Isolation, Propagation and Characterization of Breast Stem Cells

正常干细胞：根据Kuperwasser等人的描述，通过人源化NOD/ SCID小鼠的乳腺脂肪垫，建立一个强大的体内模型来研究人类动物上皮形态发生，这是我们小组的一个主要目标。为了用非转化细胞使乳腺人源化，我们使用了从Kuperwasser博士那里获得的永生化人乳腺成纤维细胞。此外，我们正在开发自己的永生化，绿色荧光蛋白（GFP）标记，人乳腺成纤维细胞。来自缩小乳房成形术的原代人成纤维细胞已成功地在体外培养。细胞被编码GFP的逆转录病毒构建物和人类端粒酶的催化亚基感染。一旦纯化和选择这些永生化乳腺成纤维细胞将用于乳腺的人源化。在注射到人源化腺体原代上皮细胞之前，在一种被报道为乳腺干细胞富集的确定培养基中培养。我们发现，在确定的培养基中加入人催乳素可以增加假定的乳腺干细胞的数量。我们的初步数据表明，细胞在注射前也需要一个3D结构，以便在整个人源腺体中成功发育。我们发现上皮细胞与原代人乳腺成纤维细胞共培养可刺激导管样结构的形成。利用最初试验中收集的腺体，我们已经成功地开发了区分人类成纤维细胞和宿主小鼠成纤维细胞的方法。这些方法包括GFP全挂载分析以及使用物种特异性免疫组织化学和基因组荧光原位杂交的组织学分析。癌症干细胞：首先，我们试图重复先前发表的数据，表明可以从4期乳腺癌妇女的胸腔积液中分离出罕见的高致瘤性细胞群。这些假定的癌症干细胞是独特的，因为它们是免疫功能低下小鼠中唯一能够启动异种移植物肿瘤的肿瘤细胞。我们还确定在已建立的乳腺癌细胞系中是否存在致瘤性的异质性，并确定新的癌症干细胞标记物。Al Hajj等人（2003）的研究表明，肿瘤干细胞可以通过以下抗原的表达从胸腔积液中富集：Lineage- CD24-/dim CD44+。血统鸡尾酒由八种抗体组成，用于确保从胸膜液中去除任何非肿瘤细胞。Al Hajj观察到，只有Lineage- CD24-/dim CD44+细胞能够在小鼠体内启动肿瘤，显示出它们的自我更新特性。这些作者还报道，由此产生的异种移植物肿瘤具有与初始胸腔积液相似的CD24 - CD44异质性，表明它们具有多能性。迄今为止，我们已经将7例胸腔积液中的细胞原位注射到免疫功能低下的NOD/SCID小鼠体内。来自SH12样本的细胞被分为两个群体：Lineage-、CD24+ CD44+和Lineage- CD24-/dim CD44+。只有200个CD24-/dim细胞产生肿瘤，而5000个24+细胞没有产生肿瘤。由此产生的异种移植物的CD24- CD44特征与最初的胸腔积液相似，大多数细胞（> 80%）落在CD24+ CD44+象限，少数含有CD24- CD44+细胞，与Al Hajj一致。该异种移植物已成功地在小鼠乳腺脂肪垫中传代多次。由此产生的SH12异种移植物已被分离并分类为四个免疫表型独特的谱系- CD24 CD44群体。将这些细胞群注射到NOD/SCID小鼠体内，比较它们与初始胸腔积液细胞群的致瘤性。这些实验目前正在进行中。在两个单独的实验中，对胸腔积液SH8进行了分类并注射到小鼠体内。在最初的实验中，注射了三个免疫表型独特的谱系-群体（CD24bright CD44+; CD24-/dim CD44+; CD24+ CD44-）。即使注射了多达25000个细胞，这些细胞群中也没有一个产生肿瘤。在第二个实验中，SH8被分为两个种群，CD24bright CD44+和CD24-/dim CD44+。与第一次实验一样，即使注射了5000多个细胞，也没有出现肿瘤。这些结果与Al Hajj报道的不一致。将胸腔积液CC4分为3个谱系群（CD24bright CD44+、CD24-/dim CD44+、CD24+ CD44-），注射到NOD/SCID小鼠体内。与SH8类似，没有形成多达5000个细胞的肿瘤。这些数据表明，胸腔积液中单纯存在Lineage- CD24-/dim CD44+细胞并不表明存在癌症干细胞，需要新的高致瘤性癌症干细胞标记物。我们也已经开始努力从原发性乳腺肿瘤中获取数据。考虑到从肿瘤切除术中获得的通常较小的标本的局限性，我们正试图在免疫功能低下的小鼠的乳腺脂肪垫中扩大这些标本。我们已经成功地从BRCA1突变载体中获得肿瘤块，并在小鼠中成功传代多次。我们最近将一个异种移植肿瘤分离并分类为两个群体（CD24bright CD44+和CD24-/dim CD44+），并将其注射到NOD/SCID小鼠体内。这个实验目前正在进行中。我们目前正在确定在已建立的细胞系中是否存在癌症干细胞。首先，我们确定了23种乳腺癌细胞系的CD44 CD24谱。我们选择了8种不同体内致瘤性的细胞系（MCF7、MCF10A-III、MCF10A-IV、ZR75-1、SUM229、SUM1315、SUM149和AU565）来测试使用CD24 CD44表达富集高致瘤性癌症干细胞的有效性。到目前为止，我们已经通过CD24 CD44表达对MCF7、MCF10A-IV、ZR75-1、SUM229和AU565细胞进行了分选，并将其注射到免疫功能低下的小鼠体内。注射后两种细胞系的小鼠没有形成肿瘤，即使注射了10,000个CD24-/dim CD44+细胞。注射分选的MCF7、MCF10A-IV和ZR75-1细胞的初步实验数据表明，随着注射细胞数量的增加（1,000至10,000），CD24 - CD44图的所有象限都具有一定的致瘤潜力。这些早期的观察结果表明，CD24的表达（或缺乏）对细胞形成肿瘤的能力几乎没有影响。然而，趋势是缺乏CD44的细胞比表达这种蛋白的细胞更不具有致瘤性。此外，我们正在努力鉴定新的标记物，以丰富高致瘤性细胞的能力。我们正在测试CD205、CD117、CD135、Cripto、SSEA4、CD87、CD200、CD133、CD29、CD49f和CD177，选择这些基因是因为它们与肿瘤侵袭性、胚胎干细胞、其他组织中的癌症干细胞和/或成体干细胞相关。

Normal stem cells: The development of a robust in vivo model to study human beast epithelial morphogenesis by humanizing the mammary fat pad of NOD/ SCID mice as described by Kuperwasser et al. 2004 has been a major objective of our group. To humanize mammary glands with non-transforming cells, we used immortalized human mammary fibroblasts obtained from Dr. Kuperwasser. Additionally, we are developing our own immortalized, green florescent protein (GFP) tagged, human breast fibroblasts. Primary human fibroblasts from reduction mammoplasties are being successfully cultured in vitro. Cells have been infected with a retroviral construct encoding GFP and the catalytic subunit of human telomerase for immortalization. Once purified and selected these immortalized breast fibroblasts will be used for humanization of the mammary gland. Preceding injection into the humanized gland primary epithelial cells are cultured in a defined media reported to enrich for mammary stem cells. We have found that the addition of human prolactin to the defined media increases the number of putative mammary stem cells. Our preliminary data suggest that the cells also require a 3D structure, prior to injection, in order to successfully develop throughout the humanized gland. We found that co-culturing the epithelial cells with primary human mammary fibroblasts stimulates the formation of duct-like structures. Using glands collected in the initial trials, we have successfully developed the methods for distinguishing human fibroblasts from host murine fibroblasts. These methods include GFP whole-mount analysis as well as histological analysis using species-specific immunohistochemistry and genomic fluorescence in situ hybridization. Cancer stem cells: First we have attempted to repeat previously published data suggesting that a rare population of highly tumorigenic cells can be isolated from pleural effusions of women with stage 4 breast cancer. These putative cancer stem cells are unique in that they are the only tumor cells capable of initiating xenograft tumors in immunocompromised mice. We are also determining if heterogeneity in tumorigenicity exists within established breast cancer cell lines and identifying novel cancer stem cell markers. Work of Al Hajj et al. (2003) demonstrated that the cancer stem cells can be enriched from pleural effusions by the expression of the following antigens: Lineage- CD24-/dim CD44+. The Lineage cocktail consist of eight antibodies employed to ensure the removal of any non-tumor cells from the pleural fluid. Al Hajj observed that only Lineage- CD24-/dim CD44+ cells were capable of initiating tumors in mice, demonstrating their self-renewal properties. These authors also reported that the resulting xenograft tumors share a heterogeneous CD24 CD44 profile similar to that of the initial pleural effusion, demonstrating their multipotent nature. To date, we have injected cells from seven pleural effusions orthotopically into immunocompromised NOD/SCID mice. Cells from sample SH12 were sorted into two populations: Lineage-, CD24+ CD44+ and Lineage- CD24-/dim CD44+. As few as 200 CD24-/dim cells gave rise to tumors whereas 5,000 24+ cells failed to do so. The resulting xenograft had a CD24 CD44 profile similar to that of the initial pleural effusion with the majority of cells (> 80%) falling in the CD24+ CD44+ quadrant and the minority containing CD24- CD44+ cells consistent with Al Hajj. This xenograft has been successfully passaged in the mammary fat pad of mice multiple times. Resulting SH12 xenografts have been dissociated and sorted into four immunophenotypically unique Lineage- CD24 CD44 populations. These populations were injected into NOD/SCID mice to compare their tumorigenicity to that of the cell populations of the initial pleural effusion. These experiments are currently ongoing. Pleural effusion SH8 has been sorted and injected into mice in two separate experiments. In the initial experiment, three immunophenotypically unique Lineage- populations were injected (CD24bright CD44+; CD24-/dim CD44+; CD24+ CD44-). None of these populations resulted in tumors even when as many as 25,000 cells were injected. In a second experiment, SH8 was sorted into two populations, CD24bright CD44+ and CD24-/dim CD44+. As in the initial experiment, no tumors resulted even after injecting as many as 5,000 cells. These results are inconsistent with those reported by Al Hajj. Pleural effusion CC4 was sorted into three Lineage- populations (CD24bright CD44+; CD24-/dim CD44+; CD24+ CD44-) and injected into NOD/SCID mice. Similarly to SH8, no tumors formed with as many as 5,000 cells. These data suggest that the simple presence of Lineage- CD24-/dim CD44+ cells in a pleural effusion does not indicate the presence of cancer stem cells and that new markers for highly tumorigenic cancer stem cells are needed. We also have initiated an effort to generate data from primary breast tumors. Given the limitations of the generally small specimens received from lumpectomies, we are attempting to expand these in the mammary fat pad of immunocompromised mice. We have been successful with a tumor chunk received from a BRCA1 mutant carrier that has been successfully passaged in mice multiple times. We recently dissociated and sorted one xenograft tumor into two populations (CD24bright CD44+ and CD24-/dim CD44+) and injected them into NOD/SCID mice. This experiment is currently ongoing. We are currently determining whether there are cancer stem cells in established cells lines. First, we determined the CD44 CD24 profile of 23 breast cancer cell lines. We chose eight cell lines of varying in vivo tumorigenicity (MCF7, MCF10A-III, MCF10A-IV, ZR75-1, SUM229, SUM1315, SUM149 and AU565) for testing the effectiveness of using CD24 CD44 expression to enrich for highly tumorigenic cancer stem cells. To date, we have sorted MCF7, MCF10A-IV, ZR75-1, SUM229, and AU565 cells by CD24 CD44 expression and injected them into immunocompromised mice. No tumors formed in mice injected with the latter two cell lines, even when 10,000 CD24-/dim CD44+ cells were injected. Preliminary data from experiments injecting sorted MCF7, MCF10A-IV and ZR75-1 cells demonstrate that with the number of cells injected (1,000 to 10,000) all quadrants of the CD24 CD44 plot possess some tumorigenic potential. These early observations suggest that expression of CD24 (or lack thereof) has little influence on the ability of cells to form tumors. However, the trend is for cells lacking CD44 to be less tumorigenic than those expressing this protein. Additionally, we are working to identify novel markers for their ability to enrich highly tumorigenic cells. We are testing CD205, CD117, CD135, Cripto, SSEA4, CD87, CD200, CD133, CD29, CD49f, and CD177, chosen because they have been associated with tumor aggressiveness, embryonic stem cells, cancer stem cells in other tissues, and/or adult stem [summary truncated at 7800 characters]