CELL DIFFERENTIATION MEDIUM COMPOSITION, HIGH SECRETION INSULIN-PRODUCING CELLS AND PREPARATION METHOD THEREOF

Abstract

The present invention relates to a cell differentiation medium composition, a high secretion insulin-producing cells and a preparation method thereof. The high secretion insulin-producing cells obtained by using the cell differentiation medium composition to induce stem cell differentiated under specific conditions can secrete a large amount of insulin in a short time, and when the high-secreting insulin-producing cells are transplanted into the human body, they are not easy to be swallowed by macrophages, which can improve the survival rate of the insulin-producing cells and prolong the time of insulin secretion thereby.

Claims

1. A cell differentiation medium composition, which is used to induce a stem cell to differentiate into a high-secreting insulin-producing cell; the cell differentiation medium composition is a serum-free DMEM/F12 medium comprising at least glucose, nicotinamide, activin-A, exendin-4, hepatocyte growth factor, pentagastrin, a B-27 serum-free supplement, and a N-2 Supplement.

2. The cell differentiation medium composition according to claim 1, wherein the cell differentiation medium composition is antibiotic-free.

3. A preparation method for a high secretion insulin-producing cell, which comprises, (a) cell attachment step: pour a solution containing stem cells into a cell culture container and let stand for at least 24 hours for cell attachment, so that the quantity of stem cells attached to the peripheral wall of the culture container is in a range of 6,000 and 15,000 cells/cm.sup.2; (b) cell differentiation step: removing the solution from the culture container, putting the cell differentiation medium composition according to claim 1 into the culture container, inducing the stem cells into high-secreting insulin-producing cells under the conditions of an ambient temperature of 35.5˜39.5° C. and a CO.sub.2 concentration of 5%, and then collecting the high-secreting insulin-producing cells after differentiating for at least 2 days; wherein the insulin secretion of each 100 thousand of the high secretion insulin-producing cells per day is above 1,000 mIU/L.

4. The preparation method for a high secretion insulin-producing cell according to claim 3, wherein before the cell attachment step (a) further comprising: culturing the stem cells at least 3 days for cell proliferation by using a proliferation medium; the proliferation medium is a keratinocyte serum-free media which comprises fetal bovine serum, N-acetyl-L-cysteine, L2 ascorbic acid, and phosphate.

5. The preparation method for a high secretion insulin-producing cell according to claim 3, wherein cell culture time in the cell differentiation step (b) is in a range of 3 to 30 days.

6. The preparation method for a high secretion insulin-producing cell according to claim 3, wherein the stem cell is at least one selected from the group consisting of an adipose-derived stem cell, a bone marrow stem cell, a peripheral blood stem cell, a cord blood stem cell.

7. The preparation method for a high secretion insulin-producing cell according to claim 3, wherein the morphology of the high secretion insulin-producing cell is spindle-shaped, and the high secretion insulin-producing cell has insulin gene, IPF-1 gene{grave over ( )}ISL-1 gene and expresses the same surface markers with the stem cells.

8. The preparation method for a high secretion insulin-producing cell according to claim 7, wherein the surface markers are CD73 (positive), CD90 (positive)and CD45 (negative).

9. A high secretion insulin-producing cell, which is spindle-shaped and has insulin gene, IPF-1 gene{grave over ( )}ISL-1 gene and expresses the same surface markers with the stem cells; the surface markers are CD73 (positive) , CD90 (positive) and CD45 (negative).

10. The high secretion insulin-producing cell according to claim 8, wherein the express level of CD47 mRNA of the high secretion insulin-producing cell is more than 3 times that of the stem cell.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0018] FIG. 1 is a diagram illustrating results of insulin content analysis for 5×10.sup.5 hADSCs and 10.sup.6 hADSCs.

[0019] FIG. 2 is a diagram illustrating cell morphological changes at day 0 and day 7 during cell differentiation.

[0020] FIG. 3A is a diagram illustrating the results of the fluorescent signal expression of cell surface markers at day 0 and day 7 during cell differentiation.

[0021] FIG. 3B is a diagram illustrating the normalized results of the expression of cell surface markers at day 0 and day 7 during cell differentiation.

[0022] FIG. 4 is a diagram illustrating results of mRNA expression of cells at day 0 and day 7 during cell differentiation.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] In order to enable those skilled in the art to better understand the objects, technical features, and advantages of the present invention and accordingly implement the present invention, the technical features and implementations of the present invention are illustrated in detail herein in conjunction with the accompanying drawings, and preferred embodiments are exemplified for further description. The drawings referenced in the following description are schematic representations for expressing the features of the present invention, and are not and need not be drawn completely based on actual situations.

[0024] Herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Further, unless otherwise clearly contradicted by context, singular terms used herein shall include pluralities and plural terms shall include the singular.

[0025] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are presented herein as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in its respective testing measurement. Herein, the term “about” generally means that an actual value is within 10%, 5%, 1%, or 0.5% above and below a particular value or range. Alternatively, the term “about” indicates that the actual value falls within an acceptable standard error of the mean when considered by one of ordinary skill in the art. Except in the Examples, or where otherwise explicitly indicated, all ranges, amounts, values, and percentages used herein (for example, for describing amounts of materials, time, temperature, operation conditions, amount ratio, and the like) are understood to be modified by the word “about”. Thus, unless expressly stated to the contrary, the numerical parameters disclosed in this specification and the appended claims are all approximations and, if required, may vary. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0026] For a more thorough and complete description of this disclosure, illustrative description for implementation aspects and specific examples of this invention is provided below; however, this is not intended to represent the only form of specific examples in which the present invention may be practiced or utilized. Features of a number of specific examples and process steps and sequences to construct and operate these specific examples are covered in the embodiments. However, the same or equivalent functions and step sequences may also be accomplished by other examples.

<Proliferation of Human Adipose Tissue-Derived MSCs (hADSCs)>

[0027] Cells used in the embodiment were Human Adipose Tissue-Derived MSCs (hADSCs).

[0028] Adipose tissue (2-5 g) was harvested from the subcutaneous fat of the abdominal wall by lipoaspiration during abdominal surgery from healthy donors. All donors gave written informed consent. Human fat tissue was placed in Ca.sup.2+/Mg.sup.2+-free phosphate-buffered saline (PBS) and immediately transferred to the laboratory.

[0029] Human adipose tissue was removed from the transport medium, placed in a Petri dish, and cut into small pieces (1-2 mm.sup.3) in the presence of Ca.sup.2+/Mg.sup.2+-free PBS. The tissues were dissociated with 0.1% collagenase I and incubated for 60 min at 37° C. After enzymatic digestion, the resultant cells were collected and cultured with a proliferation medium. The proliferation medium is a keratinocyte serum-free media (SFM) supplemented with 10% fetal bovine serum (FBS), N-acetyl-L-cysteine, L2 ascorbic acid, and phosphate. The supernatant and debris were removed from the culture dish to obtain primary hADSCs after being cultured for 2 days.

[0030] Then, in order to increase cell count, the above-mentioned primary hADSCs can be continuously cultured in a proliferation medium to the required quantity.

<Differentiation of Human Adipose Tissue-Derived MSCs (hADSCs)>

[0031] First, prepared a differentiation medium composition. The differentiation medium composition was a serum-free DMEM/F12 medium which contains 5˜25 mM glucose, 5˜15 mM nicotinamide, 1˜10 pM activin-A, 5˜20 nM exendin-4, 80˜120 fM hepatocyte growth factor (HGF), 5˜20 nM pentagastrin, 0.1˜5% B-27 serum-free supplement, and 0.1˜5% N-2 supplement. Furthermore, the differentiation medium composition of the present invention does not contain antibiotics (such as penicillin/streptomycin solution), which can avoid affecting cell growth and changing cell surface marker. The concentration of each component in the differentiation medium composition are shown in Table 1.

TABLE-US-00001 TABLE 1 Component Concentration Serum-Free DMEM/F12 Medium — Glucose 5~25 mM Nicotinamide 5~15 mM Activin-A 1~10 pM Exendin-4 5~20 nM Hepatocyte Growth Factor 80~120 fM Pentagastrin 5~20 nM B-27 Serum-Free Supplement 0.1~5% N-2 Supplement 0.1~5%

[0032] Then, after the stem cell proliferation was completed, 5×10.sup.5 hADSCs and 1×10.sup.6 hADSCs were seeded respectively in T75 flasks containing the proliferation medium and incubated 24 hours for cell attachment. The attached cells were washed twice with 10 ml of the serum-free DMEM/F12 medium (the removed liquid was used for analyzing the insulin concentration of stem cells before differentiation), and 10 ml of the differentiation medium was added there to for monolayer culture.

[0033] The flasks with the seeded hADSCs were placed in a CO.sub.2 incubator at 5% CO.sub.2 and 35.5˜39.5° C. for differentiating to obtain high secretion insulin-producing cells. The differentiation medium was replaced every 7 days, and the replaced differentiation medium was collected for insulin content analysis.

[0034] The insulin content analysis was based on Chemiluminescence (model: ADVIA CentaurXPT, SIEMENS) method, and the results were recorded in Table 2.

TABLE-US-00002 TABLE 2 example example control* 1 2 cell count in 0 5 × 10.sup.5 1 × 10.sup.6 a T75 flask cell density 0 6666.6 13333.3 (cells/cm.sup.2) Before — 716.4 87 Differentiation Insulin Day 0-7  65.8 48104.7 9034.2 concentration Day 7-14 — 18539.6 3296.1 (mIU/L) Day 14-21 — 11533.5 1791.2 Day 21-28 — 10696.4 1891.7 *Note: The control group was the differentiation medium alone.

[0035] From the results of insulin content analysis listed in Table 2 above and FIG. 1, it was clearly known that the insulin secretion of the high secretion insulin-producing cells could reach the maximum value for 7 days during cell culture. And the insulin concentration of example 1 is obviously higher than the insulin concentration of example 2, which shows that the cell density is not proportional to the amount of insulin secreted by high secretion insulin-producing cells. The higher the cell density, the lower the insulin secretion.

[0036] Therefore, the following embodiments were all using 5×10.sup.5 hADSCs in a T75 flask for differentiation.

Insulin Secretion Analysis

[0037] The above differentiation test (example 1) was repeat for 3 times and means of insulin secretion of each 100 thousand of high secretion insulin-producing cells every 7 days were calculated and recorded in Table 3.

TABLE-US-00003 TABLE 3 Insulin secretion of each 100 thousand of the insulin- Control producing cells Unit (mIU/L) (mIU/L) Days 0-7 57.63 ± 51.15 10569.66 ± 4686.63 Days 7-14 — 2526.02 ± 934.01 Days 14-21 — 2290.09 ± 631.54 Days 21-28 — 2409.42 ± 694.69 *Note: The control group was the differentiation medium alone.

[0038] As shown in Table 3, the insulin secretion of the high secretion insulin-producing cells could reach the maximum value within 7 days during cell culture, and the insulin secretion of each 10.sup.5 high secretion insulin-producing cells was 10569.66 mIU/L. In other words, the average daily insulin secretion of each 10.sup.5 high secretion insulin-producing cells were about 1510 mIU/L.

Morphology of High Secretion Insulin-Producing Cells

[0039] At day 0 and day 7 during cell culture, the cells in the flask were photographed and observed respectively by an inverted microscope (model: OLYMPUS IX71-1LL100).

[0040] As shown in FIG. 2, during the differentiation, the morphology of high secretion insulin-producing cells was spindle-shaped, and all the high secretion insulin-producing cells are separated rather than aggregated into spheres or clumps.

Expression of Surface Markers of High Secretion Insulin-Producing Cells

[0041] The cells were collected at day 0 and day 7 during cell culture, and then they were fixed and dyed with each specific antibody for cell surface marker (negative marker: CD45; positive marker: CD73 and CD90). The surface antibody was labeled with FITC conjugated secondary antibody, and the fluorescent signal expression of the cell surface protein was measured using a flow cytometer (Model: BD Accuri C6). The analysis was repeated at least twice and the average values were recorded in Table 4.

TABLE-US-00004 TABLE 4 Day 0 Day 7 Percentage of positive cells CD73 99.775 99.935 (normalized to isotype) CD90 99.985 99.985 CD45 0.095 0.095

[0042] As shown in Table 4, FIG. 3A and FIG. 3B, after 7 days during cell culture, the high secretion insulin-producing cells showed 0.095% for the negative markers CD45, whereas for CD 73 and CD 90, the stem cell positive markers, were mostly found to be 99% or higher. In other words, the culturing conditions of the present invention did not change the expression of specific surface markers of high secretion insulin-producing cells, which was still the same with hADSCs (stem cells).

Gene Expression of High Secretion Insulin-Producing Cells

[0043] The cells were collected at day 0 and day 7 during cell culture. mRNA of the cells was isolated using mRNA isolation kits (Quick-RNA™ MiniPrep, ZYMO RESEARCH), and then the isolated mRNA was reverse transcribed to cDNA using reverse transcription kit (PrimeScript™ RT reagent Kit, Takara). 100 ng of cDNA sample and proper primers for Insulin, Ipf-1, Isl-1, and CD47 were mixed with SYBR® Premix Ex Taq™ II (Takara) for Real time PCR.

[0044] The proper primers for Insulin, Ipf-1, Isl-1, and CD47 were shown in Table 5.

TABLE-US-00005 TABLE 5 specific gene Forward primer Reverse primer Ipf-1 5′-TGATACTGGATTGGCGT 5′-TCCCAAGGTGGAGTG TGTTT-3′ (SEQ ID NO: CTGTAG-3′ (SEQ ID 1) NO: 2) Isl-1 5′-CAACTGGTCAATTTTTC 5′-TTGAGAGGACATTGA AGAAGGA-3′ (SEQ ID TGCTACTTCAC-3′  NO: 3) (SEQ ID NO: 4) Insulin 5′-GCAGCCTTTGTGAACCA 5′-TTCCCCGCACACTAG ACA-3′ (SEQ ID NO: 5) GTAGAGA-3′ (SEQ ID NO: 6) CD47 5′-GGCAATGACGAAGGAGG 5′-ATCCGGTGGTATGGA TTA-3′ (SEQ ID NO: 7) TGAGA-3′ (SEQ ID NO: 8) HPRT 5′-TCAGGCAGTATAATCCA 5′-AGTCTGGCTTATATC (reference AAGATGGT-3′ (SEQ ID CAACACTTCG-3′ gene) NO: 9) (SEQ ID NO: 10)

[0045] After mRNA isolation, reverse transcription to cDNA and real-time PCR were done, each gene expression was analyzed and record in Table 6.

TABLE-US-00006 TABLE 6 Day 0 Day 7 Relative mRNA expression Insulin 1 11.47 (Normalized to HPRT mRNA) Ipf-1 1 5.89 Isl-1 1 6.13 CD47 1 3.64

[0046] As shown in Table 6, FIG. 4, after 7 days during cell culture, the expressions of the Insulin gene, Ipf-1 gene, and Isl-1 gene in the high secretion insulin-producing cells were significantly increased to at least 5 times compared with hADSCs which had not begun to be differentiated at day 0.

[0047] In addition, the expressions of CD47 in the high secretion insulin-producing cells were also significantly increased to at least 3 times compared with hADSCs at day 0. Since CD47 is a signaling molecule prevents phagocytosis of Macrophages, the result in the analysis means the high secretion insulin-producing cells of the present invention could be avoid phagocytosis of Macrophages in human body when being used in human transplantation, thereby improving the survival rate of the high secretion insulin-producing cells.

[0048] It can be seen from the above-mentioned embodiments that the present invention provides a cell differentiation medium composition and a preparation method for the high secretion insulin-producing cell, which are cultured stem cells by monolayer culturing to prevent cells from aggregating into spheres or clumps. The present can not only facilitate collection cells and measurement, also can obtain a large amount of insulin in a short time, which is helpful for industrial utilization; and when the high secretion insulin-producing cells are transplanted into the human body, they are not easy to be swallowed by macrophages, which can extend the time of insulin secretion.

[0049] The specific embodiments described above are only used to illustrate the features and effects of the present invention, and are not intended to limit the implementation scope of present invention. Any equivalent changes and modifications made based on the content disclosed in the present invention without departing from the spirit and technical scope of the present invention still fall within the patent scope described later.