Cell surface marker of subgroup with strong liver differentiation capability and application of cell surface marker

Abstract

The invention provides a cell surface marker of a subgroup with strong liver differentiation capability and application thereof. The invention discloses that the pluripotent stem cells have NAD < + > and NADH metabolic heterogeneity, and are closely related to the expression of CD157. On the basis, the content of NAD < + > / NADH or the expression of CD157 can be taken as an investigation object, and cells with strong liver differentiation capacity or cells with high self-renewal capacity can be obtained from the pluripotent stem cells. The invention also provides a method for sorting the cells, a method for preparing hepatic differentiation cells by using the cells, and the like. The invention provides a new way for the fields of stem cell differentiation and liver regeneration.

Description

Technical Field

[0001] The present invention belongs to the field of cell biology; more specifically, the present invention relates to cell surface markers of a subpopulation with strong liver differentiation ability and applications thereof. Background Art

[0002] The liver is the largest solid organ in the body and the largest digestive gland in the human body. It plays a core role in maintaining the body's metabolism and has strong detoxification and regeneration capabilities.

[0003] When the liver is damaged by pathogenic factors such as viruses, drugs, and alcohol, the remaining liver tissue can regenerate and restore its original volume and weight, ultimately achieving reconstruction of liver tissue structure and recovery of liver function. Chronic and persistent liver damage, which is common in clinical practice, significantly impairs liver function. Studies have shown that the types of cells involved in the liver damage repair process are closely related to the degree of liver damage. When the liver is mildly damaged, it is mainly the proliferation of hepatocytes that repairs the damage. When the liver is severely damaged and hepatocyte regeneration is impaired, hepatocyte necrosis increases, and repair cannot be completed by hepatocyte regeneration alone. The participation of non-parenchymal cells of the liver is often required. In this case, the liver tissue will initiate processes including differentiation of hepatic stem / progenitor cells, transdifferentiation of bile duct cells, and dedifferentiation of hepatocytes to repair the damaged liver.

[0004] Although the field has always focused on the study of liver diseases, little is known about the molecular mechanisms of how the damaged liver initiates this process. Recent studies have shown that hepatocyte transplantation can be used as an effective alternative treatment to help some patients with liver failure survive the critical period waiting for liver transplantation. In some patients with liver failure, hepatocyte transplantation can even directly achieve satisfactory therapeutic effects, thus providing a new option for the treatment of liver failure. Although hepatocytes have a strong ability to repair damage in vivo, the large-scale acquisition of liver cells in vitro is still a limiting factor, which has also formed a bottleneck in the clinical treatment of related liver damage diseases.

[0005] Therefore, the molecular mechanism of the liver injury repair process and the long-term in vitro culture of hepatocytes and further artificial intervention of hepatocyte proliferation based on this mechanism remain one of the current research focuses in life sciences. The basis of this research, that is, how to obtain more liver-differentiated cells (including liver cells) suitable for research, also needs attention.

[0006] A variety of methods have been developed to induce differentiation using pluripotent stem cells as starting cells, as well as a number of culture media and conditions for inducing differentiation. However, this field generally uses the starting cells, i.e., pluripotent stem cells, directly for differentiation without prior screening, lacking a suitable method to determine whether the starting cells are suitable for directed differentiation.

[0007] Alterations in multiple metabolic signaling pathways are currently believed to play a key role in regulating the self-renewal and directed differentiation of human pluripotent stem cells. Different human pluripotent stem cell lines also exhibit heterogeneity in certain aspects. However, several questions remain unclear, including whether metabolic heterogeneity exists between different human pluripotent stem cell lines and between cells, and whether metabolic heterogeneity plays a key role in this heterogeneity between lines or cells. Summary of the Invention

[0008] The purpose of the present invention is to provide cell surface markers of a subpopulation with strong liver differentiation ability and their applications, including using NAD+ / NADH as a marker for screening cells with strong liver differentiation ability from pluripotent stem cells; and using CD157 as a marker for screening cells with strong liver differentiation ability from pluripotent stem cells.

[0009] In the first aspect of the present invention, a method for obtaining cells (groups, including subgroups) with strong hepatodifferentiation ability or cells (groups) with high self-renewal ability is provided, comprising: isolating cells (groups) having characteristics selected from the following groups from pluripotent stem cells (including cell cultures or cell suspensions): (a) cells with low NAD+ / NADH content; or (b) cells with high expression of CD157.

[0010] In one or more embodiments, the cells with low NAD+ / NADH content are isolated or enriched by a method comprising: binding SoNar fluorescent protein probe to NAD+ and NADH, and sorting out cells with low NAD+ / NADH content according to the excitation light wavelength.

[0011] In one or more embodiments, the cells with high CD157 expression are isolated or enriched by a method comprising: using a binding molecule (such as an antibody) or a capture molecule that specifically binds to or captures CD157 to sort out the cells with high CD157 expression from pluripotent stem cells.

[0012] In one or more embodiments, the method for separating or enriching (including sorting) cells includes (but is not limited to): flow cytometry sorting, immunomagnetic bead sorting, microfluidic cell sorting, and adhesion method.

[0013] In one or more embodiments, CD157 is highly expressed in the cells with low NAD+ / NADH content.

[0014] In one or more embodiments, the SoNar fluorescent protein probe produces different conformations when bound to NAD+ and NADH, thereby responding to different excitation wavelengths. When SoNar binds to NADH, fluorescence in the 405nm channel becomes stronger; when bound to NAD+, fluorescence in the 488nm channel increases. Preferably, the ratio of the fluorescence value at the 405nm channel to the fluorescence value at the 488nm channel is used to detect changes in the NAD+ / NADH ratio in cells.

[0015] In one or more embodiments, the pluripotent stem cells comprise human pluripotent stem cells.

[0016] In one or more embodiments, the separation or enrichment (including sorting) of cells includes at least one sorting, such as 1 to 50 times, 1 to 30 times, 1 to 20 times, 1 to 10 times, 1 to 8 times, 1 to 5 times, 1 to 3 times, 1 to 2 times, etc., of enrichment / sorting / purification.

[0017] In one or more embodiments, cells (populations) having characteristics of both (a) and (b) are isolated.

[0018] In one or more embodiments, the NAD+ / NADH ratio is analyzed, and cells whose ratio belongs to the bottom 20% (bottom 20%) range are cells (populations) with low NAD+ / NADH content; preferably, cells whose ratio belongs to the bottom 15% (bottom 15%) range are cells with low NAD+ / NADH content; preferably, cells whose ratio belongs to the bottom 10% (bottom 10%) range are cells with low NAD+ / NADH content; preferably, cells whose ratio belongs to the bottom 8% (bottom 8%) range are cells with low NAD+ / NADH content; preferably, cells whose ratio belongs to the bottom 6% (bottom 6%) range are cells with low NAD+ / NADH content; preferably, cells whose ratio belongs to the bottom 5% (bottom 5%) range are cells with low NAD+ / NADH content; more preferably, cells whose ratio belongs to the bottom 4% (bottom 4%) range are cells with low NAD+ / NADH content.

[0019] In one or more embodiments, the expression of CD157 is analyzed, and cells whose CD157 signal intensity belongs to the strongest 20% (top 20%) range are CD157 high-expressing (CD157-high) cells (population); preferably, cells whose signal intensity belongs to the strongest 15% (top 15%) range are CD157 high-expressing cells; preferably, cells whose signal intensity belongs to the strongest 10% (top 10%) range are CD157 high-expressing cells; preferably, cells whose signal intensity belongs to the strongest 8% (top 8%) range are CD157 high-expressing cells; preferably, cells whose signal intensity belongs to the strongest (top 6%) range are CD157 high-expressing cells; more preferably, cells whose signal intensity belongs to the strongest 5% (top 5%) range are CD157 high-expressing cells.

[0020] In another aspect of the present invention, a method is provided for using an isolated or enriched cell (group) as a starting cell for preparing hepatotropically differentiated cells; wherein the isolated or enriched cells are cells with strong hepatotropic differentiation ability and have characteristics selected from the following groups: (a) cells with low NAD+ / NADH content; or (b) high expression of CD157.

[0021] In one or more embodiments, the hepatic differentiated cells include: endoderm cells, hepatoblasts, liver cells (including immature liver cells or mature liver cells).

[0022] In another aspect of the present invention, a method for preparing hepatotropically differentiated cells is provided, comprising: (1) preparing cells with strong hepatotropically differentiated ability using any of the methods described in requirements 1 to 2; and (2) using the cells with strong hepatotropically differentiated ability of (1) as starting cells, inducing hepatotropic differentiation, and obtaining hepatotropically differentiated cells.

[0023] In one or more embodiments, the hepatic differentiated cells include: endoderm cells, hepatoblasts, liver cells (including immature liver cells or mature liver cells).

[0024] In one or more embodiments, the endoderm cells are prepared by induction using an endoderm induction medium (such as DE differentiation medium).

[0025] In one or more embodiments, the hepatoblasts are prepared using an endoderm induction medium (such as a DE differentiation medium) and a liver differentiation medium (such as a HE differentiation medium) in sequence.

[0026] In one or more embodiments, immature hepatocytes are prepared using endoderm induction medium (such as DE differentiation medium) and liver differentiation medium (such as HE differentiation medium and IMH differentiation medium) in sequence.

[0027] In one or more embodiments, mature hepatocytes are prepared using endoderm induction medium (such as DE differentiation medium) and liver differentiation medium (such as HE differentiation medium, IMH differentiation medium and MH differentiation medium) in sequence.

[0028] In one or more embodiments, the DE differentiation medium, HE differentiation medium, IMH differentiation medium, and MH differentiation medium are based on the composition of commercially available culture media or the composition listed in the examples, wherein each component can fluctuate within a range of 50% (preferably within a range of 40%, more preferably within a range of 30%, such as 20%, 15%, 10%, 5%, 3%, 2% or 1%).

[0029] In one or more embodiments, the liver cells express (including highly express) liver cell markers (genes or proteins) Alb, Asgpr, A1at, Glut2, Cyp3a4, Afp.

[0030] In one or more embodiments, the endoderm cells express (including highly express) endoderm markers (genes or proteins) Sox17, Foxa2, Gata6, Gata4.

[0031] In one or more embodiments, the method further comprises: using the obtained liver cells to prepare liver organoids, liver tissues or organs.

[0032] In one or more embodiments, the liver cells or organoids further comprise liver cells or organoids selected from the group consisting of passaged liver cells or organoids, continuously cultured liver cells or organoids, and frozen and / or revived liver cells or organoids.

[0033] In one or more embodiments, the passaged organoids include passages 1 to 30, more specifically passages 2, 3, 5, 8, 10, 12, 15, 18, 20, and 25.

[0034] In one or more embodiments, the culture includes: three-dimensional (3D) culture, or two-dimensional (2D) culture; preferably, three-dimensional (3D) culture.

[0035] In one or more embodiments, the method further comprises: using the obtained liver cells to prepare a liver transplant, or preparing a pharmaceutical composition containing a liver transplant.

[0036] In one or more embodiments, the method further comprises: using the obtained liver cells to prepare a pharmaceutical composition for liver regeneration (a pharmaceutical composition containing active cells).

[0037] In one or more embodiments, the method is an in vitro method.

[0038] In one or more embodiments, the method is a method that is not directly intended for the diagnosis or treatment of a disease (non-diagnostic or non-therapeutic).

[0039] In another aspect of the present invention, an isolated (purified) or enriched cell (population) is provided, which is a cell with strong liver differentiation ability and has characteristics selected from the following groups: (a) low NAD+ / NADH content; or (b) high expression of CD157.

[0040] In one or more embodiments, the cell (population) is not or is different from a naturally occurring cell (population).

[0041] In one or more embodiments, the cells (population) are artificially separated cells (population).

[0042] In another aspect of the present invention, the use of NAD and NADH as markers is provided for screening cells with strong hepatodifferentiation ability or high self-renewal ability from pluripotent stem cells.

[0043] In another aspect of the present invention, the use of CD157 as a marker is provided for screening cells with strong hepatodifferentiation ability or high self-renewal ability from pluripotent stem cells.

[0044] In another aspect of the present invention, a kit for isolating or enriching cells with strong hepatotropic differentiation ability or high self-renewal ability is provided, comprising: NAD+ / NADH binding molecules or capture molecules; or, CD157 binding molecules or capture molecules.

[0045] In another aspect of the present invention, a kit for preparing hepatodifferentiated cells is provided, comprising: an NAD+ / NADH binding molecule or a capture molecule; and a reagent or culture medium for inducing pluripotent stem cells to differentiate into endoderm cells, hepatoblasts and / or liver cells (including immature liver cells or mature liver cells).

[0046] Other aspects of the present invention will be apparent to those skilled in the art in view of the disclosure herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 Human pluripotent stem cells exhibit heterogeneity in NAD+ and NADH metabolism. Green fluorescence reflects intracellular NAD+ levels (left), purple fluorescence reflects intracellular NADH levels (center), and merge fluorescence reflects intracellular NAD+ / NADH levels (right).

[0048] Figure 2, obtaining cell populations with high and low NAD+ / NADH. Flow cytometric sorting strategy for isolating high and low NAD+ / NADH cell populations (top left); flow cytometry verification of differences in NAD+ / NADH between high and low NAD+ / NADH cell populations after multiple passages (top right); NAD+ / NADH measurement results (bottom). *: P < 0.05, **: P < 0.01.

[0049] Figure 3 A significant difference in colony-forming ability was found between the two cell populations. Cells with low NAD+ / NADH ratios formed larger and more colonies. *: P < 0.05.

[0050] Figure 4 Significant differences in DE differentiation capacity were found between the two cell populations, with cells with low NAD+ / NADH ratios exhibiting greater DE differentiation potential. This includes: qPCR analysis (top left); immunofluorescence results (top right); and western blot analysis of protein levels (bottom). *: P < 0.05, **: P < 0.01. The P-values ​​for the four genes in the figure are SOX17: 0.0023; FOXA2: 0.0132; GATA6: 0.0008; and GATA4: 0.0006.

[0051] Figure 5 qPCR was used to examine the expression of liver cell marker genes (Alb, Asgpr, A1at, Glut2, and Cyp3a4) to analyze the differences in liver cell differentiation ability between cells with low NAD+ / NADH ratios and cells with high NAD+ / NADH ratios. *: P < 0.05, **: P < 0.01. The P-values ​​for the five genes in the figure are: ALB: 0.0180; ASGPR: 0.00149; A1AT: 0.00021; GLUT2: 0.00927; CYP3A4: 0.00099.

[0052] Figure 6 , qPCR was used to detect the expression of NAD metabolism-related genes. *: P < 0.05.

[0053] Figure 7 , Immunofluorescence staining analysis showed that human pluripotent stem cells had heterogeneity in CD157 expression.

[0054] Figure 8 , flow cytometry sorting was used to obtain cell populations with high and low CD157 expression.

[0055] Figure 9NAD+ / NADH measurements showed that in the cell population with high CD157 expression, NAD+ content was low, NAD+ / NADH ratio was low, and NADH content was consistent with that in the cell population with low CD157 expression. **: P < 0.01.

[0056] Figure 10 qPCR analysis was used to analyze the expression of endoderm marker genes in cells with high CD157 expression after they differentiated into endoderm. *: P < 0.05, ***: P < 0.001.

[0057] Figure 11 Cells with high CD157 expression have stronger potential for liver cell differentiation. qPCR analysis revealed that the expression of liver cell marker genes Alb, Afp, and Cyp3a4 in cells differentiated from cells with high CD157 expression was significantly higher than in cells with low CD157 expression (upper left panel). ELISA assays revealed that liver cells differentiated from cells with high CD157 expression secreted higher amounts of human albumin (middle left panel). Functional enrichment analysis of 2,534 genes (Fold change > 1.5, P-value < 0.05) with elevated expression in liver cells differentiated from cells with high CD157 expression in RNA-seq transcriptome data revealed that genes with elevated expression in liver cells with high CD157 expression were primarily enriched in metabolic pathways, cholesterol metabolism, and other pathways, all of which were related to liver function. Heat maps of liver-related genes in the RNA-seq results revealed that the vast majority of liver-related genes were expressed at elevated levels in liver cells with high CD157 expression. *:P<0.05, **:P<0.01,***:P<0.001.

[0058] Figure 12 Cells with high expression of CD157 in human pluripotent stem cells have low NAD+ / NADH and stronger liver cell differentiation potential. Therefore, CD157 can be used as a cell surface marker of human pluripotent stem cells. Subpopulations with stronger liver differentiation ability in the cell population can be enriched by flow cytometry with high expression of CD157, thereby improving the differentiation efficiency of human pluripotent stem cells into liver cells. DETAILED DESCRIPTION

[0059] The inventors have devoted their research to the differentiation of hepatocytes. Through in-depth studies, they have revealed that pluripotent stem cells exhibit heterogeneity in NAD+ and NADH metabolism, which is closely correlated with CD157 expression. Based on this, they have developed a method for isolating these cells and for preparing hepatocyte-differentiated cells using these cells. This invention provides a new approach for stem cell differentiation and liver regeneration.

[0060] NAD+ and its reduced form, NADH, are the most important cofactors in energy metabolism. The NAD+ / NADH ratio regulates the activity of various enzymes (e.g., histone deacetylases) and influences gene expression. However, it is currently unclear whether NAD+ / NADH exhibits cell-to-cell heterogeneity in human pluripotent stem cells, whether it plays a regulatory role in maintaining the self-renewal capacity and directed differentiation potential of human pluripotent stem cells, and whether it can serve as a target for improving the quality and quantity of human pluripotent stem cells. The present invention advances this work by proposing that NAD+ and NADH are heterogeneous in pluripotent stem cells.

[0061] As used herein, "isolated" means that a substance has been separated from its original environment (e.g., a native organism or natural environment). For example, a living cell is not isolated or purified while existing in vivo, but the same cell is isolated or purified when separated from other substances present in its natural state / original environment.

[0062] As used herein, "isolated cells (cultures)" include "isolated cell lines (cultures)".

[0063] As used herein, "enriching" a cell (population) refers to increasing the content of desired target cells / objective cells contained in a mixed cell population, generally referring to increasing the frequency of "target cells" in the total cells. Thus, an enriched cell population refers to a cell population with a higher frequency of target cells resulting from an enrichment step.

[0064] As used in the present invention, unless otherwise specified, the "target cells" are cells of interest and having the intended function in the present invention; more specifically, the "target cells" refer to cells with low NAD+ / NADH content, or cells with high CD157 expression (also known as CD157+).

[0065] "Strong hepatotropic differentiation capacity" refers to a significantly (statistically) high hepatotropic differentiation capacity. Pluripotent stem cells, as a known type of cell, have an "average" differentiation capacity, or at least an experimentally determined "average" value. Cells with "strong hepatotropic differentiation capacity" have a significantly (statistically) high hepatotropic differentiation capacity compared to this "average" value, for example, 5%, 10%, 20%, 30%, 50%, 80%, 100%, 120%, 150%, 200%, 300%, or more, higher in terms of differentiation efficiency or the number of ultimately differentiated hepatotropic cells obtained.

[0066] As used herein, "cell population" includes both pre-enrichment and post-enrichment cell populations. After undergoing an enrichment step, the cell population may be enriched. Each cell population can be used directly in the next step, or partially or completely frozen for long-term or short-term storage and use in subsequent steps. Furthermore, cells of a cell population can be individually suspended to obtain a single-cell suspension.

[0067] As used in the present invention, the "pluripotent stem cells" include mammalian pluripotent stem cells, such as pluripotent stem cells derived from humans, rodents, and non-human primates; preferably, the pluripotent stem cells include human pluripotent stem cells.

[0068] As used herein, the terms "marker" and "marker" are used interchangeably. In the present invention, unless otherwise specified, a "marker" is a molecule of interest (e.g., a protein surface molecule) used to identify a specific cell (or population) or its characteristics. For example, in the present invention, NAD+ / NADH is used as a marker to identify cells with low NAD+ / NADH levels; and / or CD157 is used as a marker to identify cells with high CD157 expression.

[0069] This study reveals for the first time that pluripotent stem cells possess metabolic heterogeneity. In a population of cells with a low NAD+ / NADH ratio, NAD+ levels are low, and NAD+ / NADH ratios are low, while NADH levels remain consistent with those in a population with a high NAD+ / NADH ratio. These cells exhibit enhanced self-renewal capacity, endoderm differentiation potential, and hepatocyte differentiation potential.

[0070] The present invention also reveals that CD157 is highly expressed in cells with low NAD+ / NADH, that human pluripotent stem cells have heterogeneity in CD157 expression, and that cells with high CD157 expression have stronger endoderm differentiation potential and stronger liver cell differentiation potential.

[0071] Based on the inventors' novel findings, an isolated cell (population) or cell culture is provided. The cells are isolated from pluripotent stem cells and have the following characteristics: (i) low NAD+ / NADH content, or (ii) high expression of CD157.

[0072] The amino acid sequence of CD157 can be found in GenBank accession number NM_004334.3.

[0073] In a preferred embodiment of the present invention, the cells of the present invention are isolated from hepatocytes, or are passaged cells isolated from hepatocytes.

[0074] According to the inventors' research, the cells described herein can be expanded, cultured, passaged, and preserved after isolation from their natural environment (e.g., the body), while maintaining their excellent biological activity. Therefore, the cells described herein can be cultured and applied on a large-scale or industrial scale (e.g., for the large-scale production of hepatotropic cells).

[0075] According to the new discovery of the present inventors, the present invention also provides a method for isolating or enriching cells or cell cultures, wherein the cells are cells (populations) having characteristics selected from the following groups: (a) cells with low NAD+ / NADH content; or (b) cells with high CD157 expression.

[0076] The method of the present invention can be used to enrich (or separate) at least one target cell. The method of the present invention comprises a series of culture and selection steps that can be used separately, in combination, continuously, repeatedly or cyclically.

[0077] Therefore, the method for isolating or enriching cells with specific identifiable signals can be preferably provided in the embodiments of the present invention, or a method known to those skilled in the art. Various methods for isolating or enriching cells can be used in the present invention. Some cell sorting methods include but are not limited to the following:

[0078] (a) The SoNar fluorescent protein probe method produces different conformations when bound to NAD+ and NADH, thereby responding to different excitation wavelengths. When SoNar binds to NADH, the 405nm channel fluorescence becomes stronger; when bound to NAD+, the 488nm channel fluorescence increases. Preferably, the ratio of the fluorescence value of the 405nm channel to the fluorescence value of the 488nm channel is used to detect changes in the NAD+ / NADH ratio in cells.

[0079] (b) Immunomagnetic cell sorting is a technique that uses magnetic beads to separate target cells from a cell population. First, the magnetic beads bind to specific cell surface proteins on the target cells through antibodies, biotin, or avidin. The sample is then placed in a magnetic field, where the cells labeled with the magnetic beads are adsorbed. Unlabeled cells are retained in the supernatant, thereby physically separating the target cells from non-target cells in the sample. Because it is rapid and easy to operate, immunomagnetic cell sorting is one of the commonly used methods for researchers to isolate high-purity specific cell subsets.

[0080] (c) Flow cytometry, a method for separating heterogeneous cell populations using a flow cytometer and fluorescent probes. A fluorescently stained or labeled single-cell suspension is placed in a sample tube and forced into a flow chamber at high pressure. The flow chamber is filled with sheath fluid, which, under its wrapping and propulsion, forces the cells into a single file and ejects them from the flow chamber nozzle at a constant speed. An ultrahigh-frequency piezoelectric crystal is mounted on the flow chamber nozzle, which vibrates after being charged, breaking the ejected liquid stream into uniform droplets, into which the cells to be tested are dispersed. These droplets are charged with different positive and negative charges. As the droplets pass through a deflection plate carrying several thousand volts, they are deflected by the high-voltage electric field and fall into their respective collection containers. Uncharged droplets fall into a central waste container, thereby achieving cell separation. This method is particularly suitable for single cell sorting, sorting based on intracellular markers, sorting based on the expression levels of cell surface markers, and sorting complex cell types using multiple markers when purity is high.

[0081] (d) Microfluidic cell sorting, which involves manipulating fluids at the microscopic level to isolate single cells. Microfluidics is often implemented on microchips, often referred to as "labs on a chip." Microfluidics offers significant advantages when the volume of samples and reagents used is very small. Furthermore, labs on a chip are portable and can be used almost anywhere, making them particularly suitable as tools for on-site diagnostics. Several different microfluidic cell sorting methods include: ultrasonic sorting, aqueous two-phase systems, biomimetic microfluidics sorting, affinity sorting, deterministic lateral displacement, electrophoresis sorting, field flow fractionation, gravity and sedimentation, magnetophoresis, microfiltration, and optical sorting.

[0082] (e) Adhesion method, which can separate target cells from a heterogeneous cell population based on the unique adhesion ability of different cell types; by selecting appropriate growth factors and cell culture plates to selectively promote or inhibit the adhesion ability of cells, adhered cells can be separated from the suspension. The effective use of this method also depends on the observation of the adhesion properties of the cell population separated in the present invention.

[0083] Depending on the sorting method, different types of identifiable signals can be selected. For example, when a cell population is enriched by flow cytometry to form an enriched cell population containing at least one cell of interest, the identifiable signal used is preferably a fluorescent dye, which is interpreted based on the specific excitation or emission wavelength of the fluorescent dye.

[0084] Binding molecules or capture molecules used to separate or enrich specific cells are easily prepared or obtained by those skilled in the art. Commonly used binding molecules include, but are not limited to, antibodies and ligands, which may be free or immobilized on a solid support.

[0085] In the examples of the present invention, specific cell sorting methods are provided. However, it should be understood that after the markers or cell surface molecules of interest are known in the present invention, cell sorting is not limited to those specifically listed in the examples.

[0086] After the first generation cells are artificially isolated from the natural environment, they can be expanded on a large scale under appropriate conditions, which can be achieved by using cell culture methods known in the art. Preferably, the method described in the embodiments of the present invention can be referred to, but the present invention is not limited to this preferred method.

[0087] The cells obtained by the method of the present invention can be frozen, revived, passaged, and maintained in culture for a long time.

[0088] Based on the inventors' new discovery, the present invention provides a kit for isolating or enriching cells with strong hepatotropic differentiation ability or cells with high self-renewal ability, comprising: NAD+ / NADH binding molecules or capture molecules; or, CD157 binding molecules or capture molecules.

[0089] Based on the inventors' new findings, the present invention also provides a kit for preparing hepatodifferentiated cells, comprising: NAD+ / NADH binding molecules or capture molecules; and reagents or culture media for inducing pluripotent stem cells to differentiate into endoderm cells, hepatoblasts and / or liver cells (including immature liver cells or mature liver cells).

[0090] The kit may also include reagents for hydrolyzing / decomposing cells, such as collagenase and decomposing enzymes; reagents for cell suspension; culture medium for cell culture, etc.

[0091] To facilitate use by those skilled in the art, the kits described herein may also include instructions for isolating or enriching the cells or cell cultures described herein, or for preparing liver organoids. For example, the instructions may describe the aforementioned methods of the present invention.

[0092] The present invention will be further described below with reference to specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. Experimental procedures in the following examples, where specific conditions are not specified, generally followed conventional conditions such as those described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd edition, Science Press, or according to the conditions recommended by the manufacturer.

[0093] Example 1: Metabolic Heterogeneity of Human Pluripotent Stem Cells

[0094] First, use fluorescence microscopy to analyze the localization of NAD+ and NADH in the cell. Perform the following steps:

[0095] (1) Clone the SoNar probe into the pCDH vector (System Biosciences, CD527A-1);

[0096] (2) packaging lentivirus and infecting the human pluripotent stem cell line 1016 (obtained from the Harvard Stem Cell Institute (HSCI) iPSCore Facility);

[0097] (3) Images were acquired using a fluorescence microscope (LSM880NLO-FLIM) under the conditions of excitation light of 405 nm / 488 nm and emission light of 525 nm.

[0098] SoNar (Senor of NAD(H)redox, PMID: 25955212) is a genetically encoded fluorescent protein probe that can produce different conformations when binding to NAD+ and NADH through changes in protein conformation, thereby responding to different excitation light wavelengths. When SoNar binds to NADH, the fluorescence of the 405nm channel becomes stronger; when bound to NAD+, the fluorescence of the 488nm channel is enhanced. Therefore, the ratio of the fluorescence value of the 405 channel to the fluorescence value of the 488 channel can be used to detect changes in the NAD+ / NADH ratio in cells.

[0099] When judging, the analysis of NAD+ / NADH high and low: according to Figure 2 As marked in the upper left figure, after measuring the NAD+ / NADH ratio, the top 4% of cells in the cell population are classified as the high population, the bottom 4% of cells are classified as the low population, and the remaining approximately 92% of cells are discarded.

[0100] Acquire microscope images, such as Figure 1 , green fluorescence reflects the intracellular NAD+ level (left figure), purple fluorescence reflects the intracellular NADH level (middle figure), and Merge fluorescence reflects the intracellular NAD+ / NADH level (right figure).

[0101] Therefore, there is heterogeneity in NAD+, NADH, and NAD+ / NADH within the same human pluripotent stem cell line.

[0102] Example 2: Obtaining high and low NAD+ / NADH cell populations

[0103] To isolate a heterogeneous cell line, follow these steps:

[0104] (1) High and low NAD+ / NADH human pluripotent stem cell (1016) populations were obtained by cell sorting. High and low NAD+ / NADH cells were sorted from the SoNar-infected human pluripotent stem cell line 1016 under 405nm / 488nm excitation and 525nm emission conditions. Finally, high and low NAD+ / NADH cell subpopulations were obtained after five rounds of sorting.

[0105] (2) Flow cytometry verification of high and low NAD+ / NADH cell populations: Under the conditions of 405nm / 488nm excitation light and 525nm emission light, the sorted high and low NAD+ / NADH cell populations were verified.

[0106] (3) NAD+ / NADH Assay Verification of NAD+ / NADH High and Low Cell Populations The NAD+ / NADH Assay Kit (Sigma, MAK037-1KT) was used to determine the intracellular NAD+, NADH content, and NAD+ / NADH ratio.

[0107] The results are as follows Figure 2 , obtaining cell populations with high and low NAD+ / NADH ratios. Flow cytometry analysis of the sorting strategy for high and low NAD+ / NADH cell populations (top left). Flow cytometry confirmed that the high and low NAD+ / NADH cell populations maintained significant NAD+ / NADH differences after multiple passages (top right).

[0108] The NAD+ / NADH assay showed that in the NAD+ / NADH-low cell population, the NAD+ content was low, the NAD+ / NADH ratio was low, and the NADH content remained consistent with that in the NAD+ / NADH-high cell population (Figure 2).

[0109] Example 3: Cell populations with low NAD+ / NADH have stronger self-renewal ability

[0110] Human pluripotent stem cells (10^16) with high and low NAD+ / NADH levels were seeded at a density of 1×10^4 cells per well in 12-well plates. After 7 days of culture in mTeSR1 medium (STEMCELL Technologies, 05850), the cells were fixed with 4% PFA for 10 minutes. Subsequently, the cells were stained with crystal violet (Beyotime, C0121) for 10 minutes. The colonies formed in each well were then imaged and counted.

[0111] The results are as follows Figure 3 There is a significant difference in clone-forming ability between the two cell groups, and cells with low NAD+ / NADH form more and more monoclones.

[0112] Example 4: Cells with low NAD+ / NADH have stronger endoderm differentiation potential

[0113] Human pluripotent stem cells (1016) with high and low NAD+ / NADH levels were seeded at a density of 2 × 10^5 cells per well in 12-well plates and cultured in mTeSR1 medium. After 24 hours of culture, the medium was replaced with DE differentiation medium and differentiated for three days to obtain definitive endoderm (DE) cells.

[0114] DE differentiation medium preparation: RPMI-1640 medium (Invitrogen, 11875093) supplemented with B27 (Invitrogen, 12587010) and 100 ng / mL recombinant activin A (PeproTech, AF-120-14E) and 5 μM LY-294002 (Selleck, S1105).

[0115] The cells on the third day of DE differentiation were collected, RNA was extracted, and converted into cDNA. The expression of endoderm marker genes (Sox17, Foxa2, Gata6, Gata4) was detected by qPCR. qPCR analysis found that the expression of endoderm marker genes in cells with low NAD+ / NADH was significantly higher than that in cells with high NAD+ / NADH ( Figure 4 , upper left picture).

[0116] Immunofluorescence was used to detect the expression of FOXA2 in cells on the third day of DE differentiation. Immunofluorescence results showed that the expression of FOXA2, a marker gene of endoderm, in cells with low NAD+ / NADH was significantly increased ( Figure 4 , upper right picture).

[0117] Cells were collected for different days of DE differentiation, proteins were extracted, and the expression of SOX17 and FOXA2 proteins was detected by western blot. Western blot analysis of protein levels showed that the expression of endoderm marker genes SOX17 and FOXA2 was significantly increased in cells with low NAD+ / NADH. Figure 4 , below).

[0118] Therefore, there is a clear difference in DE differentiation ability between the two groups of cells, and cells with low NAD+ / NADH have stronger DE differentiation potential.

[0119] Example 5: Cells with low NAD+ / NADH have stronger liver cell differentiation potential

[0120] Human pluripotent stem cells (1016) with high and low NAD+ / NADH were seeded at a density of 2×10^5 cells per well in a 12-well plate and cultured in mTeSR1 medium. After 24 hours of culture, the medium was replaced with DE differentiation medium for three days to obtain definitive endoderm cells; the medium was then replaced with HE differentiation medium for five days to obtain hepatoblasts. The medium was then replaced with IMH differentiation medium for five days to obtain immature hepatocytes. The medium was then replaced with MH differentiation medium for five days to obtain mature hepatocytes.

[0121] HE differentiation medium preparation: RPMI-1640 medium supplemented with B27, 20 ng / mL BMP4 (PeproTech, 120-05), 5 ng / mL FGF2 (PeproTech, AF-100-18B), and 0.5% DMSO.

[0122] Preparation of IMH differentiation medium: RPMI-1640 medium supplemented with B27, 20 ng / mL HGF (PeproTech, 100-39) and 0.5% DMSO.

[0123] Preparation of MH differentiation medium: HCM hepatocyte culture medium (Lonza, CC-3198) was supplemented with 20 ng / mL HGF, 20 ng / mL Oncostatin M (PeproTech, 300-10), 100 nM dexamethasone (Sigma, D4902), and 0.5% DMSO.

[0124] Mature liver cells were collected, RNA was extracted, and converted into cDNA. The expression of mature liver cell marker genes (Alb, Afp, Asgpr, A1at, Glut2, Cyp3a4) was detected by qPCR.

[0125] The results are as follows Figure 5 The expression of liver cell marker genes Alb, Asgpr, A1at, Glut2, and Cyp3a4 in cells with low NAD+ / NADH was significantly higher than that in cells with high NAD+ / NADH.

[0126] Therefore, there is a clear difference in liver cell differentiation ability between the two groups of cells, and cells with low NAD+ / NADH have stronger liver cell differentiation potential.

[0127] Example 6: CD157 is highly expressed in cells with low NAD+ / NADH

[0128] RNA was extracted from the two groups of cells with high and low NAD+ / NADH obtained by sorting, and converted into cDNA. The expression of NAD metabolism-related genes was detected by qPCR.

[0129] like Figure 6 qPCR analysis found that CD157 expression was significantly higher in cells with low NAD+ / NADH ratio than in cells with high NAD+ / NADH ratio. High CD157 expression in cells with low NAD+ / NADH ratio led to a decrease in NAD+ content in the cells, while maintaining the same NADH content, thus reducing the NAD+ / NADH ratio in the cells.

[0130] The CD157 gene, also known as BST1, is a multifunctional extracellular enzyme localized to the cell outer membrane. CD157 possesses both glycohydrolase and ADP-ribosyl cyclase activities. The NAD+ glycohydrolase activity catalyzes the conversion of NAD+ and water to produce NAM and ADP-ribose, while the ADP-ribosyl cyclase activity generates cyclic ADP-ribose.

[0131] Therefore, it is suggested that the difference in CD157 expression between cells with high and low NAD+ / NADH is the reason for the difference in NAD+ / NADH between the two cell populations.

[0132] Example 7: Human pluripotent stem cells exhibit heterogeneity in CD157 expression

[0133] In this example, the results of Example 7 were further analyzed and verified. Human pluripotent stem cell (1016) lines were immunofluorescently stained using CD157 antibody (ThermoFisher, 25-1579-41), and cell nuclei were stained with DAPI (Sigma, D8417). Images were captured using a microscope (OLYMPUS, IX73) under conditions of 569 nm excitation and 780 nm emission, and 340 nm excitation and 488 nm emission.

[0134] The results are as follows Figure 7 In the same human pluripotent stem cell line, there is heterogeneity in CD157 gene expression between different cells.

[0135] Example 8: Obtaining CD157 High- and Low-Expression Cell Populations and Their NAD+ / NADH Expression Characteristics

[0136] Cell populations with high and low CD157 expression were obtained by cell sorting. Human pluripotent stem cells (1016) were stained with CD157 flow cytometry antibody (ThermoFisher, 12-1579-42, 1:100) at 4°C for 30 minutes, and then 5% of cells with high CD157 expression and 5% of cells with low CD157 expression were sorted out by flow cytometry under 565nm excitation and 576nm emission conditions. This sorting process was repeated for a total of 5 rounds to further enrich cell populations with high and low CD157 expression.

[0137] Flow cytometry was used to obtain cell populations with high and low CD157 expression. Figure 8 .according to Figure 8 As shown, cells with low CD157 expression have less surface antibody binding to CD157, resulting in lower signal intensity in flow cytometry. During sorting, the top 5% of cells with the highest CD157 signal intensity are considered the CD157-high population, the bottom 5% of cells are considered the CD157-low population, and the remaining approximately 90% of cells are discarded.

[0138] Furthermore, the NAD+ / NADH assay kit (Sigma, MAK037-1KT) was used to determine the NAD+, NADH content and NAD+ / NADH ratio in CD157 high-expressing and low-expressing cell populations.

[0139] like Figure 9 ,NAD+ / NADH determination showed that in the cell population with high CD157 expression, the NAD+ content was low, the NAD+ / NADH was low, and the NADH content was consistent with that in the cell population with low CD157 expression.

[0140] Therefore, the cell population with high CD157 expression has low NAD+ / NADH.

[0141] Example 9: Cells with high CD157 expression have stronger endoderm differentiation potential

[0142] Human pluripotent stem cells (1016) with high and low CD157 expression were seeded at a density of 2×10^5 cells per well in 12-well plates and cultured in mTeSR1 medium. After 24 hours of culture, the medium was replaced with DE differentiation medium for three days. Cells were harvested, RNA was extracted, and converted to cDNA. The expression of endoderm marker genes (Sox17, Foxa2, Gata6, Gata4) was analyzed by qPCR.

[0143] like Figure 10 qPCR analysis found that the expression of endoderm marker genes in cells with high CD157 expression was significantly higher than that in cells with low CD157 expression.

[0144] Therefore, cells with high CD157 expression have stronger endoderm differentiation potential.

[0145] Example 10: Cells with high CD157 expression have stronger liver cell differentiation potential

[0146] Human pluripotent stem cells (1016) with high and low expression of CD157 were seeded in a 12-well plate at a density of 2×10^5 cells per well. After culturing for 24 hours, the culture medium was replaced with DE differentiation medium for three days, then replaced with HE differentiation medium for five days, then replaced with IMH differentiation medium for five days, and then replaced with MH differentiation medium for five days to obtain mature liver cells.

[0147] Cells were collected, RNA was extracted, and converted into cDNA. The expression of mature liver cell marker genes (Alb, Afp, Asgpr, A1at, Glut2, Cyp3a4) was detected by qPCR.

[0148] qPCR analysis found that the expression of liver cell marker genes Alb, Afp, and Cyp3a4 in cells with high CD157 expression was significantly higher than that in cells with low CD157 expression ( Figure 11 , upper left).

[0149] The supernatant of mature liver cells was collected and the concentration of human albumin was determined by ELISA (Abcam, ab108788). Total RNA was extracted from mature liver cells for RNA-seq transcriptome analysis.

[0150] ELISA test found that liver cells differentiated from cells with high CD157 expression secreted higher amounts of human albumin ( Figure 11 , middle left picture).

[0151] Functional enrichment analysis was performed on 2534 genes (Fold change>1.5, P-value<0.05) whose expression increased in liver cells differentiated from cells with high CD157 expression in RNA-seq transcriptome data. It can be seen that in liver cells with high CD157 expression, the genes with increased expression are mainly enriched in metabolic pathways, cholesterol metabolism and other related pathways, which are related to liver function; the heat map of liver-related genes in RNA-seq results showed that the expression of most liver-related genes was increased in liver cells with high CD157 expression ( Figure 11 , lower left and right).

[0152] Therefore, cells with high CD157 expression have stronger hepatocyte differentiation potential.

[0153] In summary, cells with high expression of CD157 in human pluripotent stem cells have low NAD+ / NADH and stronger liver cell differentiation potential. Therefore, CD157 can be used as a cell surface marker of human pluripotent stem cells. Subpopulations with stronger liver differentiation ability in the cell population can be enriched by flow cytometry with high expression of CD157, thereby improving the differentiation efficiency of human pluripotent stem cells into liver cells ( Figure 12 ).

[0154] Therefore, CD157 can be used as a cell surface marker to identify a subpopulation of human pluripotent stem cells with strong liver differentiation ability.

[0155] The above-described embodiments merely represent several implementation methods of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art may make several modifications and improvements without departing from the scope of the present invention, and these modifications and improvements fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be based on the appended claims. At the same time, all documents mentioned in this application are cited as references in this application, just as if each document was cited as a reference individually.

Claims

1. A method for obtaining cells with strong hepatotropic differentiation ability or high self-renewal ability, comprising: For pluripotent stem cells, cells having characteristics selected from the following group are isolated: (a) Cells with low NAD+ / NADH content; or (b) Cells with high CD157 expression.

2. The method according to claim 1, wherein The cells with low NAD+ / NADH content are isolated or enriched by the following method: using a SoNar fluorescent protein probe to bind to NAD+ and NADH, and sorting out the cells with low NAD+ / NADH content according to the wavelength of the excitation light; The cells with high expression of CD157 are isolated or enriched by the following method: using a binding molecule or a capture molecule that specifically binds to or captures CD157 to sort out the cells with high expression of CD157 from pluripotent stem cells; Preferably, the method for separating or enriching cells includes: flow cytometry sorting, immunomagnetic bead sorting, microfluidic cell sorting, and adhesion method.

3. Use of isolated or enriched cells as starting cells for preparing hepatodifferentiated cells; wherein: The isolated or enriched cells are cells with strong hepatodifferentiation ability and have characteristics selected from the following group: (a) cells with low NAD+ / NADH content; or (b) high expression of CD157; Preferably, the hepatic differentiated cells include: endoderm cells, hepatoblasts, and liver cells.

4. A method for preparing hepatotropically differentiated cells, comprising: (1) Preparing cells with strong hepatotropic differentiation ability by any of the methods described in claim 1 to 2; (2) Using the cells with strong hepatodifferentiation ability of (1) as starting cells, inducing hepatodifferentiation, and obtaining hepatodifferentiated cells.

5. The method according to claim 4, wherein The cells differentiated into liver include: endoderm cells, hepatoblasts, liver cells; preferably: Inducing using an endoderm induction medium to prepare the endoderm cells; sequentially using an endoderm induction medium and a liver differentiation medium to prepare the hepatoblasts; Sequentially using endoderm induction medium and liver differentiation medium to prepare immature hepatocytes; Endoderm induction medium and liver differentiation medium were used in sequence to prepare mature hepatocytes.

6. The method according to claim 4, wherein The liver cells express liver cell markers Alb, Asgpr, A1at, Glut2, Cyp3a4, Afp; or The endoderm cells express endoderm markers Sox17, Foxa2, Gata6, and Gata4.

7. An isolated or enriched cell having a strong hepatodifferentiation potential and having a characteristic selected from the group consisting of: (a) low NAD+ / NADH content; or (b) high expression of CD157.

8. The use of NAD and NADH as markers for screening cells with strong hepatodifferentiation ability or high self-renewal ability from pluripotent stem cells.

9. The application of CD157 as a marker for screening cells with strong hepatodifferentiation ability or high self-renewal ability from pluripotent stem cells.

10. A kit for isolating or enriching cells with strong hepatotropic differentiation ability or high self-renewal ability, comprising: NAD+ / NADH binding or capture molecules; Or, CD157 binding molecules or capture molecules.

11. A kit for preparing hepatotropically differentiated cells, comprising: NAD+ / NADH binding or capture molecules; as well as Reagents or culture media for inducing the differentiation of pluripotent stem cells into endoderm, hepatoblasts and / or hepatocytes.

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