クローン性増殖のためのCD133 +肝幹細胞の単離

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 1×10⁶ 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.