脂肪组织来源干细胞单克隆培养技术及其表面抗原的研究
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摘要
背景:
组织工程技术以少量的种子细胞经体外扩增后与生物材料结合,修复较大的组织或器官缺损,重建缺损部位的生理功能,为临床上解决组织缺损等难题提供了一条新途径,近来受到广泛关注。
如何获得来源广泛、充足的种子细胞是此项技术的关键。成体干细胞来源广泛、功能良好,是种子细胞的研究重点。成体干细胞可来源于骨髓、脂肪、皮肤、肌肉等各类组织,其中骨髓基质干细胞(BMSCs),发现早、取材方便、创伤小,被研究的最多。随着研究的普及和深入,脂肪组织来源干细胞(ADSCs)逐渐被人们认识,与BMSCs相比,ADSCs在体内分布更广,可利用的细胞量更多。ADSCs自1994年被发现以来已证实能向多种类型的细胞分化,并且具有取材方便、创伤小而细胞获得量大、病人易于接受的优势,在组织工程的研究和应用中显示出了巨大的潜力。
目前普遍认为,常用方法分离出的ADSCs是由多种分化潜能不同的细胞亚群以及大量的混杂细胞组成的混合群体,其中只有部分能够形成脂肪细胞,因而必然会影响其构建脂肪组织的成功率。如何提高从脂肪组织中提取干细胞的纯度和寻找确认干细胞的相关标记等问题一直是目前急于解决的难题之一。虽然对于ADSCs的表面抗原的研究已有诸多报道,但有关ADSCs的特异性表面标志仍无统一认识,对于特定分化潜能和标志物表达之间是否存在关联至今尚未见有报道。此外,有关用于纯化细胞群获得单细胞的克隆形成试验,国外虽有利用环克隆的方法从脂肪来源细胞中分离培养出分化潜能不同的多种细胞克隆,但未对其进行相关表面抗原的分析,国内也未见有用此种方法进行干细胞研究的报道。本实验拟通过单克隆培养的方法,分离纯化出由单一细胞形成的细胞克隆,达到纯化脂肪组织来源干细胞的目的,并检测其表面抗原的表达,同时针对不同克隆鉴定其成脂分化的潜力,并检测了成脂诱导剂对ADSCs部分相关表面抗原表达的影响,寻找ADSCs成脂分化潜能和表面抗原表达之间的关联。为ADSCs在脂肪组织工程中的应用提供实验依据。
目的:
1.提取人体皮下脂肪组织来源干细胞(ADSCs)进行单克隆培养和扩增,以获得单克隆ADSCs,并鉴定不同克隆细胞的干细胞相关表面抗原表达。
2.针对不同克隆ADSCs通过对其生长动力学、形态学、成脂分化能力等方面的特征进行鉴定比较,探索ADSCs成脂分化潜能和表面抗原表达之间的关联,寻找代表定向成脂分化亚群的细胞标志,从而提高构建脂肪组织的成功率。
3.对比成脂诱导前后ADSCs部分相关表面抗原表达的变化,研究成脂诱导剂对ADSCs部分相关表面抗原表达的影响,为ADSCs在脂肪组织工程中的应用奠定实验基础。
方法:
临床手术切取脂肪组织,经胶原酶消化法原代培养,细胞培养并传至第2代后,有限稀释法进行克隆形成实验。针对获得的单克隆ADSCs,观察其在体外培养的形态学和生物学特点,并用流式细胞仪检测不同克隆CD29、CD34、CD44、CD54、CD106、ABCG2的表达。各克隆分别进行成脂诱导,油红O染色鉴定分化潜能。第3代的ADSCs细胞分别于成脂诱导7天前、后检测其CD34、CD54、CD106的表达。第4代ADSCs进行成脂、成骨定向诱导分化,油红“O”染色、茜素红染色定性。
结果:
从皮下切取的脂肪组织中能培养出大量的ADSCs,通过克隆形成实验共获得10个单克隆细胞群,不同克隆细胞在形态及增殖活力上存在差异,细胞群扩增到第9～11代后可获得细胞数2×10~6,进行后续研究及冻存。干细胞相关标志检测显示:各个克隆群体均高表达CD29(92.9±7.4%)、CD44(94.6±6.8%):低表达ABCG2(2.5±1.4%);但CD34、CD54和CD106的表达在不同群体间有明显差异。不同克隆的成脂分化潜力明显不同,油红“O”染色可见部分含红染颗粒的阳性细胞高达60%,部分为阴性结果,说明ADSCs中存在成脂分化能力不同的细胞群。第3代ADSCs在成脂诱导7天后CD34、CD54和CD106的表达较诱导之前无明显统计学差异。ADSCs成脂诱导分化两周,可见细胞内有大量脂滴,油红“O”染色可见胞浆内有大量红染颗粒;成骨诱导分化两周可肉眼看到白色矿化的颗粒状钙盐沉积,经茜素红染色鉴定可发现成骨细胞及钙盐沉积被红染。
结论:
本实验结果证明:皮下脂肪组织经过酶消化原代培养可提取有多向分化能力的干细胞,通过克隆形成试验培养的方法可以纯化ADSCs,获得高纯度具有间充质细胞特性的细胞群,体外多次传代、长期培养并不改变细胞形态和增殖活性。不同的单克隆细胞群之间表面标志的表达和成脂分化能力存在共性和差异。差异较显著的CD34及CD54可能作为ADSCs中高成脂分化细胞群纯化和分选的标志。另外,在成脂分化后CD34、CD54和CD106的表达较之前无显著性差异,与传代培养后的下降变化不同,也提示了这些抗原可能的标识作用。但尚不能认为这些标志物和分化能力之间存在线性关系。后续研究应当对这些标志物进行鉴定,以明确其与成脂分化之间的联系,建立脂肪组织来源基质细胞的纯化技术,为ADSCs构建脂肪组织填充物实现组织工程修复缺损奠定实验基础。
BACKGROUND:
A soft tissue defect is generally defined as a large tissue void within the subcutaneous fat layer of the skin that may alter the contour of normal tissue. From cosmetic standard point, restoration of the soft tissue aesthetic function, rather than its physical function, is one of the primary goals that will minimize patients' anxiety and psychological stress associated with disfigurement. Millions of plastic and reconstructive surgeries are performed each year to repair soft tissue defects result from traumatic injury (i.e., significant burns), tumor resections (i.e., mastectomy and carcinoma removal), and congenital defects. Strategies to repair soft tissue defects, e.g. breast reconstruction procedures, collagen injections, and the use of autologous tissue transferplantation (i.e. free fat tissue grafts and tissue flaps), include the use of implants and fillers. However, no single filler material currently can meet these various clinical needs. Excess amounts of adipose tissue are present in many individuals with obesity, obtainable through liposuction, and can be used to transplant and repair soft tissue defects. However, resorption of the transplanted autologous tissue over time may result in 40 - 60% of the graft loss. This has significantly limited clinical application of the autologous fat tissue. Therefore, tissue-engineering strategies are being investigated and represent a novel and potential approach to generating adipose tissue.
The primary goal of tissue engineering is to regenerate healthy tissues or organs based on patients' need, whereby eliminating tissue or organ transplantation or mechanical devices, and their related complications. Another major challenge in organ and tissue transplantation is immunological rejection after receiving allograft tissue transplantation. Evidently, tissue-engineering strategies will not be associated with these concerns. Using engineering techniques, cells collected from a health individual can be cultured and expanded to a larger cell pool. These cells can then be seeded onto a scaffold that will support cell growth and proliferation. The cell-covered on scaffold can be implanted into a site that needs surgical repair. As the cells continue growing, the scaffold material degrades or absorbs. Eventually, a new tissue mass will develop. Tissue-engineering techniques are being investigated to develop a wide range of tissues, including bone, skin, cartilage, vascular, and adipose tissues. The development of adipose tissue-engineering strategies will be essential for revolutionizing our current practice in the restoration of tissue and repair of soft tissue defects.
Tissue engineering and regenerative medicine are an important part of the contemporary medical science that has evolved in parallel with biotechnological advances. These approaches combine biomaterials, growth factors, and stem cells to repair failing organs. Material scientists can now fabricate biocompatible scaffolds with a wide range of physical parameters, combining mechanical integrity with high porosity to promote cell infiltration and angiogenesis. Likewise, biochemists can produce highly purified, bioactive cytokines in large quantity, suitable for cell culture and in vivo applications. Despite these advances, the availability of stem cells represents the most potential approach in regenerative medicine, and technically remains very challenge before it can be used in clinical practice. By definition, a stem cell is characterized by its ability to self-renew and its ability to differentiate along multiple lineage pathways. Ideally, a stem cell for regenerative medicinal applications should meet the following criteria:
1. Can be found in abundant quantities (millions to billions of cells)
2. Can be harvested by a minimally invasive procedure
3. Can be differentiated along multiple cell lineage pathways in a regulatable and reproducible manner.
4. Can be safely and effectively transplanted to either an autologous or allogeneic host
5. Can be manufactured in accordance with current Good Manufacturing Practice guidelines.
There are several potential sources for obtaining stem cells for tissue regeneration or repair purposes. And the most commonly used cell types are adult and embryonic-origin. Embryonic stem cells are usually obtained from destroyed embryos, that has raised ethical concerns. On the other hand, use of stem cells derived from adult tissues provides an alternative, and avoids ethical issues related to embryonic stem cells. Mesenchymal stem cells may undergo self-renewal for several generations while remaining their specific characteristics. This type of stem cells is multipotent, easily isolated and cultured, and readily expanded in the laboratory setting. All these make mesenchymal stem cells an attractive and potential source in several clinical applications, including cell-based therapies for the diseases, such as Parkinson's and Alzheimer's diseases, spinal cord injuries, burns, heart disease, and osteoarthritis, among other conditions. These adult stem cells typically include hematopoietic stem cells, neural stem cells, bone marrow stromal cells, dermal stem cells, adipose-derived stem cells and fetal cord blood stem cells .
Although bone marrow is the most recognized source of mesenchymal stem cells, adipose tissue has been considered as a source of multipotent cells that have the capacity of differentiating to cells of adipogenic, chondrogenic, myogenic, and osteogenic lineages when cultured with the appropriate lineage specific stimuli. Adipose-derived stem cells (ADSCs) can be obtained from tissue harvested through liposuction [termed processed lipoaspirate cells (PLAs)], or through abdominoplasty procedures. These cells have also been identified as mesenchymal cells because they are derived from adipose tissue which is, in turn, derived from mesenchyme, like bone marrow. ADSCs have been shown to be very similar to marrow-derived stem cells in morphology and phenotype. In addition to their common multipotency, several CD antigen (or cell surface) markers on the surface of marrow stem cells have also been found on the surface of ADSCs. A wide availability, easy and safe to harvest have made ADSCs a great candidate for its clinical applications in tissue-engineering. However, genetic variation of the donors and possible contamination of ADSCs by endothelial, smooth muscle, and pericyte cell populations limit the clinical application of ADSCs. Therefore, the key issue it is critically important to purify ADSCs and identify their specific surface antigens. Although several possible cell surface antigens of ADSCs have been reported, their specificity remains to be determined. Ring cloning has been used to select clones derived from a single ADSC cell. Using lineage specific differentiation media, these clones can be used to induce adipogenesis, osteogenesis, chondrogenesis, and neurogenesis. It indicated that it is possible to purify ADSCs by clone forming.
In the present study, we established a colony unit of ADSCs, investigated the cell surface antigens of these cells, and assessed the potential of adipogenic differentiation of different ADSC clones to further determine whether adipogenesis inducement impacts the specificity of these cell surface antigens, additional assays were also performed before and after three passages of adipogenesis of the ADSCs . The present study developed a novel approach to purify the ADSCs and further characterized the cell surface antigens of these cells that will promote clinical application of ADSCs in plastic and cosmetic surgeries.
OBJECTIVES
1. To develop a colony unit of human adipose-derived stem cells(ADSCs), and investigate the cell-surface antigens.
2. To determine adipogenic differentiation potential of different clones by purifying ADSCs isolated from fatty tissues, and assessing the relation of highly adipogenic potential to cell-surface antigens.
3. To compare the changes of cell-surface antigens before and after in vitro adipogenic differentiation, find the high- adipogenic potential related cell-surface antigens
METHODS
ADSCs were isolated and cultivated from the resection of subcutaneous fat potions in healthy donors who under dermatoplasty. Fatty tissue were digested by collagenase, and the cells were isolated and seeded in primary culture. ADSCs were cultured for two passages , then subjected to limit dilution assays to form colony unit. Flow cytometry was used to identify the expression of cell-surface antigens in the clones obtained from above experiments. The antibodies to CD29、CD44、CD34、CD54、CD106, and ABCG2 were used to determine ADSCs specific antigens. Each clone was induced for adipogenesis, and determined by Oil Red O stain. Adipogenic, chondrogenic and osteogenic lineage differentiations of the 4th generation of ADSCs was assessed by Oil O Red, Alcian Blue, and Alizarin Red staining, respectively.
RESULTS
There was a large amount of ADSCs in the fatty tissue. 10 clones were obtained by colony-forming unit (CFU) assays, and after cultured for nine passages, the amount of the cells reached 2×10~6. The results of the cell-surface antigens investigation is: all of the clones were shown to be highly positive for CD29(92.9±7.4%) and CD44(94.6±6. 8%), while ABCG2 was expressed relatively low (2.5±1. 4%). The expression of CD34、CD54, and CD106 varies in different clones. Of each clone, the potential of adipogenic differentiation had a significant difference from l%～60%. Additionally, the expression of CD34, CD54, and CD106 in cells undergoing adipogenic differentiation for 7 days showed a diverse change contrast to those in cells before adipogenic differentiation. Data was analyzed by paired samples T test, and the result is no significant statistic difference. Two weeks after adipogenic differentiation of ADSCs, a significant fraction of the cells are characterized by multiple, intracellular lipid-filled droplets as indicated by Oil Red O staining, a typical function of matured fatty cells. In contrast, two weeks after chondrogenic and osteogenic induction, the cells were became positively stained by Alcian Blue and Alizarin Red, a typical function of matured chondrocyte and osteoblast cells.
CONCLUSION
ADSCs, harvested from adipose tissue by collagenase, is a mix population of multilineage stem cells. Clonal analysis could be used to successfully obtain purified ADSCs. Growth dynamics and morphology of the cells did not change with passages. There are differences and commonness of the expression of surface antigens among the clones. The potential of adipogenic differentiation of each clone also had variety. The difference in expression of cell surface antigens may attribute to the variation in potential of differentiation. Among those antigens identified, CD34, CD54, and CD106 showed markedly difference in each clone, maybe more associated with adipogenic differentiation clones. Additionally, the maintained expression of CD34, CD54, and CD106 after adipogenesis, while it reduced with passages, indicated that these markers may be considered as the high- adipogenic potential related cell-surface antigens. Our results suggest that the ADSCs, isolated from adipose tissue by collagenase can be the purified and certain cell surface antigens may be used to enhance ADSCs potential of adipogenicity.
组织工程技术以少量的种子细胞经体外扩增后与生物材料结合,修复较大的组织或器官缺损,重建缺损部位的生理功能,为临床上解决组织缺损等难题提供了一条新途径,近来受到广泛关注。
如何获得来源广泛、充足的种子细胞是此项技术的关键。成体干细胞来源广泛、功能良好,是种子细胞的研究重点。成体干细胞可来源于骨髓、脂肪、皮肤、肌肉等各类组织,其中骨髓基质干细胞(BMSCs),发现早、取材方便、创伤小,被研究的最多。随着研究的普及和深入,脂肪组织来源干细胞(ADSCs)逐渐被人们认识,与BMSCs相比,ADSCs在体内分布更广,可利用的细胞量更多。ADSCs自1994年被发现以来已证实能向多种类型的细胞分化,并且具有取材方便、创伤小而细胞获得量大、病人易于接受的优势,在组织工程的研究和应用中显示出了巨大的潜力。
目前普遍认为,常用方法分离出的ADSCs是由多种分化潜能不同的细胞亚群以及大量的混杂细胞组成的混合群体,其中只有部分能够形成脂肪细胞,因而必然会影响其构建脂肪组织的成功率。如何提高从脂肪组织中提取干细胞的纯度和寻找确认干细胞的相关标记等问题一直是目前急于解决的难题之一。虽然对于ADSCs的表面抗原的研究已有诸多报道,但有关ADSCs的特异性表面标志仍无统一认识,对于特定分化潜能和标志物表达之间是否存在关联至今尚未见有报道。此外,有关用于纯化细胞群获得单细胞的克隆形成试验,国外虽有利用环克隆的方法从脂肪来源细胞中分离培养出分化潜能不同的多种细胞克隆,但未对其进行相关表面抗原的分析,国内也未见有用此种方法进行干细胞研究的报道。本实验拟通过单克隆培养的方法,分离纯化出由单一细胞形成的细胞克隆,达到纯化脂肪组织来源干细胞的目的,并检测其表面抗原的表达,同时针对不同克隆鉴定其成脂分化的潜力,并检测了成脂诱导剂对ADSCs部分相关表面抗原表达的影响,寻找ADSCs成脂分化潜能和表面抗原表达之间的关联。为ADSCs在脂肪组织工程中的应用提供实验依据。
目的:
1.提取人体皮下脂肪组织来源干细胞(ADSCs)进行单克隆培养和扩增,以获得单克隆ADSCs,并鉴定不同克隆细胞的干细胞相关表面抗原表达。
2.针对不同克隆ADSCs通过对其生长动力学、形态学、成脂分化能力等方面的特征进行鉴定比较,探索ADSCs成脂分化潜能和表面抗原表达之间的关联,寻找代表定向成脂分化亚群的细胞标志,从而提高构建脂肪组织的成功率。
3.对比成脂诱导前后ADSCs部分相关表面抗原表达的变化,研究成脂诱导剂对ADSCs部分相关表面抗原表达的影响,为ADSCs在脂肪组织工程中的应用奠定实验基础。
方法:
临床手术切取脂肪组织,经胶原酶消化法原代培养,细胞培养并传至第2代后,有限稀释法进行克隆形成实验。针对获得的单克隆ADSCs,观察其在体外培养的形态学和生物学特点,并用流式细胞仪检测不同克隆CD29、CD34、CD44、CD54、CD106、ABCG2的表达。各克隆分别进行成脂诱导,油红O染色鉴定分化潜能。第3代的ADSCs细胞分别于成脂诱导7天前、后检测其CD34、CD54、CD106的表达。第4代ADSCs进行成脂、成骨定向诱导分化,油红“O”染色、茜素红染色定性。
结果:
从皮下切取的脂肪组织中能培养出大量的ADSCs,通过克隆形成实验共获得10个单克隆细胞群,不同克隆细胞在形态及增殖活力上存在差异,细胞群扩增到第9～11代后可获得细胞数2×10~6,进行后续研究及冻存。干细胞相关标志检测显示:各个克隆群体均高表达CD29(92.9±7.4%)、CD44(94.6±6.8%):低表达ABCG2(2.5±1.4%);但CD34、CD54和CD106的表达在不同群体间有明显差异。不同克隆的成脂分化潜力明显不同,油红“O”染色可见部分含红染颗粒的阳性细胞高达60%,部分为阴性结果,说明ADSCs中存在成脂分化能力不同的细胞群。第3代ADSCs在成脂诱导7天后CD34、CD54和CD106的表达较诱导之前无明显统计学差异。ADSCs成脂诱导分化两周,可见细胞内有大量脂滴,油红“O”染色可见胞浆内有大量红染颗粒;成骨诱导分化两周可肉眼看到白色矿化的颗粒状钙盐沉积,经茜素红染色鉴定可发现成骨细胞及钙盐沉积被红染。
结论:
本实验结果证明:皮下脂肪组织经过酶消化原代培养可提取有多向分化能力的干细胞,通过克隆形成试验培养的方法可以纯化ADSCs,获得高纯度具有间充质细胞特性的细胞群,体外多次传代、长期培养并不改变细胞形态和增殖活性。不同的单克隆细胞群之间表面标志的表达和成脂分化能力存在共性和差异。差异较显著的CD34及CD54可能作为ADSCs中高成脂分化细胞群纯化和分选的标志。另外,在成脂分化后CD34、CD54和CD106的表达较之前无显著性差异,与传代培养后的下降变化不同,也提示了这些抗原可能的标识作用。但尚不能认为这些标志物和分化能力之间存在线性关系。后续研究应当对这些标志物进行鉴定,以明确其与成脂分化之间的联系,建立脂肪组织来源基质细胞的纯化技术,为ADSCs构建脂肪组织填充物实现组织工程修复缺损奠定实验基础。
BACKGROUND:
A soft tissue defect is generally defined as a large tissue void within the subcutaneous fat layer of the skin that may alter the contour of normal tissue. From cosmetic standard point, restoration of the soft tissue aesthetic function, rather than its physical function, is one of the primary goals that will minimize patients' anxiety and psychological stress associated with disfigurement. Millions of plastic and reconstructive surgeries are performed each year to repair soft tissue defects result from traumatic injury (i.e., significant burns), tumor resections (i.e., mastectomy and carcinoma removal), and congenital defects. Strategies to repair soft tissue defects, e.g. breast reconstruction procedures, collagen injections, and the use of autologous tissue transferplantation (i.e. free fat tissue grafts and tissue flaps), include the use of implants and fillers. However, no single filler material currently can meet these various clinical needs. Excess amounts of adipose tissue are present in many individuals with obesity, obtainable through liposuction, and can be used to transplant and repair soft tissue defects. However, resorption of the transplanted autologous tissue over time may result in 40 - 60% of the graft loss. This has significantly limited clinical application of the autologous fat tissue. Therefore, tissue-engineering strategies are being investigated and represent a novel and potential approach to generating adipose tissue.
The primary goal of tissue engineering is to regenerate healthy tissues or organs based on patients' need, whereby eliminating tissue or organ transplantation or mechanical devices, and their related complications. Another major challenge in organ and tissue transplantation is immunological rejection after receiving allograft tissue transplantation. Evidently, tissue-engineering strategies will not be associated with these concerns. Using engineering techniques, cells collected from a health individual can be cultured and expanded to a larger cell pool. These cells can then be seeded onto a scaffold that will support cell growth and proliferation. The cell-covered on scaffold can be implanted into a site that needs surgical repair. As the cells continue growing, the scaffold material degrades or absorbs. Eventually, a new tissue mass will develop. Tissue-engineering techniques are being investigated to develop a wide range of tissues, including bone, skin, cartilage, vascular, and adipose tissues. The development of adipose tissue-engineering strategies will be essential for revolutionizing our current practice in the restoration of tissue and repair of soft tissue defects.
Tissue engineering and regenerative medicine are an important part of the contemporary medical science that has evolved in parallel with biotechnological advances. These approaches combine biomaterials, growth factors, and stem cells to repair failing organs. Material scientists can now fabricate biocompatible scaffolds with a wide range of physical parameters, combining mechanical integrity with high porosity to promote cell infiltration and angiogenesis. Likewise, biochemists can produce highly purified, bioactive cytokines in large quantity, suitable for cell culture and in vivo applications. Despite these advances, the availability of stem cells represents the most potential approach in regenerative medicine, and technically remains very challenge before it can be used in clinical practice. By definition, a stem cell is characterized by its ability to self-renew and its ability to differentiate along multiple lineage pathways. Ideally, a stem cell for regenerative medicinal applications should meet the following criteria:
1. Can be found in abundant quantities (millions to billions of cells)
2. Can be harvested by a minimally invasive procedure
3. Can be differentiated along multiple cell lineage pathways in a regulatable and reproducible manner.
4. Can be safely and effectively transplanted to either an autologous or allogeneic host
5. Can be manufactured in accordance with current Good Manufacturing Practice guidelines.
There are several potential sources for obtaining stem cells for tissue regeneration or repair purposes. And the most commonly used cell types are adult and embryonic-origin. Embryonic stem cells are usually obtained from destroyed embryos, that has raised ethical concerns. On the other hand, use of stem cells derived from adult tissues provides an alternative, and avoids ethical issues related to embryonic stem cells. Mesenchymal stem cells may undergo self-renewal for several generations while remaining their specific characteristics. This type of stem cells is multipotent, easily isolated and cultured, and readily expanded in the laboratory setting. All these make mesenchymal stem cells an attractive and potential source in several clinical applications, including cell-based therapies for the diseases, such as Parkinson's and Alzheimer's diseases, spinal cord injuries, burns, heart disease, and osteoarthritis, among other conditions. These adult stem cells typically include hematopoietic stem cells, neural stem cells, bone marrow stromal cells, dermal stem cells, adipose-derived stem cells and fetal cord blood stem cells .
Although bone marrow is the most recognized source of mesenchymal stem cells, adipose tissue has been considered as a source of multipotent cells that have the capacity of differentiating to cells of adipogenic, chondrogenic, myogenic, and osteogenic lineages when cultured with the appropriate lineage specific stimuli. Adipose-derived stem cells (ADSCs) can be obtained from tissue harvested through liposuction [termed processed lipoaspirate cells (PLAs)], or through abdominoplasty procedures. These cells have also been identified as mesenchymal cells because they are derived from adipose tissue which is, in turn, derived from mesenchyme, like bone marrow. ADSCs have been shown to be very similar to marrow-derived stem cells in morphology and phenotype. In addition to their common multipotency, several CD antigen (or cell surface) markers on the surface of marrow stem cells have also been found on the surface of ADSCs. A wide availability, easy and safe to harvest have made ADSCs a great candidate for its clinical applications in tissue-engineering. However, genetic variation of the donors and possible contamination of ADSCs by endothelial, smooth muscle, and pericyte cell populations limit the clinical application of ADSCs. Therefore, the key issue it is critically important to purify ADSCs and identify their specific surface antigens. Although several possible cell surface antigens of ADSCs have been reported, their specificity remains to be determined. Ring cloning has been used to select clones derived from a single ADSC cell. Using lineage specific differentiation media, these clones can be used to induce adipogenesis, osteogenesis, chondrogenesis, and neurogenesis. It indicated that it is possible to purify ADSCs by clone forming.
In the present study, we established a colony unit of ADSCs, investigated the cell surface antigens of these cells, and assessed the potential of adipogenic differentiation of different ADSC clones to further determine whether adipogenesis inducement impacts the specificity of these cell surface antigens, additional assays were also performed before and after three passages of adipogenesis of the ADSCs . The present study developed a novel approach to purify the ADSCs and further characterized the cell surface antigens of these cells that will promote clinical application of ADSCs in plastic and cosmetic surgeries.
OBJECTIVES
1. To develop a colony unit of human adipose-derived stem cells(ADSCs), and investigate the cell-surface antigens.
2. To determine adipogenic differentiation potential of different clones by purifying ADSCs isolated from fatty tissues, and assessing the relation of highly adipogenic potential to cell-surface antigens.
3. To compare the changes of cell-surface antigens before and after in vitro adipogenic differentiation, find the high- adipogenic potential related cell-surface antigens
METHODS
ADSCs were isolated and cultivated from the resection of subcutaneous fat potions in healthy donors who under dermatoplasty. Fatty tissue were digested by collagenase, and the cells were isolated and seeded in primary culture. ADSCs were cultured for two passages , then subjected to limit dilution assays to form colony unit. Flow cytometry was used to identify the expression of cell-surface antigens in the clones obtained from above experiments. The antibodies to CD29、CD44、CD34、CD54、CD106, and ABCG2 were used to determine ADSCs specific antigens. Each clone was induced for adipogenesis, and determined by Oil Red O stain. Adipogenic, chondrogenic and osteogenic lineage differentiations of the 4th generation of ADSCs was assessed by Oil O Red, Alcian Blue, and Alizarin Red staining, respectively.
RESULTS
There was a large amount of ADSCs in the fatty tissue. 10 clones were obtained by colony-forming unit (CFU) assays, and after cultured for nine passages, the amount of the cells reached 2×10~6. The results of the cell-surface antigens investigation is: all of the clones were shown to be highly positive for CD29(92.9±7.4%) and CD44(94.6±6. 8%), while ABCG2 was expressed relatively low (2.5±1. 4%). The expression of CD34、CD54, and CD106 varies in different clones. Of each clone, the potential of adipogenic differentiation had a significant difference from l%～60%. Additionally, the expression of CD34, CD54, and CD106 in cells undergoing adipogenic differentiation for 7 days showed a diverse change contrast to those in cells before adipogenic differentiation. Data was analyzed by paired samples T test, and the result is no significant statistic difference. Two weeks after adipogenic differentiation of ADSCs, a significant fraction of the cells are characterized by multiple, intracellular lipid-filled droplets as indicated by Oil Red O staining, a typical function of matured fatty cells. In contrast, two weeks after chondrogenic and osteogenic induction, the cells were became positively stained by Alcian Blue and Alizarin Red, a typical function of matured chondrocyte and osteoblast cells.
CONCLUSION
ADSCs, harvested from adipose tissue by collagenase, is a mix population of multilineage stem cells. Clonal analysis could be used to successfully obtain purified ADSCs. Growth dynamics and morphology of the cells did not change with passages. There are differences and commonness of the expression of surface antigens among the clones. The potential of adipogenic differentiation of each clone also had variety. The difference in expression of cell surface antigens may attribute to the variation in potential of differentiation. Among those antigens identified, CD34, CD54, and CD106 showed markedly difference in each clone, maybe more associated with adipogenic differentiation clones. Additionally, the maintained expression of CD34, CD54, and CD106 after adipogenesis, while it reduced with passages, indicated that these markers may be considered as the high- adipogenic potential related cell-surface antigens. Our results suggest that the ADSCs, isolated from adipose tissue by collagenase can be the purified and certain cell surface antigens may be used to enhance ADSCs potential of adipogenicity.
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