Method for efficiently inducing adult adipose tissue into insulin secretion tissue

Abstract

The invention discloses a method for efficiently inducing an adult adipose tissue into an insulin secretion tissue, a culture medium used in the method and application of the insulin secretion tissue obtained by the method. The method for efficiently inducing the adult adipose tissue into the insulin secretion tissue comprises the following steps: constructing the adult adipose tissue into a self-aggregation adipose tissue; the self-aggregation adipose tissue is dynamically cultured for 5 days to 10 days in the following culture medium IV: DMEM (Dulbecco Modified Eagle Medium) based, 0.5% to 5% of PSG (Phosphosilicate Sodium Glycol), 0.5% to 5% of B27, 0.1% to 5% of Ascobic Acid, 5 ng / ml to 200 ng / ml of FGF-2, 1 to 100 [mu] M of CHIR999021, 0.5% to 50 [mu] M of Retinoic Acid, 0.1 to 10 [mu] M of DZNe, 0.1 mM to 10 mM of 5-AZA and 0.1 to 10 [mu] M of Y-27632; and carrying out induced differentiation on the cultured product to pancreas islet organs. According to the method disclosed by the invention, the number of living cells and beta-like cells in a single islet organ is remarkably increased.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a method for efficiently inducing adult adipose tissue to become insulin-secreting tissue. Background Technology

[0002] Diabetes mellitus is a chronic metabolic disease characterized by hyperglycemia. In 2021, there were 537 million adults worldwide with diabetes, and this number is projected to exceed 783 million by 2045. The situation regarding diabetes prevention and control in my country is also severe: the number of patients surged by 56% from 2011 to 2021, and is projected to exceed 174 million by 2045. Diabetes is mainly divided into two types: Type 1 diabetes mellitus (T1DM) and Type 2 diabetes mellitus (T2DM). A common feature of these two types of diabetes is insufficient insulin in the peripheral blood, which disrupts glucose metabolic homeostasis and ultimately leads to serious complications such as cardiovascular disease, chronic renal failure, blindness, or lower limb amputation. Over the past century, treatment strategies for diabetes have focused on lifestyle interventions and oral hypoglycemic agents, but the fundamental problem of insulin deficiency has not been addressed. While injecting exogenous insulin can effectively and rapidly lower blood sugar, the body still cannot detect a hyperglycemic state. Allogeneic islet transplantation holds the potential to address the issues of glucose perception and insulin-responsive secretion, but low graft survival rates, a severe shortage of islet donors, and the need for lifelong immunosuppressants hinder its widespread clinical application. Therefore, the development of novel treatment strategies is urgently needed.

[0003] Currently, islet organoids derived from stem cells hold the potential to completely cure diabetes and have demonstrated promising therapeutic effects in various diabetic animal models and clinical trials. In current research on islet organoid construction, the most commonly used pluripotent stem cells are embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). The construction process of islet organoids mainly simulates several key stages of islet development: first, pluripotent stem cells are induced to differentiate into definitive endoderm (DE), then through the primitive gut tube (PGT), posterior foregut, pancreatic endoderm (PE), and pancreatic endocrine progenitors (PEP), ultimately differentiating into β-like cells with insulin-secreting function. Although the reported differentiation efficiency of using ESCs and iPSCs to induce differentiation into β-like cells is as high as 60-80%, the ethical and legal controversies, tumorigenic risks, and immune rejection associated with the application of ESCs and iPSCs have brought great uncertainty to their clinical application. Summary of the Invention

[0004] This invention provides a method for efficiently inducing adult adipose tissue to become insulin-secreting tissue.

[0005] The technical solution of the present invention is as follows: A method for efficiently inducing adult adipose tissue to become insulin-secreting tissue includes the following steps: Step (1): Construct adult adipose tissue into self-aggregated adipose tissue (also known as RMF (Reaggregated microfat)); Step (2): The self-aggregated adipose tissue was dynamically cultured in the following No. 4 medium for 5-10 days: DMEM basic + 0.5-5% PSG + 0.5-5% B27 + 0.1-5 mM Ascorbic Acid + 5-200 ng / ml FGF-2 + 1-100μM CHIR999021 + 0.5-5μM Retinoic Acid + 0.1-10μM DZNep + 0.1-10mM 5-AZA + 0.1-10μM Y-27632; Step (3): Inducing the product of dynamic culture in step (2) to differentiate into pancreatic islet organoids, including sequentially inducing directional endoderm, foregut, pancreatic progenitor cells 1, pancreatic progenitor cells 2, endocrine progenitor cells, and pancreatic islet organoids in their respective culture media.

[0006] In a preferred embodiment, the method for constructing adult adipose tissue into self-aggregating adipose tissue in step (1) includes the following steps: 1.1 Adult adipose tissue was mechanically prepared into microparticle adipose tissue; 1.2 Self-aggregating adipose tissue was obtained by culturing microparticle adipose tissue using the following method: First, perform in vitro proliferation suspension culture for 2-3 weeks, followed by extended proliferation culture for 1-2 weeks. The in vitro proliferation suspension culture method is as follows: first, culture in medium I for 1-3 days, then culture in medium II for 11-20 days. The composition of medium I is: α-MEM + 1-20% FBS + 1-20 mM HEPES + 0.5-5% PSG + 5-50 ng / ml FGF-2 + 5-50 ng / ml PDGF-BB + 0.1-10 μM Y-27632; the composition of medium II is: α-MEM + 1-20% FBS + 1-20 mM HEPES + 0.5-5% PSG + 5-50 ng / ml FGF-2 + 5-50 ng / ml PDGF-BB; the extended proliferation culture is carried out in medium III as follows: α-MEM + 1-20% FBS + 1-20 mM HEPES + 0.5-5% PSG + 5-50 ng / ml FGF-2 + 5-50 ng / ml PDGF-BB. mMHEPES + 0.5-5% PSG+ 5-50 ng / ml FGF-2 + 5-50 ng / ml PDGF-BB + 0.1-10 μMCHIR999021+ 50-200 ng / mL Noggin + 0.5-50μM A-83-01 + 0.5-50μM Forskolin+ 0.1-10 μM Y-27632.

[0007] More preferably, self-aggregating adipose tissue is obtained by culturing microparticle adipose tissue using the following method: First, in vitro proliferation suspension culture was performed for 2 weeks, followed by a 1-week extended proliferation culture. The in vitro proliferation suspension culture method was as follows: cultured for 1 day in medium I, then for 13 days in medium II. The composition of medium I was: α-MEM + 10% FBS + 10 mM HEPES + 1% PSG + 10 ng / ml FGF-2 + 20 ng / ml PDGF-BB + 10 μM Y-27632; the composition of medium II was: α-MEM + 10% FBS + 10 mM HEPES + 1% PSG + 10 ng / ml FGF-2 + 20 ng / ml PDGF-BB; the extended proliferation culture was performed in medium III as follows: α-MEM + 10% FBS + 10 mM HEPES + 1% PSG + 10 ng / ml FGF-2 + 20 ng / ml PDGF-BB + 3 μM Y-27632. CHIR999021 + 100 ng / mL Noggin + 0.5 μM A-83-01 + 1 μM Forskolin + 10 μM Y-27632.

[0008] In a preferred embodiment, the method for inducing the product of dynamic culture in step (2) into the directional endoderm in step (3) includes: first, dynamic culture in medium 5 for 1-3 days, and then dynamic culture in medium 6 for 3-6 days; the composition of medium 5 is: MCDB131 + 0.5-5% GlutaMAX + 1-25 mM Glucose + 1-20 mM NaHCO3 + 0.5-5% PSG + 0.1-5% BSA + 50-200 ng / mL Activin A + 1-10 μM CHIR99021 + 10-100 ng / mL Wnt-3a; the composition of medium 6 is: MCDB131 + 0.5-5% GlutaMAX + 1-25 mM Glucose + 1-20 mM NaHCO3 + 0.5-5% PSG + 0.1-5% BSA + 10-200 ng / mL Activin A + 10-100 ng / mL Wnt-3a. ng / mL Activin A. If the culture time is longer than one day, change the culture medium daily.

[0009] In a preferred embodiment, the method for inducing the product from the previous step into the foregut in step (3) includes: dynamically culturing in medium No. 7 for 1-3 days, wherein the composition of medium No. 7 is: MCDB131 + 0.5-5% GlutaMAX + 1-25 mM Glucose + 1-20 mM NaHCO3 + 0.5-5% PSG + 0.1-5% BSA + 0.1-5 mM Ascorbic Acid + 1-100 ng / mL KGF. If the culturing time exceeds one day, the medium is changed daily.

[0010] In a preferred embodiment, the method for inducing the product from the previous step into pancreatic progenitor cells 1 in step (3) includes: dynamically culturing in medium 8 for 1-3 days, wherein the composition of medium 8 is: MCDB131 + 0.5-5% GlutaMAX + 1-25 mM Glucose + 2-40 mM NaHCO3 + 0.5-5% PSG + 0.1-5% BSA + 0.1-5mM Ascorbic Acid + 0.1-5% ITS-X + 1-100 ng / mL KGF + 0.1-50 μM TPPB + 0.1-50 μM MSANT1 + 0.1-50 μM LDN193189 + 0.1-50 μM RA + 1-100 μM Taurine. If the culture time is longer than one day, the medium is changed daily.

[0011] In a preferred embodiment, the method for inducing the product from the previous step into pancreatic progenitor cells 2 in step (3) includes: dynamic culture in medium 9 for 2-6 days, wherein the composition of medium 9 is: MCDB131 + 0.5-5% GlutaMAX + 1-25 mM Glucose + 2-40 mM NaHCO3 + 0.5-5% PSG + 0.1-5% BSA + 0.1-5mM Ascorbic Acid + 0.1-5% ITS-X + 1-100 ng / mL KGF + 0.1-50 μM TPPB + 0.1-50 μM MSANT1 + 0.1-50 μM LDN193189 + 0.1-50 μM RA + 10-100 μM Taurine + 1-50 μM MWIKI4. The present invention adds the small molecule drug WIKI4 to the No. 9 culture medium, which can promote the expression of NKX6.1.

[0012] In a preferred embodiment, the method for inducing the product from the previous step into endocrine progenitor cells in step (3) includes: dynamic culture in medium 10 for 5-10 days, wherein the composition of medium 10 is: MCDB131 + 0.5-5% GlutaMAX + 0.1-25 mM Glucose + 2-40 mM NaHCO3 + 0.5-5% PSG + 0.1-5% BSA + 0.1-5 mM Ascorbic Acid + 0.1-5% ITS-X + 1-50 μg / mL heparin + 1-50 mM Nicotinamide + 0.1-50 μM SANT1 + 0.1-50 μM ALK5i II + 0.1-10 μM XXI + 1-50 ng / mL Betacellulin + 0.01-1 μM RA + 0.1-50 μM T3 + 10-100 μM Taurine + 0.1-10 μM Y-27632. This invention adds Nicotinamide to culture medium No. 10, which can stimulate the WNT signaling pathway, promote cell proliferation, inhibit methylation and apoptosis, and maintain stemness.

[0013] In a preferred embodiment, the method for inducing the product from the previous step into pancreatic organoids in step (3) includes: dynamic culture in medium 11 for 7-28 days, wherein the composition of medium 11 is: MCDB131 + 0.5-5% GlutaMAX + 1-25 mM Glucose + 0.1-5% BSA + 0.5-5% Pen / Strep + 1-5% NEAA + 1-50 μg / mL heparin + 0.1-5 mM Ascorbic Acid + 1-50 µg / mL ZnSO4•7H2O + 0.1-50 μM ALK5i II + 1-50 μM T3.

[0014] This invention also provides a culture medium for efficiently inducing adult adipose tissue into insulin-secreting tissue. The culture medium comprises: DMEM basic + 0.5-5% B27 + 0.5-5% PSG + 0.1-5 mM MAscorbic Acid + 5-200 ng / ml FGF-2 + 1-100 μM CHIR999021 + 0.5-50 μM Retinoic Acid + 0.1-10 μM DZNep + 0.1-10 μM 5-AZA + 0.1-10 μM Y-27632. This culture medium is the aforementioned fourth culture medium.

[0015] This invention stimulates the WNT signaling pathway by adding components such as CHIR999021, Retinoic Acid, DZNep, 5-AZA, and MY-27632 to culture medium No. 4, thereby promoting cell proliferation, inhibiting methylation and apoptosis, and maintaining stemness.

[0016] This invention also provides an application of the insulin-secreting tissue prepared by the aforementioned method in the fields of diabetes treatment models, pancreatic disease simulation models, pancreatic islet-related drug screening, and pancreatic organ development models.

[0017] In this invention, all percentages of the components in the various culture media are volume percentages.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows: First, the method provided by the present invention adds step (2) before inducing differentiation, which is the key to improving the efficiency of inducing differentiation in subsequent steps.

[0019] Secondly, the method provided by this invention employs a new self-aggregating adipose tissue proliferation culture protocol, including a new culture medium and new culture steps, which significantly increases the proportion of proliferating cells (Ki67 positive cells) and can shorten the original 4-week self-aggregating proliferation culture time to 3 weeks.

[0020] Third, the method provided by this invention employs a novel scheme for inducing differentiation of self-aggregating adipose tissue into pancreatic islet organoids, including the setting of steps and the selection of culture media in each step, which significantly improves the induction differentiation efficiency at each stage: In the directional endoderm stage, FOXA2 is used... + / SOX17 + The proportion of cells increased from (58.68 ± 9.77)% in the conventional protocol to (85.18 ± 6.60)%; in the pancreatic progenitor cell stage, PDX1 was used... + / NKX6.1 + The proportion of cells increased from (46.82 ± 12.11)% in the traditional protocol to (61.11 ± 11.78)%; in the pancreatic islet organoid stage, CP... + / PDX1 + The cell percentage increased from (15.78 ± 2.00)% in the traditional protocol to (45.95 ± 13.26)%, which improved CP. + / NKX6.1 + The proportion of cells increased from (20.15 ± 5.72)% in the conventional protocol to (44.00 ± 11.46)%.

[0021] Fourth, the method provided by this invention employs a novel scheme for inducing differentiation of self-aggregating adipose tissue into pancreatic islet organoids, significantly increasing the number of viable cells and β-like cells (CPs) within a single islet organoid. + / NKX6.1 + Number of live cells: The number of live cells was calculated from (1.94 ± 0.21) × 10⁻⁶. 4 The organoid production rate increased to (2.73 ± 0.51) × 10⁻⁶. 4 / organoids, CP + / NKX6.1 + Cells increased from (0.57 ± 0.19) × 10 4 The organoid yield increased to (1.12 ± 0.23) × 10⁻⁶. 4 / Organoids.

[0022] Of course, any product implementing this invention does not necessarily need to achieve all of the advantages described above at the same time. Attached Figure Description

[0023] Figure 1 This is a schematic diagram of the process for obtaining self-aggregating adipose tissue through microparticle adipose tissue culture in this invention; Figure 2 This is a flowchart illustrating the process of inducing differentiation from self-aggregated adipose tissue into pancreatic islet organoids in this invention. Figure 3 This is a comparison diagram of the self-aggregation culture scheme in Embodiment 1 of the present invention and the traditional self-aggregation culture scheme in the proliferation state of adipose tissue cells; Figure 4 The differentiation induction scheme in Embodiment 1 of this invention differs from the traditional scheme in the directional endoderm stage (FOXA2). + / SOX17 + Comparison of the induced differentiation efficiency of cells; Figure 5 The differentiation induction protocol in Example 1 of this invention differs from the traditional protocol in the pancreatic progenitor cell (PDX1) stage. + / NKX6.1 + Comparison of the induced differentiation efficiency of cells; Figure 6 The differentiation induction protocol in Example 1 of this invention differs from the traditional protocol in the pancreatic endocrine cell stage (CP). + / NKX6.1 + Comparison of the induced differentiation efficiency of cells; Figure 7 The differentiation induction protocol in Example 1 of this invention differs from the traditional protocol in the pancreatic endocrine cell stage (CP). + / PDX1 + Comparison of the induced differentiation efficiency of cells; Figure 8 This is a comparison of the function of β-like cells in the pancreatic endocrine cell stage between the differentiation induction scheme in Example 1 of the present invention and the traditional scheme. Detailed Implementation

[0024] Addressing the existing challenges in constructing pancreatic islet organoids, the inventors' team pioneered a novel approach in their earlier research. Through in vitro self-aggregation culture and reprogramming, they directly transformed adult adipose tissue blocks into insulin-secreting islet organoids. After transplantation into type 1 diabetic mice, these organoids reversed hyperglycemia within two weeks and maintained stable blood glucose levels for up to three months. This novel paradigm of directly converting abundant and readily available human adipose tissue into functional islet organoids offers a new pathway for diabetes treatment. However, despite the team's initial success in directly converting adult adipose tissue into functional islet organoids, the efficiency of converting mesodermal adipose tissue into endoderm β cells remains relatively low compared to ESCs and iPSCs. In the inventors' earlier pancreatic organoids constructed from adult adipose tissue, the final proportion of functional β-like cells (20.15% of cells co-expressing C-peptide and PDX1, and 15.78% of cells co-expressing C-peptide and NKX6.1) was still lower than the proportion of functional β-like cells (60-80%) reported in the literature for pancreatic organoids constructed from ESCs and iPSCs. This may affect the feasibility of its clinical translation.

[0025] Based on the aforementioned previous research results, the technical solution provided by this invention significantly increases the proportion of functional β-like cells and improves the insulin secretion level of the constructed pancreatic islet organoids by adjusting the induction differentiation strategy.

[0026] This invention provides a method for efficiently inducing adult adipose tissue to become insulin-secreting tissue, typically adult adipose tissue.

[0027] The invention is further characterized by a novel culture medium and culture steps designed for the construction of self-aggregating adipose tissue, which proliferate microparticle adipose tissue prepared from adult adipose tissue to obtain self-aggregating adipose tissue. This new culture medium and steps not only significantly increase the proportion of proliferating cells (Ki67-positive cells) but also shorten the original 4-week self-aggregating proliferation culture time to 3 weeks.

[0028] In this document, the range expressed as "from one value to another" is a concise way of representing a range to avoid listing all the values ​​in that range in the specification. Therefore, the description of a particular range of values ​​covers any value within that range as well as the smaller range of values ​​defined by that value, just as if the arbitrary value and the smaller range of values ​​were explicitly stated in the specification.

[0029] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of protection of the present invention. Improvements and adjustments made by those skilled in the art based on the present invention in practical applications still fall within the scope of protection of the present invention.

[0030] For the sake of brevity, not all possible combinations of the technical features in each implementation scheme or embodiment are described herein. Therefore, as long as the combinations of these technical features do not contradict each other, the technical features in each implementation scheme or embodiment can be combined arbitrarily, and all possible combinations should be considered within the scope of this specification. Example 1 In this embodiment, the characteristic design part is referred to... Figure 1 and Figure 2 ,in, The self-aggregating adipose tissue construction stage, such as Figure 1 As shown: In the first stage, from days 1 to 3, microparticle adipose tissue was statically cultured in medium No. 1 (components: α-MEM + FBS + HEPES + PSG + FGF-2 + PDGF-BB + Y-27632). From days 4 to 14 of the first stage, the culture was changed to medium No. 2 (components: α-MEM + FBS + HEPES + PSG + FGF-2 + PDGF-BB) to continue culturing and obtain RMF tissue. In the second stage, the RMF tissue was cultured in medium No. 3 (components: α-MEM + FBS + HEPES + PSG + FGF-2 + PDGF-BB + CHIR999021 + Noggin + A-83-01 + Forskolin + Y-27632) for 7 days to obtain RMF microspheres.

[0031] Differentiation-inducing strategies such as Figure 2As shown, RMF pellet 1 was cultured in medium 4 (DMEM basic + PSG + B27 + Ascorbic Acid + FGF-2 + CHIR999021 + Retinoic Acid (RA) + DZNep + 5-AZA + Nicotinamide + Y-27632) for 8 days to obtain RMF pellet 2. RMF pellet 2 was then cultured in medium 5 (MCDB131 + GlutaMAX + Glucose + NaHCO3 + PSG + BSA + Activin A + CHIR99021 + Wnt-3a) for one day, followed by 3 days in medium 6 (MCDB131 + GlutaMAX + Glucose + NaHCO3 + PSG + BSA + Activin A) to induce directional endoderm formation. Finally, it was cultured in medium 7 (MCDB131 + GlutaMAX + Glucose + NaHCO3 + PSG + BSA + Ascorbic Acid + Pancreatic progenitor cells were cultured for 2 days in KGF (KG) to induce the foregut, then cultured for 2 days in medium 8 (MCDB131 + GlutaMAX + Glucose + NaHCO3 + PSG + BSA + Ascorbic Acid + ITS-X + KGF + TPPB + SANT1 + LDN193189 + RA + Taurine) to induce pancreatic progenitor cells 1. They were then cultured for 4 days in medium 9 (MCDB131 + GlutaMAX + Glucose + NaHCO3 + PSG + BSA + Ascorbic Acid + ITS-X + KGF + TPPB + SANT1 + LDN193189 + RA + Taurine + WIKI4) to induce pancreatic progenitor cells 2. Finally, they were cultured for 4 days in medium 10 (MCDB131 + GlutaMAX + Glucose + NaHCO3 + PSG + BSA + Ascorbic Acid + ITS-X + Heparin + Nicotinamide + SANT1 + ALK5i II + XXI + ... After culturing in Betacellulin + RA + T3 + Taurine + Y-27632 for 7 days, endocrine progenitor cells were induced.Then, they were cultured in medium 11 (MCDB131 + GlutaMAX + Glucose + BSA + Pen / Strep + NEAA + Heparin + Ascorbic Acid + ZnSO4 + ALK5i II + T3) for 7-14 days to obtain pancreatic islet organoids.

[0032] A specific and complete example of cultivation is as follows: The method for inducing adult adipose tissue globules to differentiate into pancreatic islet organoids mainly includes the following steps: Step (1) involves the processing of adult adipose tissue, specifically including: S1, Take the human fat tissue obtained from liposuction surgery and cut it into pieces with scissors; S2, the adipose tissue was placed into a 50 mL centrifuge tube, rinsed with an equal volume of physiological saline, centrifuged at 1200g for 3 minutes, and repeated 3 times. S3. Collect the adipose tissue from the middle layer into a 20 mL syringe. Connect another empty 20 mL syringe using a connector with an inner diameter of 2 mm. Push the two syringes repeatedly 30 times. Then replace it with another connector with an inner diameter of 1 mm and push the two syringes repeatedly 30 times to fully fragment the adipose tissue. S4. Transfer the fragmented adipose tissue to a 50 mL centrifuge tube and centrifuge at 1600g for 3 minutes.

[0033] S5, carefully remove the upper oil layer and the lower liquid layer, retaining the middle micro-fatty tissue.

[0034] Step (2) involves the self-aggregation culture of microparticle adipose tissue suspension, the procedure of which can be found in [reference needed]. Figure 1 As shown, the specific operation is as follows: S1. Take 1.5 mL of the microparticle adipose tissue obtained in step (1), inoculate it into a 6-well culture plate, add 3 mL of medium No. 1, place it in a 5% CO2 incubator, and statically culture for 1 day. Then replace it with medium No. 2 and continue culturing for 2 weeks. Change the medium 2-3 times a week. After 2 weeks of in vitro proliferation and suspension culture in this step, the microparticle adipose tissue can gradually re-aggregate to form a new adipose tissue mass.

[0035] The composition of medium No. 1 is: α-MEM + 10% FBS + 10 mM HEPES + 1% PSG + 10 ng / ml FGF-2 + 20 ng / ml PDGF-BB + 10 μM Y-27632.

[0036] The composition of medium No. 2 is: α-MEM + 10% FBS + 10 mM HEPES + 1% PSG + 10 ng / ml FGF-2 + 20 ng / ml PDGF-BB.

[0037] In S2, using a 1 mm diameter biopsy drill, adipose tissue spheres approximately 1 mm in diameter are extracted from the aggregated adipose tissue mass obtained in S1. These spheres are placed in a low-adhesion 96-well culture plate, and 0.2 mL of medium (Type III) is added. Culture is continued for one week, with medium changes 2-3 times per week, to obtain adipose tissue spheres, also known as RMF spheres. Extending the proliferation culture by one week allows the adipose tissue spheres to become more compact and rounded.

[0038] The composition of medium 3 is as follows: Medium 3: α-MEM + 10% FBS + 10 mM HEPES + 1% PSG + 10 ng / ml FGF-2 + 20 ng / ml PDGF-BB + 3 μM CHIR999021 + 100 ng / mL Noggin + 0.5 μM A-83-01 + 1 μM Forskolin + 10 μM Y-27632.

[0039] This embodiment also includes a comparative example (Scheme 1.0), which is the cultivation method provided by the inventor in patent document CN118048293 A. After the culture in step (2) is completed, samples are taken from the adipose tissue globules, and the morphology and cell proliferation status of the adipose tissue after self-aggregation are evaluated by HE staining and Ki67 staining, and compared with the inventor's previous scheme 1.0. The results are as follows: Figure 3 As shown. Visual observation reveals that the morphology of the adipose tissue after self-aggregation in embodiment 2.0 of the present invention is denser and more rounded compared to embodiment 1.0; Figure 3 In Figure A (first row), the confocal microscopy results can be used to observe changes in KI67 expression; and, in embodiment (Scheme 2.0) of the present invention, Ki67 + The proportion of cells was significantly higher than that of the control group (Program 1.0), such as Figure 3 As shown in B (second row).

[0040] Step (3) involves the induction of adipose tissue globules into pancreatic islet organoids; the procedure is described in the following reference. Figure 2 As shown, the specific operation is as follows: S1, Preparation stage: Transfer the adipose tissue spheres obtained in step (2) from the 96-well plate to the low adhesion 6-well culture plate, add 3 mL of culture medium No. 4, place it on the shaker in the 5% CO2 incubator, and culture dynamically for 8 days at a speed of 90 rpm, changing the medium 2-3 times a week. The composition of medium No. 4 is: DMEM basic + 1% PSG + 1% B27 + 0.2 mM AscorbicAcid + 100 ng / ml FGF-2 + 6 μM CHIR999021 + 2 μM RA (Retinoic Acid) + 2 μM DZNep + 1 μM 5-AZA + 6 μM Y-27632.

[0041] S2, the induction of adipose tissue globules into oriented endoderm cells: Culture medium No. 4 was carefully aspirated, and after washing with PBS, 3 mL of culture medium No. 5 was added, and the cells were dynamically cultured for 1 day. Then, culture medium No. 5 was carefully aspirated, and after washing with PBS, culture was replaced with culture medium No. 6, and dynamic culture continued for 3 days, changing the culture medium daily, to obtain oriented endoderm cells. After this stage of culture, samples were taken, and the expression levels of FOXA2 and SOX17 were assessed by immunofluorescence staining and flow cytometry. The assessment results are shown below. Figure 4 The results of confocal microscopy are as follows: Figure 4 As shown in Figure A, it can be clearly observed that FOXA2 in the directional endoderm cells obtained in embodiment 2.0 of this invention... + / SOX17 + The number of cells was significantly increased compared to the traditional protocol 1.0.

[0042] Meanwhile, flow cytometry analysis results, such as Figure 4 As shown in Figure B, the highly efficient induction culture medium provided by embodiment 2.0 of the present invention significantly improves the efficiency of induction differentiation of adipose tissue globules into directional endoderm, and FOXA2... + / SOX17 + The proportion of cells increased from (58.68 ± 9.77)% in the conventional protocol 1.0 to (85.18 ± 6.60)%. Figure 4 C is a representative graph of FOXA2 and SOX17 expression levels analyzed by flow cytometry.

[0043] In this step, The composition of medium No. 5 is: MCDB131 + 1% GlutaMAX + 8 mM Glucose + 14 mM NaHCO3 + 1% PSG + 0.1% BSA + 100 ng / mL Activin A + 3μM CHIR99021 + 20 ng / mL Wnt-3a.

[0044] The composition of medium No. 6 is: MCDB131 + 1% GlutaMAX + 8 mM Glucose + 14 mM NaHCO3 + 1% PSG + 0.1% BSA + 100 ng / mL Activin A.

[0045] S3, Directional endoderm induction stage for foregut: Carefully aspirate culture medium 6, wash with PBS, add 3 mL of medium 7, and continue dynamic culture for 2 days, changing the culture medium daily to obtain foregut cells; The composition of medium No. 7 is: MCDB131 + 1% GlutaMAX + 4 mM Glucose + 14 mM NaHCO3 + 1% PSG + 0.1% BSA + 0.25 mM MAscorbic Acid + 50 ng / mL KGF.

[0046] S4, the induction stage of pancreatic progenitor cells 1 from the foregut: carefully aspirate culture medium 7, wash with PBS, add 3 mL of medium 8, and continue dynamic culture for 2 days, changing the culture medium every day to obtain pancreatic progenitor cells 1; this stage can enhance the expression of PDX1.

[0047] The composition of medium No. 8 is as follows: MCDB131 + 1% GlutaMAX + 2 mM Glucose + 24 mM NaHCO3 + 1% PSG + 2% BSA + 0.25 mM Ascorbic Acid + 0.5% ITS-X + 50 ng / mL KGF + 0.2μM TPPB + 0.25μM SANT1 + 0.2μM LDN193189 + 2μM RA + 50μM Taurine.

[0048] Induction of pancreatic progenitor cells 1 into pancreatic progenitor cells 2: Carefully aspirate culture medium 8, wash with PBS, add 3 mL of medium 9, and continue dynamic culture for 4 days, changing the culture medium daily to obtain pancreatic progenitor cells 2; this stage can enhance the expression of pancreatic precursor cells NKX6.1.

[0049] The composition of medium 9 is as follows: MCDB131 + 1% GlutaMAX + 2 mM Glucose + 24 mM NaHCO3 + 1% PSG + 2% BSA + 0.25 mM Ascorbic Acid + 0.5% ITS-X + 50 ng / mL KGF + 0.2 μM TPPB + 0.25 μM SANT1 + 0.2 μM LDN193189 + 0.1 μM RA + 50 μM Taurine + 9 μM IKI4.

[0050] Samples were taken after this stage of culture, and the expression levels of PDX1 and NKX6.1 were assessed by immunofluorescence staining and flow cytometry.

[0051] Confocal microscopy results as follows Figure 5 As shown in Figure A, it can be clearly observed in the figure that PDX1 is present in the pancreatic progenitor cells obtained by Scheme 2.0 of this embodiment. + / NKX6.1 + The number is significantly increased compared to the traditional scheme 1.0; among them, such as Figure 5 As shown in Figure B, the use of WIKI4 can significantly increase the expression of NKX6.1 in the obtained pancreatic progenitor cells.

[0052] Meanwhile, the flow cytometry analysis results are as follows Figure 5 As shown in C, the highly efficient induction medium provided by this invention significantly improves the efficiency of directional differentiation of endoderm into pancreatic progenitor cells, and PDX1 + / NKX6.1 + The cell percentage increased from (46.82 ± 12.11)% in the traditional protocol to (61.11 ± 11.78)%. Figure 5 D is a representative plot of PDX1 and NKX6.1 expression levels analyzed by flow cytometry.

[0053] S5, the induction stage of pancreatic progenitor cells 2 into pancreatic endocrine cells: carefully aspirate culture medium 9, wash with PBS and add 3 mL of medium 10, continue dynamic culture for 7 days, change the culture medium every day to obtain pancreatic endocrine cells. The composition of medium 10: MCDB131 + 1% GlutaMAX + 20 mM Glucose + 24 mM NaHCO3 + 1% PSG + 2% BSA + 0.25 mM Ascorbic Acid + 0.5% ITS-X + 10 μg / mL heparin + 10 mM Nicotinamide + 0.25 μM SANT1 + 10 μM ALK5i II + 1 μM XXI + 20 ng / mL Betacellulin + 0.1 μM RA + 1 μM T3 + 50 μM Taurine + 10 μM Y-27632.

[0054] After this stage of culture, pancreatic endocrine cells were sampled, and the amount of CP / PDX1 co-expressing cells and CP / NKX6.1 co-expressing cells was assessed by immunofluorescence staining and flow cytometry.

[0055] Confocal microscopy results as follows Figure 6 As shown in Figure A, it can be clearly observed that the number of pancreatic endocrine cells co-expressing CP / PDX1 obtained by Scheme 2.0 of Embodiment 1 of the present invention is significantly increased compared with that of the traditional Scheme 1.0; at the same time, the flow cytometry analysis results are as follows. Figure 6 As shown in B, Scheme 2.0 of Embodiment 1 of the present invention contains CP in glandular endocrine cells. + / PDX1 + The cell percentage increased from (15.78 ± 2.00)% in the conventional protocol 1.0 to (45.95 ± 13.26)%. Figure 6 C is a representative graph representing the expression levels of CP and PDX1 analyzed by flow cytometry.

[0056] Similarly, immunofluorescence staining was used on NKX6.1 + Cells were observed, and the ratio of CP+ / NKX6.1+ cells was analyzed using flow cytometry. The results are as follows: Figure 7 As shown in A, B, and C, Figure 7 As can be seen from the above, the construction scheme 2.0 provided in Embodiment 1 of the present invention significantly improves the performance of pancreatic endocrine cells PDX1. + The expression, at the same time, will CP + / NKX6.1 + The proportion of cells increased to (44.00 ± 11.46)%.

[0057] In addition, the results of in vitro glucose-stimulated insulin secretion experiments, such as Figure 8As shown in Figures A and B, the results indicate that the insulin secretion capacity of the pancreatic endocrine cells obtained from Scheme 2.0 of Embodiment 1 of the present invention is not significantly different from that of the pancreatic endocrine cells from the conventional Scheme 1.0. However, the total cell activity results are as follows: Figure 8 As shown in C, the number of live cells and CP obtained by Scheme 2.0 of Embodiment 1 of the present invention are shown. + / NKX6.1 + The number of cells significantly exceeded that obtained by the conventional culture protocol 1.0.

[0058] S6, Induction of pancreatic endocrine cells into pancreatic islet organoids: Carefully aspirate 10 mL of culture medium, rinse with PBS, add 3 mL of medium No. 11, and continue dynamic culture for 7-14 days, changing the culture medium every two days to obtain pancreatic islet organoids.

[0059] The composition of medium 11 is as follows: MCDB131 + 1% GlutaMAX + 2 mM Glucose + 2% BSA + 1% Pen / Strep + 1% NEAA + 10 μg / mL heparin + 0.25 mM Ascorbic Acid + 10 µg / mL ZnSO4•7H2O + 10 μM ALK5i II + 1 μM T3.

[0060] As can be seen from the above embodiments, the solution of the present invention has the following effects and advantages: First, the method provided by the present invention adds step (2) before inducing differentiation, which is the key to improving the efficiency of inducing differentiation in subsequent steps.

[0061] Secondly, the method provided by this invention employs a new self-aggregating adipose tissue proliferation culture protocol, including a new culture medium and new culture steps, which significantly increases the proportion of proliferating cells (Ki67 positive cells) and can shorten the original 4-week self-aggregating proliferation culture time to 3 weeks.

[0062] Third, the method provided by this invention employs a novel scheme for inducing differentiation of self-aggregating adipose tissue into pancreatic islet organoids, including the setting of steps and the selection of culture media in each step, which significantly improves the induction differentiation efficiency at each stage: In the directional endoderm stage, FOXA2 is used... + / SOX17 + The proportion of cells increased from (58.68 ± 9.77)% in the conventional protocol to (85.18 ± 6.60)%; in the pancreatic progenitor cell stage, PDX1 was used... + / NKX6.1 +The proportion of cells increased from (46.82 ± 12.11)% in the traditional protocol to (61.11 ± 11.78)%; in the pancreatic islet organoid stage, CP... + / PDX1 + The cell percentage increased from (15.78 ± 2.00)% in the traditional protocol to (45.95 ± 13.26)%, which improved CP. + / NKX6.1 + The proportion of cells increased from (20.15 ± 5.72)% in the conventional protocol to (44.00 ± 11.46)%.

[0063] Fourth, the method provided by this invention employs a novel scheme for inducing differentiation of self-aggregating adipose tissue into pancreatic islet organoids, significantly increasing the number of viable cells and β-like cells (CPs) within a single islet organoid. + / NKX6.1 + Number of live cells: The number of live cells was calculated from (1.94 ± 0.21) × 10⁻⁶. 4 The organoid production rate increased to (2.73 ± 0.51) × 10⁻⁶. 4 / organoids, CP + / NKX6.1 + Cells increased from (0.57 ± 0.19) × 10 4 The organoid yield increased to (1.12 ± 0.23) × 10⁻⁶. 4 / Organoids.

[0064] This invention also provides an application of the insulin-secreting tissue prepared by the above-mentioned method for efficiently inducing adult adipose tissue into insulin-secreting tissue in the fields of diabetes treatment models, pancreatic disease simulation models, screening of islet-related drugs, and pancreatic organ development models.

[0065] Meanwhile, under the teachings of this invention and the above embodiments, those skilled in the art can easily foresee that all the raw materials or their equivalents and processing methods listed or exemplified in this invention can achieve this invention, and that the upper and lower limits and range values ​​of the parameters of each raw material and processing method can also achieve this invention. Examples are not listed one by one here.

Claims

1. A method for efficiently inducing adult adipose tissue to become insulin-secreting tissue, characterized in that, Includes the following steps: Step (1): Construct adult adipose tissue into self-aggregating adipose tissue; Step (2): The self-aggregated adipose tissue was dynamically cultured in the following No. 4 medium for 5-10 days: DMEM basic + 0.5-5% PSG + 0.5-5% B27 + 0.1-5 mM Ascorbic Acid + 5-200 ng / ml FGF-2 + 1-100μM CHIR999021 + 0.5-50μM Retinoic Acid + 0.1-10μM DZNep + 0.1-10 mM 5-AZA + 0.1-10μM Y-27632; Step (3): Inducing the product of dynamic culture in step (2) to differentiate into pancreatic islet organoids, including sequentially inducing directional endoderm, foregut, pancreatic progenitor cells 1, pancreatic progenitor cells 2, endocrine progenitor cells, and pancreatic islet organoids in their respective culture media.

2. The method for efficiently inducing adult adipose tissue into insulin-secreting tissue as described in claim 1, characterized in that, The method for constructing self-aggregating adipose tissue from adult adipose tissue in step (1) includes the following steps: 1.1 Adult adipose tissue was mechanically prepared into microparticle adipose tissue; 1.2 Self-aggregating adipose tissue was obtained by culturing microparticle adipose tissue using the following method: First, perform in vitro proliferation suspension culture for 2-3 weeks, followed by extended proliferation culture for 1-2 weeks. The in vitro proliferation suspension culture method is as follows: first, culture in medium I for 1-3 days, then culture in medium II for 11-20 days. The composition of medium I is: α-MEM + 1-20% FBS + 1-20 mM HEPES + 0.5-5% PSG + 5-50 ng / ml FGF-2 + 5-50 ng / ml PDGF-BB + 0.1-10 μM Y-27632; the composition of medium II is: α-MEM + 1-20% FBS + 1-20 mM HEPES + 0.5-5% PSG + 5-50 ng / ml FGF-2 + 5-50 ng / ml PDGF-BB; the extended proliferation culture is carried out in medium III as follows: α-MEM + 1-20% FBS + 1-20 mM HEPES + 0.5-5% PSG + 5-50 ng / ml FGF-2 + 5-50 ng / ml PDGF-BB. mM HEPES+ 0.5-5% PSG+ 5-50 ng / ml FGF-2 + 5-50 ng / ml PDGF-BB + 0.1-10 μM CHIR999021+50-200 ng / mL Noggin + 0.5-50μM A-83-01 + 0.5-50μM Forskolin+ 0.1-10 μM Y-27632.

3. The method for efficiently inducing adult adipose tissue into insulin-secreting tissue as described in claim 2, characterized in that, Self-aggregating adipose tissue was obtained by culturing microparticle adipose tissue using the following method: First, in vitro proliferation suspension culture was performed for 2 weeks, followed by a 1-week extended proliferation culture. The in vitro proliferation suspension culture method was as follows: cultured for 1 day in medium I, then for 13 days in medium II. The composition of medium I was: α-MEM + 10% FBS + 10 mM HEPES + 1% PSG + 10 ng / ml FGF-2 + 20 ng / ml PDGF-BB + 10 μM Y-27632; the composition of medium II was: α-MEM + 10% FBS + 10 mM HEPES + 1% PSG + 10 ng / ml FGF-2 + 20 ng / ml PDGF-BB; the extended proliferation culture was performed in medium III as follows: α-MEM + 10% FBS + 10 mM HEPES + 1% PSG + 10 ng / ml FGF-2 + 20 ng / ml PDGF-BB + 3 μM Y-27632. CHIR999021 + 100 ng / mL Noggin + 0.5μM A-83-01 + 1μM Forskolin + 10μM Y-27632.

4. The method for efficiently inducing adult adipose tissue into insulin-secreting tissue as described in claim 1, characterized in that, The method for inducing the product of dynamic culture in step (2) into the directional endoderm in step (3) includes: first, dynamic culture in medium No. 5 for 1-3 days, and then dynamic culture in medium No. 6 for 3-6 days; the composition of medium No. 5 is: MCDB131 + 0.5-5% GlutaMAX + 1-25mM Glucose + 1-20 mM NaHCO3 + 0.5-5% PSG + 0.1-5% BSA + 50-200 ng / mL Activin A + 1-10 μM CHIR99021 + 10-100 ng / mL Wnt-3a; the composition of medium No. 6 is: MCDB131 + 0.5-5% GlutaMAX + 1-25mM Glucose + 1-20 mM NaHCO3 + 0.5-5% PSG + 0.1-5% BSA + 10-200 ng / mL Activin A.

5. The method for efficiently inducing adult adipose tissue into insulin-secreting tissue as described in claim 1, characterized in that, The method for inducing the product from the previous step into the foregut in step (3) includes: dynamic culture in medium No. 7 for 1-3 days, wherein the composition of medium No. 7 is: MCDB131 + 0.5-5% GlutaMAX + 1-25mM Glucose + 1-20 mM NaHCO3 + 0.5-5% PSG + 0.1-5% BSA + 0.1-5 mM Ascorbic Acid + 1-100 ng / mL KGF.

6. The method for efficiently inducing adult adipose tissue into insulin-secreting tissue as described in claim 1, characterized in that, The method for inducing the product from the previous step into pancreatic progenitor cells 1 in step (3) includes: dynamic culture in medium 8 for 1-3 days, wherein the composition of medium 8 is: MCDB131 + 0.5-5% GlutaMAX + 1-25mM Glucose + 2-40 mM NaHCO3 + 0.5-5% PSG + 0.1-5% BSA + 0.1-5 mM Ascorbic Acid + 0.1-5% ITS-X + 1-100ng / mL KGF + 0.1-50μM TPPB + 0.1-50μM SANT1 + 0.1-50μM LDN193189 + 1-50μM RA + 1-100 μM Taurine.

7. The method for efficiently inducing adult adipose tissue into insulin-secreting tissue as described in claim 1, characterized in that, The method for inducing the product from the previous step into pancreatic progenitor cells 2 in step (3) includes: dynamic culture in medium 9 for 2-6 days, wherein the composition of medium 9 is: MCDB131 + 0.5-5% GlutaMAX + 1-25 mM Glucose + 2-40 mM NaHCO3 + 0.5-5% PSG + 0.1-5% BSA + 0.1-5 mM Ascorbic Acid + 0.1-5% ITS-X + 1-100 ng / mL KGF + 0.1-50 μM TPPB + 0.1-50 μM SANT1 + 0.1-50 μM LDN193189 + 0.1-50 μM RA + 10-100 μM Taurine + 1-50 μM WIKI4.

8. The method for efficiently inducing adult adipose tissue into insulin-secreting tissue as described in claim 1, characterized in that, The method for inducing the product from the previous step into endocrine progenitor cells in step (3) includes: dynamic culture in medium 10 for 5-10 days, wherein the composition of medium 10 is: MCDB131 + 0.5-5% GlutaMAX + 0.1-25 mM Glucose + 2-40 mM NaHCO3 + 0.5-5% PSG + 0.1-5% BSA + 0.1-5 mM Ascorbic Acid + 0.1-5% ITS-X + 1-50 μg / mL heparin + 1-50 mM Nicotinamide + 0.1-50 μM SANT1 + 0.1-50 μM ALK5i II + 0.1-10 μM XXI + 1-50 ng / mL Betacellulin + 0.01-1 μM RA + 0.1-50 μMT3 + 10-100 μM Taurine + 0.1-10 μM Y-27632.

9. The method for efficiently inducing adult adipose tissue into insulin-secreting tissue as described in claim 1, characterized in that, The method for inducing the product from the previous step into pancreatic organoids in step (3) includes: dynamic culture in medium 11 for 7-28 days, wherein the composition of medium 11 is: MCDB131 + 0.5-5% GlutaMAX + 1-25 mM glucose + 0.1-5% BSA + 0.5-5% Pen / Strep + 1-5% NEAA + 1-50 μg / mL heparin + 0.1-5 mM MAscorbic Acid + 1-50 µg / mL ZnSO4•7H2O + 0.1-50 μM ALK5i II + 1-50 μM T3.

10. A culture medium for the method of efficiently inducing adult adipose tissue into insulin-secreting tissue as described in any one of claims 1-9, characterized in that, The culture medium consists of: DMEM basic + 0.5-5% PSG + 0.5-5% B27 + 0.1-5 mM Ascorbic Acid + 5-200 ng / ml FGF-2 + 1-100 μM CHIR999021 + 0.5-50 μM Metinoic Acid + 0.1-10 μM DZNep + 0.1-10 μM 5-AZA + 0.1-10 μM Y-27632.

11. The application of insulin-secreting tissue prepared by the method for efficiently inducing adult adipose tissue into insulin-secreting tissue as described in any one of claims 1-9 in the fields of diabetes treatment models, pancreatic disease simulation models, screening of islet-related drugs, and pancreatic organ development models.

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