WO2022042700A1 - Method for reprogramming umbilical cord mesenchymal stem cells into liver cells and liver organoid prepared therefrom - Google Patents

Abstract

Provided is a method for reprogramming umbilical cord mesenchymal stem cells into liver cells. The method comprises obtaining umbilical cord mesenchymal stem cells; pretreating the umbilical cord mesenchymal stem cells for 2 days; using a hepatic differentiation-inducing composition to conduct a hepatic differentiation of the pretreated umbilical cord mesenchymal stem cells for 7 days; after the hepatic differentiation induction treatment, using a first maturation inducing composition to conduct a first maturation induction of umbilical cord mesenchymal stem cells for 7 days to obtain liver cells; and using a second maturation inducing composition to conduct a second maturation induction of the obtained liver cells for 12-20 days to obtain functional liver cells. A type of liver organoids: acellular matrix hydrogel, stem cells and liver cells. The stem cells and liver cells are distributed in the acellular matrix hydrogel. Cultivation time is prolonged, and the induction efficiency is significantly increased by adding the additional step of second maturation induction.

Description

Method for reprogramming umbilical cord mesenchymal stem cells into liver cells and prepared liver organoids technical field

The present application relates to a cell culture technology, in particular to a method for reprogramming umbilical cord mesenchymal stem cells into liver cells and the prepared liver organoids.

Background technique

Liver tumors usually require a major hepatectomy, and the remaining liver will regenerate to compensate, which can easily lead to post-hepatectomy liver failure (PHLF), which is one of the most worrying complications that must be managed by transplantation and life-long immunization. inhibition to treat. The shortage of donor livers, high cost, and immunosuppression limit the use of this treatment modality. Treatment of liver failure can also be improved with temporary liver support methods. For example, bioartificial livers, continuous hemodiafiltration through plasma exchange and albumin dialysis can improve some liver function parameters, but it is difficult to prove that it can improve patient survival. And the technology is limited by the lack of a safe source of hepatocytes. Potential alternative sources of hepatocytes include porcine hepatocytes, immortalized human hepatocytes, ES cell-derived hepatocytes and various adult stem cells, but they still cannot solve the problem of the lack of primary hepatocytes in the current artificial liver therapy. Further, studies have shown that transplantation of mesenchymal stem cells (MSCs) or MSCs-derived hepatocyte-like cells can improve liver function in rodents or patients with liver injury. However, several conventional approaches used so far have had little success, and these hepatocyte-like cells exhibit only a subset of the labeling and function of primary hepatocytes.

SUMMARY OF THE INVENTION

The purpose of this application is to overcome the deficiencies of the prior art and provide a method for reprogramming into liver cells from umbilical cord mesenchymal stem cells that are simple, easy to repeat, have high reprogramming efficiency, and can be used for clinically obtaining a large amount of functional liver cells. The obtained liver cells were used to prepare liver organoids.

In order to achieve the above technical purpose, the technical scheme adopted in this application is as follows:

The first is a method for reprogramming umbilical cord mesenchymal stem cells into liver cells, which includes the following steps:

Obtain umbilical cord mesenchymal stem cells;

The umbilical cord mesenchymal stem cells are pretreated for 2 days;

The pretreated umbilical cord mesenchymal stem cells were induced to induce hepatogenesis for 7 days using the hepatogenesis induction composition;

The umbilical cord mesenchymal stem cells subjected to hepatogenesis induction were subjected to the first maturation induction for 7 days with the first maturation inducing composition to obtain liver cells;

The liver cells are subjected to a second maturation induction for 12-20 days by using the second maturation inducing composition to obtain functional liver cells.

Application of the method as previously described in the preparation of liver organoids.

Second is a liver organoid comprising: an acellular matrix hydrogel, stem cells and liver cells distributed in the acellular matrix hydrogel.

Again, it is a preparation method of liver organoids, which comprises the following steps:

Mix the acellular matrix hydrogel diluent, stem cells and liver cells evenly with serum-free medium;

In a culture vessel, statically cultivate until the acellular matrix hydrogel is solidified;

After confirming the survival of cells in the acellular matrix hydrogel after coagulation, the culture was continued to obtain liver organoids.

Preferably, the dilution of the acellular matrix hydrogel is adjusted to pH 7-7.5 before mixing.

Compared with the prior art, the present application has the following advantages:

(1) The method for reprogramming umbilical cord mesenchymal stem cells into liver cells of the present application, the used hepatogenesis inducing composition, the first maturation inducing composition and the second maturation inducing composition only contain factors and small molecule compounds , the formula is simple and easy to prepare.

(2) The method for reprogramming umbilical cord mesenchymal stem cells into liver cells of the present application adds a second maturation induction step, prolongs the culture time, and significantly improves the induction efficiency, especially the functional liver cells obtained. Albumin expression was significantly increased.

(3) In the method for reprogramming umbilical cord mesenchymal stem cells into liver cells of the present application, an inhibitor is added to the second maturation inducing composition, which inhibits the signaling of the pathway and promotes the differentiation of mesenchymal stem cells into liver cells. cell.

(4) The liver organoids of the present application use cells derived from umbilical cord mesenchymal stem cells and acellular matrix hydrogels, so that the obtained liver organoids have ultra-low immunogenicity and the possibility of cross-species transplantation , experiments have confirmed that human-derived liver organoids can be maintained and grown in rats.

Description of drawings

FIG. 1 is a morphological view of the umbilical cord mesenchymal stem cells obtained by the application under an optical microscope.

FIG. 2 is a morphological view under an optical microscope at various stages of differentiation from umbilical cord mesenchymal stem cells to functional liver cells using the method of reprogramming umbilical cord mesenchymal stem cells into liver cells according to the present application.

FIG. 3 is a graph showing the results of immunofluorescence expression of functional liver cells obtained in the present application.

FIG. 4 is a comparison diagram of the expression levels of liver-specific genes in liver cells and functional liver cells obtained by the present application.

Fig. 5 is a diagram showing the identification of glycogen staining of functional liver cells obtained in the present application.

FIG. 6 is a graph showing the identification of cell survival in the liver organoids obtained in the present application.

FIG. 7 is a morphological diagram of the liver organoids obtained in this application under an electron microscope.

Figure 8 is a graph showing the maintenance of the liver organoids obtained in the application after transplantation in rats.

detailed description

The present application will be further described in detail below with reference to the accompanying drawings and specific embodiments.

Example 1

Method for reprogramming umbilical cord mesenchymal stem cells into liver cells

Umbilical cords are derived from humans, but are usually disposed of as medical waste. They are widely sourced and do not involve ethical issues. They are an excellent tissue source for obtaining large quantities of mesenchymal stem cells in clinical and research. Umbilical cord mesenchymal stem cells (UC-MSCs) have strong proliferative activity and have been used in the treatment of various diseases.

In this application, UC-MSCs were cultured to 6 points under serum-free conditions, that is, UC-MSCs were cultured in vitro until they entered the logarithmic growth phase. As shown in Figure 1, when UC-MSCs were cultured in vitro to the third generation, the morphology The UC-MSCs were uniformly grown in a long spindle-shaped dimple-like shape. The subsequent identification of cell surface markers and differentiation ability of UC-MSCs confirmed that the extracted UC-MSCs met the requirements of subsequent reprogramming steps.

Subsequently, the induction differentiation treatment was started, and the following reagents were added to the serum-free medium in sequence to induce the reprogramming of UC-MSCs:

Pretreatment stage: epidermal growth factor (EGF) 20ng/mL (Gibco; PHG0315), basic fibroblast growth factor (bFGF) 10ng/mL (PeproTech; 100-31-25), pretreatment for 2d;

Hepatogenesis induction composition: basic fibroblast growth factor (bFGF) 30ng/mL, hepatocyte growth factor (HGF) 20ng/mL (PeproTech; 100-39-10), nicotinamide (nicotinamide) 0.61g/L ( Sigma; N0636), induction 7d;

The first maturation induction composition: ITS+Premix 40ng/mL (Gibco; 51500056), recombinant human osteoprotegerin-M (OSM) 10ng/mL (PeproTech; 300-10-2), dexamethasone (Dexamethasone) 1umol/L (Invitrogen; D1383), induction 7d;

The second maturation induction composition: ITS+Premix 40ng/mL, recombinant human osteoprotegerin-M (OSM) 10ng/mL, dexamethasone (Dexamethasone) 1umol/L, human γ-secretase complex inhibitor (γ- secretase inhibitor, or DAPT for short) 10umol/L (MCE; HY-13027), small molecule inhibitor SB431542 10umol/L (abcam; ab146590), induced for 12-20 days.

The reprogrammed cells were identified during the induction of UC-MSCs and the results were as follows:

1. Observe cell morphology under inverted microscope: cells grow well during the induction reprogramming process, after pretreatment (day1-day2) and hepatogenesis induction stage (day3-day9), MSCs become more slender, and hepatocyte maturation stage (day10) -day16), UC-MSCs differentiated into liver cells, the cell shape gradually changed from long spindle to flat and rounded, and the liver cells obtained by reprogramming at the secondary maturation stage of liver cells (day17-day32) were further matured into functional liver cells. When , the cells turned into irregular circles or polygons, with obvious nucleoli and no apoptosis (as shown in Figure 2).

2. Identification of liver-specific genes by immunofluorescence: albumin (ALB, abcam kit: 207327), alpha-fetoprotein (AFP, abcam kit: ab169552) and cytokeratin (CK19, abcam kit: ab52625) , the identification results are AFP(+) (see the first column of pictures in Figure 3), ALB(+) (see the first column of pictures in Figure 3), and CK19(+) (see the second column of pictures in Figure 3). Further, the fluorescence value was statistically analyzed. Compared with the 16th day on the 32nd day of the induction stage, that is, the functional liver cells and liver cells were compared, the expression rates of AFP, ALB and CK19 were significantly increased (Fig. The programmed liver cells matured further and were closer to liver cells with liver function.

3. Determination of glycogen accumulation function (using Solarbio kit: G285): As shown in Figure 5, the liver cells were stained with glycogen, and the identification result of glycogen accumulation function was positive.

In conclusion, after the process of pretreatment, hepatogenesis, and two maturation inductions, functional liver cells reprogrammed and differentiated from UC-MSCs were successfully obtained. Compared with the prior art, the second maturation induction stage was added. The induction time of liver cells was prolonged, and the success rate of induction was greatly improved.

Embodiment 2

Liver organoids and preparation method thereof

Organoids are three-dimensional cell cultures that contain some of the key properties of the organs they represent. Organoids are also an in vitro culture system that includes a population of self-renewing stem cells that can differentiate into multiple organ-specific cell types, have a similar spatial organization to their counterparts and reproduce some of their functions, thereby providing an Highly physiologically relevant systems. Organoid cultures have been used in a variety of tissues, including the gut, liver, pancreas, kidney, prostate, lung, optic cup, and brain. Organoids are also a tool with applications in developmental biology, disease pathology, cell biology, regenerative mechanisms, precision medicine, and drug toxicity and efficacy testing, among others. For these and other applications, organoid culture enables a highly informative complement to existing 2D culture methods and animal model systems.

In addition to their use in clinical liver failure transplantation, liver organoids can be used as an emergency bridge in the transition from liver failure to liver regeneration, or as a complement to extensive liver resection and temporary liver maintenance during the transplant waiting period. .

Using the aforementioned method for reprogramming umbilical cord mesenchymal stem cells into liver cells and the obtained functional liver cells, liver organoids are prepared. Specifically, the method for constructing liver organoids is as follows:

(1) Take 3ml of umbilical cord acellular matrix pregel on ice, and balance it with 300ul of 10×PBS to form a hydrogel;

(2) Adjust pH to 7-7.5 with NaOH for subsequent use;

(3) Add 1 × 10 ⁷ UC-MSCs, 1 × 10 ⁶ liver cells, and 3 ml of the balanced hydrogel to 12 ml of serum-free medium, and mix well by pipetting thoroughly;

(4) Take a 6-well plate, add 2.5ml of the above mixed liquid to each well, let it stand for 2-4h in the incubator, and add 2ml of serum-free medium after gelation. The growth of cells in the hydrogel was observed under a microscope, and the validity of the cells cultured in the hydrogel was identified: the 1d, 3d, and 7d of culture were stained to identify the survival of the cells.

(5) The formed "3D-hOs" (liver organoids, see Figure 7) can be obtained in 6-7 days.

Among them, the umbilical cord acellular matrix hydrogel is prepared from the umbilical cord tissue, which not only maintains low immunogenicity relative to the human body, but also preserves the integrity of the internal structure of the ECM to the greatest extent, which is beneficial to stem cells and liver cells. Proliferation and survival within the gel, even after transplantation into the body. As shown in Figure 6, the cells in the hydrogel were cultured for 1d, 3d, and 7d, and the density of living cells became higher and higher, which proved that the cells grew well in the voids of the hydrogel.

The UC-MSCs can also be replaced with other types of stem cells. From the perspective of developmental potential, totipotent stem cells, pluripotent stem cells and unipotent stem cells can all have the potential to form liver organoids.

The hepatocytes can be obtained by inducing the differentiation of umbilical cord mesenchymal stem cells as described above, or the hepatocytes are derived from primary cultured liver cells or subcultured liver cell lines.

The preparation process of the above-mentioned liver organoids uses serum-free and xeno-free medium throughout the whole process, which conforms to GMP standards. All cells and tissues are obtained from human beings, which are easy to obtain in large quantities and do not involve medical ethics. Due to the immunomodulatory properties of MSCs, umbilical cord decellularized ECM hydrogels have ultra-low immunogenicity. The constructed 3D-hOs will not cause immune responses when transplanted between different individuals, and can even be transplanted across species, with low cost and a wide range of sources. , It can be mass-produced in large scale, the system is mature and easy to replicate, and the effect is stable, which lays a foundation for future clinical trials. Further, the liver organoids were transplanted into rats, and the rats were dissected 10 days after transplantation. It was found that the grafts migrated to the surface of the liver, and the 3D-hOs were covered with blood vessels (Fig. 8), indicating that the human 3D-hOs It can be maintained and grown in rats without causing immune rejection.

To sum up, the method for reprogramming umbilical cord mesenchymal stem cells into liver cells of the present application adds the second maturation induction step, prolongs the culture time, and significantly improves the induction efficiency, especially the obtained functional liver. The albumin expression of the cells was significantly increased. The liver organoids of the present application use cells derived from umbilical cord mesenchymal stem cells and acellular matrix hydrogels, so that the obtained liver organoids have ultra-low immunogenicity and the possibility of cross-species transplantation.

The above-mentioned embodiments are the preferred embodiments of the application, but are not only limited by the above-mentioned embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principle of the application should be The equivalent substitution methods are all included in the protection scope of the present application.

Claims (16)

1. A method for reprogramming umbilical cord mesenchymal stem cells into liver cells, characterized in that it comprises the following steps:

   Obtain umbilical cord mesenchymal stem cells;

   The umbilical cord mesenchymal stem cells are pretreated for 2 days;

   The pretreated umbilical cord mesenchymal stem cells were induced to induce hepatogenesis for 7 days using the hepatogenesis induction composition;

   The umbilical cord mesenchymal stem cells subjected to hepatogenesis induction were subjected to the first maturation induction for 7 days with the first maturation inducing composition to obtain liver cells;

   The liver cells are subjected to a second maturation induction for 12-20 days by using the second maturation inducing composition to obtain functional liver cells.
2. The method of claim 1, wherein before the umbilical cord mesenchymal stem cells are pretreated, they are cultured in vitro to a logarithmic growth phase.
3. The method of claim 1, wherein the umbilical cord mesenchymal stem cells are cultured in vitro under serum-free conditions before pretreatment.
4. The method of claim 1, wherein the pretreated reagent comprises 20ng/mL epidermal growth factor and 10ng/mL basic fibroblast growth factor;

   The hepatogenesis inducing composition comprises 30ng/mL basic fibroblast growth factor, 20ng/mL hepatocyte growth factor and 0.61g/L nicotinamide;

   The first maturation induction composition comprises a mixture of 40ng/mL ITS Premix, 10ng/mL recombinant human osteoprotegerin-M and 1umol/L dexamethasone;

   The second maturation induction composition comprises 40ng/mL ITS+Premix, 40ng/mL recombinant human osteoprotegerin-M, 1umol/L dexamethasone, 10um/L human γ-secretase complex inhibitor and 10umol/L small molecule inhibitor SB431542.
5. The method of claim 1, wherein, under an optical microscope, the functional liver cells are irregular circles or polygons, and the nucleoli have clear boundaries.
6. The method of claim 1, wherein the functional liver cells have a higher liver-specific gene expression rate than the liver cells.
7. The method of claim 6, wherein the liver-specific genes include alpha-fetoprotein, albumin, and cytokeratin.
8. The method of claim 1, wherein the glycogen accumulation function of the functional liver cells is identified as positive.
9. Application of the method according to claims 1 to 8 in the preparation of liver organoids.
10. A liver organoid, characterized in that it comprises: an acellular matrix hydrogel, stem cells and liver cells, and the stem cells and liver cells are distributed in the acellular matrix hydrogel.
11. The liver organoid of claim 10, wherein the acellular matrix hydrogel is prepared from umbilical cord tissue; the stem cells are totipotent stem cells, pluripotent stem cells or unipotent stem cells; the liver cells are induced by umbilical cord tissue The mesenchymal stem cells are obtained by differentiation, or the liver cells are derived from primary cultured liver cells or subcultured liver cell lines.
12. A method for preparing liver organoids, characterized in that it comprises the following steps:

    Mix the acellular matrix hydrogel diluent, stem cells and liver cells evenly with serum-free medium;

    In a culture vessel, statically cultivate until the acellular matrix hydrogel is solidified;

    After confirming the survival of cells in the acellular matrix hydrogel after coagulation, the culture was continued to obtain liver organoids.
13. The method of claim 12, wherein the pH of the diluent of the acellular matrix hydrogel is adjusted to 7-7.5 before mixing.
14. The method of claim 12, wherein the mixing ratio of the acellular matrix hydrogel diluent, stem cells and liver cells is: 1×10 ⁷ stem cells, 1×10 10 ⁶ liver cells and 3 ml of acellular matrix hydrogel dilution.
15. The method of claim 12, wherein the survival of reprogrammed liver cells and normal liver cells in the decellularized matrix hydrogel is determined by staining.
16. The method of claim 12, wherein after the cells in the decellularized matrix hydrogel survive, continue to culture for 6-7 days to obtain liver organoids.

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