the term “comprising” in a claim and / or specification, but also coincides with the meanings of “one or more,” “at least one,” and “one or more than one.” The use of the term “or” in a claim is used to mean “and / or” unless it is explicitly indicated that only options are being referred to, or that the options are mutually exclusive; however, this disclosure supports definitions that refer only to options and “and / or.”

[0115] Unless otherwise indicated, the term “at least” preceding a set of elements should be understood to refer to all elements within that set. Those skilled in the art can recognize or confirm, by mere conventional experimentation, many equivalents to the specific embodiments of the books described herein. Such equivalents are intended to be encompassed by the following claims.

[0116] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which the present invention pertains. Any methods and materials similar or equivalent to those described herein may be used in carrying out or testing the present invention, but preferred methods and materials are described herein.

[0117] Other embodiments of the present invention will be apparent to those skilled in the art from consideration of this specification and from the practice of the invention disclosed herein. This specification and the examples are for illustrative purposes only, and the true scope and spirit of the invention are intended to be shown by the following claims. [Examples]

[0118] The present invention is described herein with reference to the following embodiments. These embodiments are provided for illustrative purposes only, and the present invention is not limited to these embodiments, but rather encompasses all variations that are evident as a result of the teachings provided herein.

[0119] In several embodiments for generating mature and differentiated hepatocytes, exemplary parameters, including starting cells, culture vessels, coatings, dissociation agents, culture medium names and major components, seeding density for an exemplary 6-well plate, and oxygen levels, are provided in Table I. [Table 1]

[0120] Example 1: Generating endodermal cells from iPSCs iPSCs are stored in standard-sized 6-well cell culture plates (approximately 9.5 cm²). 2 Growth area per well) or standard size 12-well cell culture plate (approximately 3.8 cm²) 2Plate the cultures into growth area / wells and begin differentiation. Other sizes of culture vessels are equally applicable as needed, and sometimes plates with more than 6 or 12 wells may be preferable for greater efficiency in reagent and time use.

[0121] In a 6-well plate (a standard commercially available one), 1 × 10 5 ~4×10 5 We successfully used a population-sized number of cells per well. iPSCs were considered ready for differentiation if they were well-represented iPSC colonies with clear, well-defined margins (in this case, the cells were densely packed and the colonies were not excessively large). The quality of the iPSCs produced using these criteria proved to be decisive for differentiation when comparing the iPSC lines of the present invention with iPSC lines produced by others using other methods.

[0122] iPSCs at this stage were induced to differentiate into mesoendodermal cell lines. Even though most modern protocols for differentiation prefer the use of adhered monolayer cells, suspension culture systems proved highly useful for scaling up at this stage. iPSCs grown in suspension for induction were found to be resistant to chemical toxicity and more readily plated at later stages. Ultra-low adhesion plates (Sigma-Aldrich) or other low-adhesion plates were used to promote iPSC suspension cell growth.

[0123] When iPS cells need to be passaged, it was important to dissociate the iPSC colonies using a protocol that induces low cytotoxicity and produces smaller clusters of iPSCs (which can rapidly form spheres if suspension culture is desired). Dissociating iPSCs in 0.1 mM, sometimes 0.5 mM, or 1 mM EDTA in DPBS (Fisher Scientific) at 37°C for 5 minutes was performed using TripLE. TMDissociation was performed using ThermoFisher and Accutase (Life Technology). For this process, various dissociation times were successfully used, including 1 to 2 minutes and sometimes up to 10 to 20 minutes. In some embodiments, the dissociation time may be approximately 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes, 16 minutes, 17 minutes, 18 minutes, 19 minutes, 20 minutes, or 25 minutes, or any time range between any two of the times described.

[0124] Regarding the culture media, MEMa, DMEM / F12, and DMEM B27 were tested with 10–50 μM insulin and 5% KSR, and the desired results were achieved at this stage of differentiation. Subsequently, iPSCs were induced to differentiate towards mesoendoderm by the presence of GSK3 inhibitors (e.g., CHIR99021, CHIR98014, BIO, or GSK inhibitor IX) and SB-216763, which desuppress the function of Wnt, BMP4, and Activin A pathway genes, thus moving away from the pluripotency stage. In some cases, the insulin concentration may be, for example, approximately 10 μM, 11 μM, 12 μM, 13 μM, 14 μM, 15 μM, 16 μM, 17 μM, 18 μM, 19 μM, 20 μM, 25 μM, 30 μM, 35 μM, 40 μM, 45 μM, or 50 μM, or any value or range between any two of the concentrations listed. The GSK3 inhibitor was tested over time periods of 1 day, 2 days, and 3 days, and the number of FoxA2, ​​CXCR4-positive cells was used as a quality analysis when selecting the time and the inhibitor concentration, such as 5 mM, 8 mM, or 10 mM.

[0125] In one experiment, cells were then transfected with FoxA2 and / or Sox17 mRNA at a dose of approximately 20 ng / well with Stemgent Transfection Reagent (Stemgent). This transfection was also performed with GATA4 mRNA and / or GATA6 mRNA and repeated 3, 4, 5, and 6 times, sometimes at 10-fold higher mRNA doses, using Stemgent Transfection Reagent or other commercially available transfection reagents. At this stage, the cells exhibited morphology closer to epithelial cells than mesenchymal cells derived from iPSCs from various mRNA transfection times and starting densities (Figure 1).

[0126] In another experiment, as an alternative to the process outlined in Table 1, iPSC cells grown as spheres in suspension were directly transfected using electroporation (e.g., using a MaxCyte STX electroporator) without plated onto a plate surface. In one embodiment, 2 million starting iPSCs in spheres were transfected in suspension with various mRNAs (e.g., Sox17 or Pax6) or mock-transfected. The amount of mRNA tested in Figure 8 was 2500 ng. The cells were then grown in NBM in the case of Sox17 transfection, or in MEMalpha with KSR in the case of Pax6 transfection. Transfection could be repeated one, two, three, four, five, or even more times if the transition took a longer period. As a result, after the first transfection with Sox17 mRNA, the cell clusters became noticeably smaller and the spheres became less dense, losing their distinct "edges" or external boundaries. In contrast, mock-transfected spheres were well-preserved and showed clearly visible outer "margins" in 2D images. The smaller spheres of the untransfected or mock-transfected iPSCs had a transparent appearance, while the larger spheres did not appear as transparent due to their thicker cell layers. For comparison, iPSC spheres transfected with Pax6 (neuronal differentiation TF) mRNA progressed towards the ectoderm, i.e., neural progenitor cells, and these spheres had darker and less defined "margins" than the mock-transfected ones, but were larger in size and had more clearly defined boundaries than those transfected with Sox17.

[0127] By the same principles and similar methods, germ-layer-specific intermediate cells (e.g., endodermal cells) and further downstream intermediate cells (e.g., hepatic progenitor cells, pancreatic progenitor cells, etc.) can also be transfected with additional TF mRNA in spheres. Cells thus transfected are resistant to toxicity derived from small molecules, growth factors, or other elements in the cell culture and should generally be more efficiently differentiated than 2D transfection using chemical reagents. This observation has not yet been found in scientific publications and was made incidentally during testing of an electroporation apparatus, and has served as a feasible method as part of this disclosure.

[0128] Example 2: Generating liver progenitor cells from endodermal cells Endoderm cells are plated onto a commercially available cell culture vessel. A 6-well plate was used in the experiment shown in Figure 2, but other well sizes are also applicable. The plate is pre-coated with Matrigel (BD Biosciences), and then 1 × 10⁶ 5 ~1 × 10 6 Cells were plated in DMEM / F12 or MCDB131 supplemented with 8 mM D-glucose. Occasionally, the addition of 1% DMSO at this stage was observed to be useful in increasing the efficiency of hepatic progenitor cell generation.

[0129] In one experiment, cells were then transfected with Hex and / or Tbx3 mRNA. Furthermore, for stronger effects, these cells were transfected or co-transfected with GATA4 mRNA and GATA6 mRNA at a dose of 50 ng / well, along with Stemgent Transfection Reagent (Stemgent) in the culture medium, and this was repeated two, three, or more times with Stemgent Transfection Reagent or other commercially available transfection reagents at low doses of approximately 10 ng and high doses of approximately 200 ng / well. In some phases, the Stemgent concentration may be, for example, approximately 10 ng / well, 20 ng / well, 30 ng / well, 40 ng / well, 50 ng / well, 60 ng / well, 70 ng / well, 80 ng / well, 90 ng / well, 100 ng / well, 110 ng / well, 120 ng / well, 130 ng / well, 140 ng / well, 150 ng / well, 160 ng / well, 170 ng / well, 180 ng / well, 190 ng / well, or 200 ng / well, or any amount between any two of the amounts listed. This amount may also be adjusted for other well volumes. Cells at this stage appear darker than endodermal cells and tend to form clusters of hepatic progenitor cells (Figure 2).

[0130] Example 3: Generating hepatocytes from hepatic progenitor cells Hepatic progenitor cells were cultured in 6-well plates or other plates pre-coated with Matrigel (BD Biosciences) or Collagen I (Sigma) in DMEM / F12, MEMa, or DMEM B27. Other similar adherent cell culture media are also suitable for use.

[0131] Hepatic progenitor cells were further transfected with 10–200 ng / well doses of HNF1a, HNF4a, HNF6, CEB / Pa, or CEB / Pb mRNA along with Stemgent Transfection Reagent (Stemgent) in culture medium supplemented with 200 ng / mL B18R. The dose of Stemgent may also be approximately 10 ng / well, 20 ng / well, 30 ng / well, 40 ng / well, 50 ng / well, 60 ng / well, 70 ng / well, 80 ng / well, 90 ng / well, 100 ng / well, 110 ng / well, 120 ng / well, 130 ng / well, 140 ng / well, 150 ng / well, 160 ng / well, 170 ng / well, 180 ng / well, 190 ng / well, or 200 ng / well, or any amount between any two of the amounts described. This amount may also be adjusted for other well volumes. Most commercially available transfection reagents can also be used. Hepatocytes were obtained at this stage by passage the progenitor cells to a low density in hepatocyte culture medium (Figure 3).

[0132] Example 4: Hepatocytes in a 3D sphere Instead of dissociating and re-platering, liver progenitor cells were allowed to continue growing for 1 week to 2 months, or even longer. During this period, the clustered progenitor cells continued to form three-dimensional (3D) spheres and transferred to the suspension (Figure 4).

[0133] At this stage, cells in 3D were examined by glycogen expression (Figure 5) and antibody-based staining of liver cell markers (Figure 6). Positive staining for glycogen, AFP, and trypsin confirmed that the cells had reached the mature stage of liver cells.

[0134] These hepatocytes in the 3D spheres were dissociated with Accutase, TrypLE, or other dissociation reagents and re-plated on a coated surface as in Example 3. They immediately exhibited the final differentiated morphology of hepatocytes without further division in the monolayer culture (Figure 7).

[0135] Example 5: iPSC-derived hepatocyte function in an animal model To further test the function of mature hepatocytes produced according to the present invention, the liver function of iPSC-derived hepatocytes is tested in the following mouse models of liver disease or injury: 1) a surgical bile duct ligation (BDL) mouse model of cholestatic liver injury, 2) an MDR2 / Tgfbr2 / Il2ra genetically modified mouse model of cholestatic liver injury, 3) a DDC-modified diet, ANIT-modified diet, or d-galactosamine-inducible mouse model as an alternative to cholestatic liver injury, 4) a high-calorie diet-inducible mouse model of NASH liver injury, 5) an ob / ob, nSREBP-1c, or PTEN genetically modified mouse model of NASH liver injury, 6) an MCDD / CDAA mouse model as an alternative to NASH liver injury, or 7) a CCl4 / TAA / DEN / DMN-inducible mouse model of toxic liver injury.

[0136] Furthermore, alcohol-induced hepatitis (ALD), autoimmune hepatitis (AIH), and viral infectious liver diseases are all major public health issues that can be addressed through transplantation, including the use of animal models, by the hepatocytes produced by the present invention.

[0137] Non-human primate models (e.g., 1) high-calorie diet-induced monkey liver injury model, 2) CCl4-induced monkey liver injury model, 3) BDL-induced monkey liver injury model) are used to test the function of hepatocytes to be disclosed.

[0138] Delivery route: Hepatocytes or spheres are injected into the liver.

[0139] Tests: Measure blood levels of albumin, AST, ALT, bilirubin, and hyaluronan (weeks 2, 4, 8, 12, 16, and 24); measure blood levels of pro-inflammatory cytokines such as IL-8, TNFα, and MCP-1 (weeks 2, 4, 8, 12, 16, and 24); perform IHC at the transplant site for fibrosis, hepatocytes, Kupffer cells / macrophages, and HCC markers.

[0140] Example 6: iPSC-derived hepatocytes in the treatment of human patients with liver disease such as chronic liver failure Clinical trials using human iPSC-derived hepatocytes, administered according to animal studies with reference to other cell therapies, are conducted using disclosed protocols adapted to suit the cGMP procedures. Mini-organs based on the fabricated hepatocytes or 3D hepatocytes are delivered to the liver or other parts of the human body (e.g., muscle, connective tissue, or certain parts of other organs) to achieve efficacy.

Claims

[Claim 1] The method described in the specification.

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