人胚胎来源多潜能干细胞向脂肪和神经细胞分化的研究
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摘要
人胚胎肝组织来源的多潜能干细胞是从胎儿肝组织中分离的一类干细胞,与较之前报道的成体干细胞(如骨髓间充质干细胞)有很多相似的特性,具有多分化潜能并能够形成新的组织和器官,具有重要的医学治疗作用。
本研究对人胚胎肝组织来源的多潜能干细胞进行分离和扩增,对其生物学特性进行了研究,探讨了人胚胎来源多潜能干细胞向脂肪细胞和神经细胞方向诱导分化的潜能。采用胶原酶消化法分离人胚胎肝组织来源多潜能干细胞,经过体外培养和扩增,对人胚胎肝组织来源多潜能干细胞的生长经时变化和表面标志CD105、CD166、CD73和HLA-DR进行检测。应用成脂诱导培养液(地塞米松、3-异丁基-1-甲基黄嘌呤、吲哚美辛、胰岛素)诱导其向脂肪细胞分化,使用化学诱导法诱导其向神经细胞方向分化,然后进行鉴定。结果表明,人胚胎肝组织来源多潜能干细胞的生物学特性与骨髓间充质干细胞类似,免疫荧光化学染色及RT-PCR结果说明人胚胎肝组织来源多潜能干细胞经诱导可分化成为脂肪细胞及神经细胞。本研究结果提示,人胚胎来源多潜能干细胞具有分化成脂细胞和神经细胞的潜能,有望应用于组织工程及细胞治疗的种子细胞。
Pluripotent stem cells, which are the descendants of totipotent cells, can differentiate into all cells derived from the three germ layers. Human embryonic pluripotent stem cells can differentiate into many cells of the body and construct tissues and organs. So they play an important role in medical treatment. The differentiation potential of embryonic pluripotent stem cells is larger than adult stem cells. There is less ethical restriction than human embryonic stem cells because they are derived from aborted fetus. However, in relation to BMSCs, studies on embryonic pluripotent stem cells are in the initial phase. The biological characteristics of embryonic pluripotent stem cells are comprehensive understanding. More researches are needed to confirm the results of the differentiation potential in vivo and vitro and induced to other germ layer cells.
Based on this research background, isolation of the pluripotent stem cells derived from human fetal liver; to reveal its basic biological characteristics; identification to their adipogenic and neurogenic differentiation potential.
1. Studies on the basic biological characteristics of human embryonic pluripotent stem cells
Firstly to obtain the human fetal liver derived from aborted fetus(8w~18w), then digest with typeⅡcollagenase. 5×106cells/ml cells seed in culture dish. Cells were cultured at 37℃, with 5% CO2 and 95% air. The separated cells got attached after 3 days and the adherent cells were round with little protrusions; After 6 days the cells have cytoplasmic processes with spindle shape. Through subculture, the cells grew in parallel or as whirlpool, similar to the bone marrow-derived MSCs in morphology.
Detection of cell growth by the time change and cumulative growth of multiple were tested. Human embryonic pluripotent stem cells of P3 were in static phase and proliferation was not obvious in the first 3 days. Then it was rapid growth period and reached the peak at the 7th day. It grew slowly and reached plateau phase. The amount of human embryonic pluripotent stem cells was amplified to 2125.25±146.93 times of the original amount when it was P7. The amount of each generation remained stable. The average double number is 2.99±0.21. The amount of P7 cells is 2.1×109. The amplification is up to 2000 times. This illustrated that human embryonic pluripotent stem cells remain good proliferation after long-term passage.
The cell surface markers such as CD166, CD105, CD73 and HLA-DR were detected by flow cytometry. Our results demonstrated that the isolated and amplified cells showed positive expression for CD105, CD166, CD73, but did not express the HLA-DR, confirming that the separated cells were similar with mesenchymal stem cells.
2. Studies on human embryonic pluripotent stem cells differentiating into adipocyte and neurons
Take the P3 cells from logarithmic phase, digest them with separation medium, adjust the cell density to 1×105cells/ml, inoculate in 6-well plates, and culture them in 37℃with 5% CO2 and 95% air. When the cells adhered and grew to occupation of 80% of the bottom of the culture dish, add inducing medium to induce the differentiation.
2.1 Adipogenic differentiation
Adipogenic induced medium: dexamethasone+IBMX+indometacin+insulin
Inducing human embryonic pluripotent stem cells with AM, cell morphology were short spindle, oval and round. There was lipid droplet in intracellular at the 5th day and a time-dependent increasing. It was proved to be adipose liquid by Oil Red O staining which is an established lipid dye. Induction of human embryonic pluripotent stem cells with AM resulted in expression of the adipose-specific transcription factor peroxisome-proliferating activated receptor PPAR-γby RT-PCR. It demonstrated that human embryonic pluripotent stem cells can differentiate into adipocyte.
2.2 Neurogenic differentiation
Neurogenic induced medium: 1mmol/Lβ-mercaptoethanol + 10% FBS, 24h later 10mmol/Lβ-mercaptoethanol
Changing the induced medium to 10mmol/Lβ-mercaptoethanol, cell morphology changed significantly after 2h. The cytoplasm retracted to form a tight, spherical cell body around the nucleus. The cell body was multipolar and most cells were woven into mesh structure. But neurite was tiny and nucleus was indefinite. There was more vacuoles in the cytoplasm and cells detached from the dish bottom gradually with the time passing by. Most cells were dead within 10 hours. Immunocytochemistry showed positive staining of NSE. RT-PCR results also were confirmed by the induction of cell expression NSE.
In a word, human embryonic pluripotent stem cells are similar with BMSCs in morphology, growing characteristics and cell surface marker. Human embryonic pluripotent stem cells can differentiate into adipocyte and neurons after inducing. Both immunocytochemistry and PT-PCR results support the conclusion. So human embryonic pluripotent stem cells can apply to tissue engineering and cell therapy.
本研究对人胚胎肝组织来源的多潜能干细胞进行分离和扩增,对其生物学特性进行了研究,探讨了人胚胎来源多潜能干细胞向脂肪细胞和神经细胞方向诱导分化的潜能。采用胶原酶消化法分离人胚胎肝组织来源多潜能干细胞,经过体外培养和扩增,对人胚胎肝组织来源多潜能干细胞的生长经时变化和表面标志CD105、CD166、CD73和HLA-DR进行检测。应用成脂诱导培养液(地塞米松、3-异丁基-1-甲基黄嘌呤、吲哚美辛、胰岛素)诱导其向脂肪细胞分化,使用化学诱导法诱导其向神经细胞方向分化,然后进行鉴定。结果表明,人胚胎肝组织来源多潜能干细胞的生物学特性与骨髓间充质干细胞类似,免疫荧光化学染色及RT-PCR结果说明人胚胎肝组织来源多潜能干细胞经诱导可分化成为脂肪细胞及神经细胞。本研究结果提示,人胚胎来源多潜能干细胞具有分化成脂细胞和神经细胞的潜能,有望应用于组织工程及细胞治疗的种子细胞。
Pluripotent stem cells, which are the descendants of totipotent cells, can differentiate into all cells derived from the three germ layers. Human embryonic pluripotent stem cells can differentiate into many cells of the body and construct tissues and organs. So they play an important role in medical treatment. The differentiation potential of embryonic pluripotent stem cells is larger than adult stem cells. There is less ethical restriction than human embryonic stem cells because they are derived from aborted fetus. However, in relation to BMSCs, studies on embryonic pluripotent stem cells are in the initial phase. The biological characteristics of embryonic pluripotent stem cells are comprehensive understanding. More researches are needed to confirm the results of the differentiation potential in vivo and vitro and induced to other germ layer cells.
Based on this research background, isolation of the pluripotent stem cells derived from human fetal liver; to reveal its basic biological characteristics; identification to their adipogenic and neurogenic differentiation potential.
1. Studies on the basic biological characteristics of human embryonic pluripotent stem cells
Firstly to obtain the human fetal liver derived from aborted fetus(8w~18w), then digest with typeⅡcollagenase. 5×106cells/ml cells seed in culture dish. Cells were cultured at 37℃, with 5% CO2 and 95% air. The separated cells got attached after 3 days and the adherent cells were round with little protrusions; After 6 days the cells have cytoplasmic processes with spindle shape. Through subculture, the cells grew in parallel or as whirlpool, similar to the bone marrow-derived MSCs in morphology.
Detection of cell growth by the time change and cumulative growth of multiple were tested. Human embryonic pluripotent stem cells of P3 were in static phase and proliferation was not obvious in the first 3 days. Then it was rapid growth period and reached the peak at the 7th day. It grew slowly and reached plateau phase. The amount of human embryonic pluripotent stem cells was amplified to 2125.25±146.93 times of the original amount when it was P7. The amount of each generation remained stable. The average double number is 2.99±0.21. The amount of P7 cells is 2.1×109. The amplification is up to 2000 times. This illustrated that human embryonic pluripotent stem cells remain good proliferation after long-term passage.
The cell surface markers such as CD166, CD105, CD73 and HLA-DR were detected by flow cytometry. Our results demonstrated that the isolated and amplified cells showed positive expression for CD105, CD166, CD73, but did not express the HLA-DR, confirming that the separated cells were similar with mesenchymal stem cells.
2. Studies on human embryonic pluripotent stem cells differentiating into adipocyte and neurons
Take the P3 cells from logarithmic phase, digest them with separation medium, adjust the cell density to 1×105cells/ml, inoculate in 6-well plates, and culture them in 37℃with 5% CO2 and 95% air. When the cells adhered and grew to occupation of 80% of the bottom of the culture dish, add inducing medium to induce the differentiation.
2.1 Adipogenic differentiation
Adipogenic induced medium: dexamethasone+IBMX+indometacin+insulin
Inducing human embryonic pluripotent stem cells with AM, cell morphology were short spindle, oval and round. There was lipid droplet in intracellular at the 5th day and a time-dependent increasing. It was proved to be adipose liquid by Oil Red O staining which is an established lipid dye. Induction of human embryonic pluripotent stem cells with AM resulted in expression of the adipose-specific transcription factor peroxisome-proliferating activated receptor PPAR-γby RT-PCR. It demonstrated that human embryonic pluripotent stem cells can differentiate into adipocyte.
2.2 Neurogenic differentiation
Neurogenic induced medium: 1mmol/Lβ-mercaptoethanol + 10% FBS, 24h later 10mmol/Lβ-mercaptoethanol
Changing the induced medium to 10mmol/Lβ-mercaptoethanol, cell morphology changed significantly after 2h. The cytoplasm retracted to form a tight, spherical cell body around the nucleus. The cell body was multipolar and most cells were woven into mesh structure. But neurite was tiny and nucleus was indefinite. There was more vacuoles in the cytoplasm and cells detached from the dish bottom gradually with the time passing by. Most cells were dead within 10 hours. Immunocytochemistry showed positive staining of NSE. RT-PCR results also were confirmed by the induction of cell expression NSE.
In a word, human embryonic pluripotent stem cells are similar with BMSCs in morphology, growing characteristics and cell surface marker. Human embryonic pluripotent stem cells can differentiate into adipocyte and neurons after inducing. Both immunocytochemistry and PT-PCR results support the conclusion. So human embryonic pluripotent stem cells can apply to tissue engineering and cell therapy.
引文
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[2]Siminovitch L, McCulloch EA, Till JE. The distribution of colony-forming cells among spleen colonies[J]. J Cell Physiol, 1963, 62: 327-336.
[3]Beckmann J, Scheitza S, Wernet P, et al. Asymmetric cell division within the human hematopoietic stem and progenitor cell compartment: identification of asymmetrically segregating proteins[J].Blood, 2007, 109 (12): 5494-5501.
[4]Watt FM, Driskell RR. The therapeutic potential of stem cells[J]. Philos Trans R Soc Lond B Biol Sci, 2010, 365(1537): 155-163.
[5]Mitalipov S, Wolf D.Totipotency, pluripotency and nuclear reprogramming[J]Adv Biochem Eng Biotechnol,2009, 114: 185-199.
[6]Ulloa-Montoya F, Verfaillie CM, Hu WS. Culture systems for pluripotent stem cells[J]. J Biosci Bioeng, 2005, 100(1): 12-27.
[7]Pierce GB Jr, Beals TF. The ultrastructure of primordial germinal cells of the fetal testes and of embryonal carcinoma cells of mice[J]. Cancer Res, 1964, 24: 1553-1567.
[8]Evans M, Kaufman M. Establishment in culture of pluripotential cells from mouse embryos[J]. Nature, 1981, 292 (5819): 154–156.
[9]Martin G. Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells[J]. Proc Natl Acad Sci USA, 1981, 78 (12): 7634–7638.
[10]Thomson J, Itskovitz-Eldor J, Shapiro S, et al. Embryonic stem cell lines derived from human blastocysts[J]. Science, 1998, 282 (5391): 1145–1147.
[11]Adewumi O, Aflatoonian B, Ahrlund-Richter L, et al. Characterization of human embryonic stem cell lines by the International Stem Cell Initiative[J]. Nature Biotechnology, 2007, 25 (7): 803-816.
[12]Xie T, Spradling AC. Decapentaplegic is essential for the maintenance and division of germline stem cells in the Drosophila ovary[J]. Cell, 1998, 94 (2): 251-260.
[13]Song X, Zhu C, Doan C, et al. Germline stem cells anchored by adherens junctions in theDrosophila ovary niches[J]. Science, 2002, 296 (5574): 1855–1857.
[14]Boyer LA, Lee TI, Cole MF, et al. Core transcriptional regulatory circuitry in human embryonic stem cells[J]. Cell, 2005, 122 (6): 947–956.
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