Method Article
Here we describe the isolation of CD133 expressing liver stem cells and cancer stem cells from whole murine liver, a process that requires tissue digestion, cell enrichment, and flow cytometry isolation. We include methods for advanced single cell isolation and clonal expansion.
Liver stem cell, or oval cells, proliferate during chronic liver injury, and are proposed to differentiate into both hepatocytes and cholangiocytes. In addition, liver stem cells are hypothesized to be the precursors for a subset of liver cancer, Hepatocellular carcinoma. One of the primary challenges to stem cell work in any solid organ like the liver is the isolation of a rare population of cells for detailed analysis. For example, the vast majority of cells in the liver are hepatocytes (parenchymal fraction), which are significantly larger than non-parenchymal cells. By enriching the specific cellular compartments of the liver (i.e. parenchymal and non-parenchymal fractions), and selecting for CD45 negative cells, we are able to enrich the starting population of stem cells by over 600-fold.The proceduresdetailed in this report allow for a relatively rare population of cells from a solid organ to be sorted efficiently. This process can be utilized to isolateliver stem cells from normal murine liver as well as chronic liver injury models, which demonstrate increased liver stem cell proliferation. This method has clear advantages over standard immunohistochemistry of frozen or formalin fixed liver as functional studies using live cells can be performed after initial co-localization experiments. To accomplish the procedure outlined in this report, a working relationship with a research based flow-cytometry core is strongly encouraged as the details of FACS isolation are highly dependent on specialized instrumentation and a strong working knowledge of basic flow-cytometry procedures. The specific goal of this process is to isolate a population of liver stem cells that can be clonally expanded in vitro.
すべてのソリューション、メディア、楽器、フィルタ、およびチューブは滅菌および汚染のリスクを減らすために無菌操作で処理する必要があります。 4で、事前とストア内のすべてのバッファとメディア24時間を準備℃に
1。全体の肝臓からの実質及び非実質分離
2。赤血球溶解
層流フードで働く、細胞は冷たいまま、および使用のソリューションは、4℃に冷却した。
3。非実質画分からCD45造血細胞枯渇
層流フードで働く、細胞は冷たいまま、および使用のソリューションは、4℃に冷却した。
4。 CD133陽性細胞のフローサイトメトリー分離
5。細胞培養法
6。 RT - PCRを用いて双方向性のステータスの確認
この手順をRNeasyキットに同梱さをRNeasyプロトコルハンドブック、に詳述されている。
7。 CD133 +幹細胞の腫瘍性の確認
8。代表的な結果:
正常な、健康なマウスの肝臓から、CD133 +肝臓の幹細胞の予想される細胞の収率は、肝臓あたり1,000〜5,000です。これらの細胞は静止肝臓では比較的まれであり、文化の中でうまく展開しません。我々は、正常な肝臓の単一細胞解析を使用しないことをお勧めして拡大する生細胞の収率は極めて低いです。
/ - -または肝臓特異的PTEN - / -マウス、2-4セル数の期待値などMAT1aなどの6週間1または慢性的な損傷につながる遺伝的改変のためのDDC 0.1%食として重要な慢性的なけが、、と肝臓のための最大100,000個の細胞を持つLS孤立増加、孤立/肝臓(図2)。遺伝的モデルを使用している場合は肝臓の幹細胞を分離するためのこれらの手順は腫瘍フリー動物を用いて実施されるべきであるとして、自発的な腫瘍の率の知識は、非常に重要です。 / - - MAT1aはたとえば、モデルが18ヶ月で自然発生腫瘍を形成する、我々は前に報告された自然発生腫瘍に、遅くとも15から16ヶ月以上の動物を使用しないことをお勧めします。
慢性的な傷害のモデルからの単一細胞の単離は、手続きがマスターされている、と細胞の生存率が確保されると、単一の細胞から拡大(3-9 colonies/96ウェルプレート)いくつかのコロニーが得られます。 7日後に拡大単一のCD133 +細胞由来のコロニーの3現在の代表的な位相コントラスト像を示しています。
双方向性のステータスの確認は、 アルブミンとKrt19 RT - PCRを使用して行われている。拡張された単一細胞からコロニーが肝細胞のマーカー( アルブミン )と胆管(Krt19)のための表現の両方を紹介します。図4は、7日間で約25セル/コロニーでは、3つの分離されたコロニーから双方向の潜在的な発現を示しています。
CD133 +正常な肝臓と化学的に誘発された肝臓損傷(6週間用などDDC 0.1%食)からの幹細胞はヌードマウスで腫瘍を形成しません。 CD133 +特定の遺伝的モデル(MAT1a - / -または肝臓特異的PTEN - / -マウス)由来の幹細胞は、後半にプレ腫瘍慢性傷害期後期に分離された場合、ヌードマウスで腫瘍を形成します。この腫瘍形成表現型は、現在、癌幹細胞として識別される2〜4を例えば、MAT1a - 。/ -マウスは生後18週で自然発生肝腫瘍を形成する。肝疾患の後期慢性傷害の段階で、15〜16週で分離されたCD133 +肝臓の幹細胞は、ヌードマウスで腫瘍を形成します。図5は、単一のCD133 +肝臓の幹細胞からin vitroで拡大1 × 10 6細胞の二国間の注入から代表的な腫瘍を示しています。
遺伝子 | フォワードプライマー | リバースプライマー |
β-アクチン | 5' - TGTTACCAACTGGGACGACA - 3' | 5' - GGGGTGTTGAAGGTCTCAAA - 3' |
アルブミン | 5' - CATGCCAAATTAGTGCAGGA - 3' | 5' - GCTGGGGTTGTCATCTTTGT - 3' |
ケラチン19 | 5' - TGCTGGATGAGCTGACTCTG - 3' | 5' - AATCCACCTCCACACTGACC - 3' |
表1:RT - PCR用プライマーの設計
図1:メソッドの全体の概略図。
図2:野生型マウスから高度に濃縮されたCD45枯渇非実質割合で識別されたCD133 +肝臓の幹細胞制御無傷肝臓は高濃縮CD45枯渇非実質分数内で0.4パーセントCD133 +細胞を示しています。遺伝的にMAT1aノックアウト- / - 、慢性肝障害のモデルである、CD133の人口は、同じ高濃縮画分の10倍以上拡大する。
図3:単一のCD133 +クローンからのコロニーを拡大ラミニンコート96ウェルプレート上に展開単一のCD133 +細胞に由来するfourコロニーの位相コントラスト像が。。
図4:。CD133 +肝臓の幹細胞クローン拡大コロニーからのBi -潜在的な遺伝子発現は、7日目では、コロニーは約25細胞である。遺伝子発現は、CD133 +細胞の双方向性の状態を確認し、肝細胞マーカーアルブミンとcholangiocyteマーカーKrt19の発現を示しています。
図5: - / -モデルCD133 +肝臓の幹細胞はヌードマウスで腫瘍を形成する矢印は、4週間MAT1aから注入された1 × 10 6 CD133 +細胞の後に増殖する腫瘍を示している。 CD133 +などのCCl 4(0.1%)DDC食事などの毒素誘発される慢性肝障害から細胞、腫瘍を形成しない。腫瘍形成は悪性の可能性、または幹細胞集団内のがん幹細胞の表現型を識別するために使用されます。
Unlike the hematopoietic system, in which hematopoietic stem cells are responsible for maintaining a cellular differentiation system that replacesnormal physiologic turn-over of leukocytes, red blood cells, and platelets, liver stem cell, or adult liver progenitor cells, do not participate in normal liver homeostasis.5,6 After acute liver injury or partial hepatectomy, hepatocytes, as differentiated liver epithelium, undergo several rounds of proliferation to replace the lost liver mass.5,6Only during chronic injury are liver stem cells observed to proliferate.1,5-11 These adult, organ specific stem cells are proposed to differentiate into both hepatocytes and cholangiocytes.1,5-8Interestingly, the vast majority of liver cancer develops on the background of chronic injury, and thus liver stem cells are also hypothesized to be the precursors for a subset of liver cancer.2-4,12-17
One of the major challenges to stem cell work in the liver is isolation of rare populations of cells for functional analysis. For example, the vast majority of cells in the liver are hepatocytes, which are significantly larger than cholangiocytes and other smaller non-parenchymal cells. By breaking the whole liver into cell compartments (large hepatocytes - parenchymal fraction and smaller cells - non-parenchymal fraction), and further selecting for CD45 negative cells (non-hematopoietic cells), we are able to enrich the starting population of stem cells by over 600-fold.1,18-21Of note, we are by no means indicating that the CD133+ population is a 100% pure stem cell population, but clearly represents a heterogeneous population of cells, with different lineage and repopulation potential. One of the major limitations of the field is definitions of stem cells and progenitor cells. We use the term "stem cell" more broadly in this work, but in strict definition, CD133+ non-parenchymal cells represent a bi-lineage progenitor population. Given the state of current stem and progenitor research in the liver, this report does provide a starting place for investigators who are interested in this field. As new markers emerge, such as EpCAM,22,23 or transcription factors, such as Sox9,24 they can be incorporated. For example, we have found a fairly high rate of overlap between EpCAM+ cells and CD133+ cells.
In this report, we detail a process for stem cell isolation from normal murine liver as well as chronic liver injury models, which demonstrate increased liver stem cell proliferation. This method has clear advantages over standard immunohistochemistry of frozen or formalin fixed liver as functional studies using live cells can be performed after isolation.1,3,4 The specific goal of this process is to isolate a relatively pure population of liver stem cells that can be clonally expanded in vitro.
The primary limitation to this procedure is that the majority of cells isolated will not be viable after flow cytometry (see Trouble Shooting section). This is the result of the hours required to prepare the cells, and the numerous procedures required to refine the population prior to isolation. If the liver is digested in single cell suspension for immediate FACS analysis, the size difference between hepatocytes and other non-parenchymal cells will make effective FACS gate creation impossible. If the liver non-parenchymal cells are utilized, without elimination of hematopoietic cells, there is a risk that a significant number of CD133+ cells may be of hematopoietic origin may contaminate the fraction. Furthermore, by processing the cells through the Miltenyi filter, only single cells and very small clusters of cells are collected. This ensures that the sample will not clog the FACS intake needle.
Alternatives for this procedure include modification for any alternative cell surface marker, such as CD49f, EpCAM, or combination of markers, such as CD133+EpCAM+A second published report utilizes a density gradient to isolate liver stem cells from a non-parenchymal fraction. This procedure requires an Ultra-centrifuge (8000 x g), adds significant time to the procedure, and in our experience, significantly reduced pre-FACS cellular yield and post-FACS viability.8
Future experiments include a broader range of gene expression analysis, including fetal liver genes, such as HNF3, HNF4α, and αfp.In addition, Western blot analysis and immunocytochemistry of expressed proteins can be utilized to confirm RT-PCR results of cells in culture.
One issue to note is recent work that identified CD133 expression on hepatic stellate cells.25 We now routinely screen our samples for markers of stellate cells (see Trouble Shooting section), and have not identified significant contamination in our fractions. This may be related to different techniques of liver digestion and cell isolation.2,3,26
Based on the fact that the majority of cells will not be viable immediately after FACS isolation, we recommend that the cells be plated, either as bulk CD133+ cells or single cells, prior to use in animals. 5-7 days in vitro will significantly improve results of tumor analysis. In addition, given the rigors of single cell isolation, this should only be conducted once colonies can be expanded from bulk CD133+ isolated cells. A more thorough discussion of the various culture conditions and scaffold proteins which may be utilized to culture liver stem and progenitor cells has been well characterized by Lola Reid. This work provides alternative conditions and modifications, which investigators may incorporate into their research program once basic isolation techniques are mastered.27,28 Dr. Reid's work also provides a more detailed analysis of the lineage biology and maturation between hepatic stem cells and committed progenitors.
In terms of in vivo tumor analysis, we have had success using freshly isolated cells and cells from clonally expanded CD133+ cells in vitro, primarily using 1x106 cells. We have focused ontwo genetic strains of chronic liver injury, the MAT1a-/- and liver specific Pten-/- mice, and we have utilized both nude mice and wild-type mice as hosts for tumor growth. In our experience, CD133+ liver stem cells will only form tumors if they are isolated from significant liver injury models that are pre-malignant. Note that the tumors formed from CD133+ cells general have both hepatocellular carcinoma and cholangiocarcinoma features, suggesting a stem cell or progenitor cell origin to the tumors.2-4,29
Follow-up work after tumors are documented includes standard pathologic analysis of tumor tissue (H&E staining) and immunohistochemical staining. In addition, tumors can be minced and digested for FACS analysis or re-culture.2-4,30
In conclusion, we have detailed a procedure for the isolation, expansion, and basic characterization of CD133+ liver stem cells and CD133+ cancer stem cells.
Trouble Shooting:
CD45 contamination:
For Step 3, if there is contamination of CD45+ cells, which can be assessed by adding a CD45-FITC Ab prior to FACS analysis and isolation, check to ensure that the CD45 microbead antibody is not expired and that the filtrate was collected only while the filter was in the magnetic holder. Any filtrate collected while the filter is not in the magnetic holder will contain CD45+ cells.
Low cell number:
For the uninjured liver, the total number of CD133+ non-parenchymal isolated may be less than 10,000. These cells are rare in the quiescent liver. For a chronic injury model, such as the DDC 0.1% diet, the number will increase greatly to 100,000 cells. If the total cell number is significantly below these numbers, one issue to consider is the FACS Ab staining. Check to ensure that the CD133 Ab is not expired, as poor staining will result in a poor yield. Also, we recommend performing a FACS analysis of liver non-parenchymal cells to determine the relative population prior to attempting FACS isolation.
Low cell viability after FACS:
One of the issues of viability may be related to how the cells are processed and over what time. Ideally, the entire cell isolation procedure, steps 1-4, should be conducted without any delays between steps and completed in the same day. Any significant delay between steps 1-4 will greatly reduce the cell viability. A second issue related to cell viability may be related to the sheath pressure used for FACS isolation. We recommend using a lower sheath pressure. Lastly, once the cells are isolated, they should be immediately plated, as any significant storage on ice after sorting will also reduce viability.
CD133+ non-parenchymal heterogeneity:
Recent reports indicate that hepatic stellate cells may also have CD133 expression and have some plasticity.25 Therefore, in addition to verifying bi-potency genes with Albumin and Krt19, additional verification can include genes associated with stellate cells, such as glial fibrillary acidic protein in quiescent stellate cells and alpha-smooth muscle actin and desmin in activated stellate cells.3,4,26 In addition, the CD133+ population represents a broader progenitor population. The addition of a second marker, such as EpCAM, may help to refine the population further, and limit heterogeneity.
Dr. Rountree receives research support from Bayer Pharmaceutics. Drs. Ding and Crooks and Ms. Dang and Ms. VanKirk have no disclosures to report.
Dr. Rountree acknowledges current support from the Children's Miracle Network, National Institute of Health, K08DK080928 and R03DK088013, and the American Cancer Society Research Scholar Award, MGO-11651. Dr. Rountree acknowledges that this procedure was initially developed and refined while funded by the Pediatric Scientist Development Program (NICHD Grant Award K12-HD00850).
Name | Company | Catalog Number | Comments |
DMEM:F12 | Invitrogen | 10565-018 | With phenol red |
CD45 microbeads | Miltenyi Biotec | 130-052-301 | |
Hepatocyte Growth Factor | Sigma-Aldrich | H1404 | |
Epidermal Growth Factor | Sigma-Aldrich | E4127 | |
DNase | Sigma-Aldrich | DN25 | 1 gram |
Collagenase D | Roche Group | 1088874 | |
Pronase | Roche Group | 0165921 | |
70 micron mesh strainer | Fisher Scientific | 352350 | |
Omniscript RT | Qiagen | 205111 | 50 reactions |
HotStarTaq | Qiagen | 203203 | |
Miltenyi LD column | Miltenyi Biotec | 130-042-901 | |
CD133-PE FACS Ab | eBioscience | 12-1331-82 | |
Laminin coated plates | BD Biosciences | 354410 | 96 well |
Trypsin 0.05% EDTA | Invitrogen | 25300-354 | 100 mL |
RNeasy Micro Kit | Qiagen | 74004 | 50 columns for 5x105 cells or less |
Pharm Lyse | BD Biosciences | 555899 | 10X concentration |
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