通过成熟的脂肪细胞在天花板文化去分化代人脂肪干细胞

摘要

Mature adipocytes may represent an abundant source of stem cells through dedifferentiation, which leads to a homogenous population of fibroblast-like cells. Collagenase digestion is used to isolate mature adipocytes from human fat. The goal of our protocol is to obtain multipotent, dedifferentiated fat cells from human mature adipocytes.

摘要

Mature adipocytes have been shown to reverse their phenotype into fibroblast-like cells in vitro through a technique called ceiling culture. Mature adipocytes can also be isolated from fresh adipose tissue for depot-specific characterization of their function and metabolic properties. Here, we describe a well-established protocol to isolate mature adipocytes from adipose tissues using collagenase digestion, and subsequent steps to perform ceiling cultures. Briefly, adipose tissues are incubated in a Krebs-Ringer-Henseleit buffer containing collagenase to disrupt tissue matrix. Floating mature adipocytes are collected on the top surface of the buffer. Mature cells are plated in a T25-flask completely filled with media and incubated upside down for a week. An alternative 6-well plate culture approach allows the characterization of adipocytes undergoing dedifferentiation. Adipocyte morphology drastically changes over time of culture. Immunofluorescence can be easily performed on slides cultivated in 6-well plates as demonstrated by FABP4 immunofluorescence staining. FABP4 protein is present in mature adipocytes but down-regulated through dedifferentiation of fat cells. Mature adipocyte dedifferentiation may represent a new avenue for cell therapy and tissue engineering.

引言

In vitro dedifferentiation of mature adipocytes is achieved through a technique called ceiling culture¹. Because of their natural tendency to float in aqueous solutions, isolated mature adipocytes adhere to the surface of an inverted flask fully filled with culture medium. Over a few days, cells modify their spherical morphology and become fibroblast-like cells. The resulting cells, called dedifferentiated fat (DFAT) cells, are multipotent². Research articles on adipocyte dedifferentiation, especially on human cells, are limited. However, they have already provided interesting information regarding multipotency, cell phenotype and replicative capacity of DFAT cells². Mature adipocytes originating from various fat compartments have been successfully dedifferentiated including those originating from human visceral and subcutaneous adipose tissues^2-4. In addition to these depots, Kishimoto and collaborators sampled adipose tissue from the buccal fat pads and dedifferentiated adipocytes into DFAT cells⁵. Matsumoto and collaborators successfully generated subcutaneous DFAT cells from patients covering a wide range of ages, and the majority of cells had a high proliferative rate and less than 6% of senescence even after 10 passages in culture².

DFAT cells have been successfully re-differentiated into several lineages, including adipogenic, osteogenic, chondrogenic and neurogenic lineages^2,3,6. These cells express several embryonic stem cell markers such as Nanog and the four identified pluripotent factors Oct4, c-myc, Klf4 and Sox2³. They also express markers specific to each of the three germ layers⁷. In addition, DFAT cells are similar to Bone Marrow-derived Mesenchymal Stem Cells (BM-derived MSC) based on their epigenetic signature³. Exploiting the stem cell capacity of DFAT cells, many groups have investigated their potential to treat or improve various diseases^8,9. Improvements of pathologic conditions, such as infracted cardiac tissue, spinal cord injury and urethral sphincter dysfunction, have been observed with DFAT cell injections in rat models of disease^10-12.

In addition to the stem cell properties of DFAT cells, they may represent a new cellular model for adipocyte physiology studies. The 3T3-L1 cell line is often used for this purpose as these cells differentiate into adherent, lipid-storing adipocytes under adipogenic stimulation¹³. However, these cells originate from mouse embryo tissue¹³. Also, depot-specificity cannot be investigated with this model and it may not fully reflect human adipocyte physiology¹⁴. Other laboratories work with isolated adipose cells from murine fat depots, but fat distribution is not dimorphic in mice and anatomical configuration of the rodent's abdominal cavity prevents from extrapolating directly to humans¹⁵. In order to study adipocytes in the context of the physiopathology of human obesity, consideration of body fat distribution and fat depot-specific differences has become essential¹⁶. Some limitations of primary preadipocyte cultures, including cell quantities obtained from adipose tissue biopsy samples and their senescence after a few passages in culture, created the need for alternate models. Perrini and collaborators investigated depot-specificity in gene expression of DFAT cells originating from visceral and subcutaneous fat and compared them to adipose-derived stem cells (ASC) from the same fat depot. They demonstrated that differences in gene expression and function where mainly found between depots than between cell types, suggesting that DFAT cells are physiologically close to ASC from the same depot. DFAT cells may represent an interesting alternative to available models for studies on fat distribution in the pathophysiology of human obesity. Moreover, ceiling culture is a promising method to obtain adult stem cells for tissue engineering purposes.

Here, we describe collagenase digestion, a widely-used technique to isolate mature adipocytes from the subcutaneous and/or visceral fat samples¹⁷, and the subsequent steps to perform ceiling culture and dedifferentiate these cells into multipotent, fibroblast-like cells.

研究方案

伦理学声明:该项目已通过IUCPQ的研究伦理委员会之前，患者招募。在这篇文章中/视频的目的，我们从2例患者获得组织:1）一名62岁的男性患者，50.7公斤/米²和2）35岁的女性患者体重指数与57公斤体重/米^2。实验可以与脂肪车厢做，但都被限制在一个隔脂肪这部影片的目的。视频技术方面进行与患者1，并与去分化细胞来自患者2进行FABP4免疫。

1.样品处理

1. 问医生从大网膜和皮下脂肪车厢收集脂肪组织，在腹腔镜减肥手术的时间。
2. 迅速将脂肪样品送到实验室RT，并立即处理。
3. 在实验室中进行消化，在非无菌的气氛中。该细胞，最终会转嫁到培养室并在无菌条件下培养。为了避免污染，准备KRH缓冲用蒸馏水，过滤水，并按照过滤（0.22μm的过滤器）之前消化。彻底清洁管与烧瓶和板制备在细胞培养罩乙醇之前传输。
4. 放置脂肪组织上的预加权盘和记录重量。固定在10％福尔马林缓冲液中的小片的每个组织样本（小于1厘米^2）的在RT石蜡包埋前至少24小时。使用免疫组织化学实验，这种嵌入式样品（未显示技术）。
5. （ -技术未示出例如 ，基因表达）在-80℃下储存于整个脂肪组织进一步研究之前放置另一块在一个50毫升管和闪冻在液氮中。

2.胶原酶消化

1. 将剩余的脂肪组织片在50毫升涂对于消化。
2. 添加4毫升KRH-WB的补充有胶原酶（350单位/毫升）每克样品中的消化管。
3. 直言不讳的脂肪组织用剪刀。
4. 放置切碎的脂肪组织悬浮液在摇动器，37℃，90转最大，为45分钟的温育（最多1个小时）。

3.净化脂肪细胞和前脂肪细胞的

1. 倾的脂肪少的块通过一个400微米的尼龙半透明溶液啮合到塑料烧杯中。
2. 用镊子，擦上尼龙网细胞制剂和洗用5毫升KRH-WB的。
3. 该过滤细胞悬液微妙转移到50毫升管的塑料管中，并附着在管末端一60毫升注射器。
4. 让该悬浮液与成熟脂肪细胞静置约10分钟，使细胞通过浮选到达缓冲器的顶部。
5. 使用60毫升syrin慢慢吸出缓冲在管的底部GE吸力。
6. 添加20毫升KRH-WB的洗。从2额外的洗涤步骤3.4中重复。
7. 收集在缓冲带来的脂肪细胞悬浮液中的5％或10 ml的终体积，这取决于细胞的数量。寻求与在第5个步骤。
8. 从如果需要收集与离心进一步初级细胞培养物的60毫升注射器（3000转，室温，5分钟）缓冲恢复基质血管级分（技术未示出）。

4.成熟的脂肪细胞计数

1. 负载10微升轻轻摇动脂肪细胞悬液在计数室（血球）。一式四份进行细胞计数。
2. 计算分离的成熟细胞的数目。

5.成熟的脂肪细胞去分化为T-25瓶

1. 填充的25cm ²组织培养瓶到¾用DMEM / F12-20％小牛血清的体积。
2. 根据细胞计数，倾500000成熟细胞到烧瓶中。
3. 填完全使用50毫升管介质烧瓶中并除去尽可能多的气泡尽可能。
4. 拧上瓶不通风帽。
5. 清洗之前用温育，以避免污染乙醇的烧瓶中。
6. 孵育瓶倒了一个星期没有接触它，以避免可能破坏细胞粘附的文化运动。
7. 前扭转烧瓶在第7天倒置培养，轻轻操纵烧瓶中并通过抽吸除去所有介质中的烧瓶中，避免了突然的动作。
8. 添加12毫升DMEM-F12-20％小牛血清培养细胞的标准技术。滤波后，排出帽可以加入到该烧瓶中。

6.成熟脂肪细胞脱分化成6孔板

1. 放置盖玻片上的6孔板的每个孔的底部
2. 添加½"塑料套管每个盖玻片上。
3. 填孔用8ml 20％小牛血清的DMEM培养基。
4. 把盖玻片上的每个塑料套管。
5. 插入幻灯片和管到滑动下注射细胞（每孔50,000个细胞）之间吸管尖。
6. 孵育板在标准细胞培养孵化器在37℃，5％CO ₂的一个星期。
7. 反向盖玻片与附着细胞到每个孔中含有2毫升培养基补充有20％小牛血清和追求培养。
8. 用盖玻片与细胞发生去分化为几个目的，包括免疫荧光（技术未示出）。

结果

主要形态变化发生去分化（ 图1）中成熟脂肪细胞。 如图2中 ，细胞经历去分化染色用抗FABP4抗体进行荧光分析。细胞与圆形形态表达的FABP4蛋白，而大多数的成纤维细胞样细胞的没有。去分化后，DFAT细胞可以培养使用标准程序几个传代。我们已经能够达到超过15代对人网膜和皮下DFAT细胞系（数据未示出）。

讨论

成熟的脂肪细胞与天花板培养技术去分化是一种新的方法来从本地脂肪组织的一个小样本的获取脂肪干细胞。根据我们的经验和其他人^2，一克组织足以在板25-cm ²的烧瓶中并以获得DFAT细胞的量的均匀性已被证实由波洛尼和合作者³群。脂肪细胞分化似乎独立他们的年龄，性别和其他特征可以与来自任何供体细胞。间所产生的DFAT获得人口，仍然存在一些圆形或部分细长的细?...

材料

Name	Company	Catalog Number	Comments
Bovine serum albumine	Sigma	A7906
Adenosine	Sigma	A4036
Ascorbic acid	Sigma	A0278
NaCl			Any brand can be used
KCl			Any brand can be used
CaCl2			Any brand can be used
MgCl2			Any brand can be used
KH2PO4			Any brand can be used
HEPES			Any brand can be used
Glucose			Any brand can be used
Type I collagenase	Worthington Biochemical Corp	LS-004196
DMEM/F-12, HEPES, no phenol red	Gibco-Life Technologies	11039-021	Add to medium : 20% calf serum, gentamicin (50µg/ml) and fungizone (2.5µg/ml)
Calf Serum, iron supplemented, from formula-fed calves	Sigma	C8056-500ml
1/2 In plastic bushing	Iberville	2704-CP	SKU:1000120918 (Home Depot)
Liquid nitrogen	Linde
Formalin soluton, neutral buffered, 10%	SIGMA	HT501128
Sterile tweezers
Sterile scissors
60cc syringes	BD Syringe
Plastic tubing
Krebs-Ringer-Henseleit stock buffer (KRH)			Prepare stock buffer as following: 25mM HEPES pH7.6, 125mM NaCl, 3.73mM KCl, 5mM CaCl2.2H2O, 2.5mM MgCl2.6H2O, 1mM K2HPO4. Adjust pH to 7.4.
Krebs-Ringer-Henseleit-Working Buffer (KRH-WB)			Add the following components freshly to KRH buffer: 4% bovine serum albumin, 5mM glucose, 0.1µM adenosine, 560 µM ascorbic acid
KRH-WB supplemented with Type I collagenase			Add 350U/ml of Type I collagenase
T25 unvented cap tissue culture flask	Sarsted or other brand
6-well tissue culture plate	BD Falcon or other brand
Microscope cover glass 22x22	Fisherbrand	12-542-B
Sterile beakers