1Department of Pediatrics and Pharmacology, Pennsylvania State College of Medicine, 2Department of Pharmacology, Pennsylvania State College of Medicine, 3Department of Pediatrics, University of California Los Angeles, School of Medicine
Rountree, C. B., Ding, W., Dang, H., VanKirk, C., Crooks, G. M. Isolation of CD133+ Liver Stem Cells for Clonal Expansion. J. Vis. Exp. (56), e3183, doi:10.3791/3183 (2011).
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.
All solutions, media, instruments, filters, and tubes should be sterile and handled with sterile technique to reduce the risk of contamination. Prepare all buffers and media 24 hours in advance and store at 4°C.
1. Parenchymal and non-parenchymal separation from whole liver
2. Red cell lysis
Work in laminar flow hood, keep cells cold, and use solutions cooled to 4°C.
3. CD45 hematopoietic cell depletion from non-parenchymal fraction
Work in laminar flow hood, keep cells cold, and use solutions cooled to 4°C.
4. Flow cytometry isolation of CD133 positive cells
5. Cell culture methods
6. Confirmation of bi-potential status using RT-PCR
This procedure is detailed in the RNeasy protocol handbook, which is supplied with the RNeasy Kit.
7. Confirmation of tumor potential of CD133+ stem cells
8. Representative Results:
From normal, healthy murine liver, the expected cellular yield of CD133+liver stem cells is 1,000 to 5,000 per liver. These cells are relatively rare in quiescent liver and will not expand well in culture. We do not recommend using single cell analysis on normal liver and the yield of viable cells that will expand is extremely low.
For livers with significant chronic injury, such as the DDC 0.1% diet for 6 weeks1 or genetic modification resulting in chronic injury, such as the MAT1a-/- or liver specific Pten-/-mice,2-4the expected number of cells isolated increases, with up to 100,000 cells isolated/liver (Figure 2). If using genetic models, knowledge of spontaneous tumor rate is critical, as these procedures for liver stem cell isolation should be conducted in tumor free animals. For example, if the MAT1a-/- model forms spontaneous tumors at 18 months, we recommend using animals no later than 15-16 months, prior to any reported spontaneous tumors.
Isolation of single cells from chronic injury models will yield several (3-9 colonies/96 well plate) colonies that expand from single cells once the procedure is mastered, and cell viability is ensured. Figure 3 present representative phase contrast images of colonies derived from single CD133+ cells expanded after 7 days.
Confirmation of bi-potential status is conducted using Albumin and Krt19 RT-PCR. Colonies from expanded single cells will demonstrate both expression for markers of hepatocytes (Albumin) and cholangiocytes (Krt19). Figure 4 demonstrates bi-potential expression from three isolated colonies, with approximately 25 cells/colony at 7 days.
CD133+ stem cells from normal liver and chemically induced liver injury (e.g. DDC 0.1% diet for 6 weeks) will not form tumors in nude mice. CD133+ stem cells from specific genetic models (MAT1a-/- or liver specific Pten-/- mice) will form tumors in nude mice if isolated late in late pre-tumor chronic injury phase. This tumor forming phenotype is currently identified as a cancer stem cell.2-4For example, the MAT1a-/- mice form spontaneous liver tumors at 18 weeks of age. CD133+ liver stem cells isolated at 15-16 weeks, during a late chronic injury phase of liver disease, will form tumors in nude mice. Figure 5 demonstrates representative tumors from bilateral injection of 1 x 106 cells expanded in vitro from single CD133+ liver stem cells.
|Gene||Forward Primer||Reverse Primer|
Table 1: Primer design for RT-PCR
Figure 1: Overall schematic of methods.
Figure 2: CD133+ liver stem cells identified in highly enriched CD45 depleted non-parenchymal fraction. Control uninjured liver from wild-type mice demonstrates 0.4% CD133+ cells within the highly enriched CD45 depleted non-parenchymal fraction. In the genetic knock-out MAT1a-/-, which is a model of chronic liver injury, the CD133 population expands 10 fold or more in the same highly enriched fraction.
Figure 3: Expanding colonies from single CD133+ clones. Phase-contrast images of four colonies derived from single CD133+ cells expanded on laminin coated 96 well plates.
Figure 4: Bi-potential gene expression from CD133+ liver stem cell clonally expanded colonies. At Day 7, colonies are approximately 25 cells. Gene expression demonstrates expression of hepatocyte marker Albumin and cholangiocyte marker Krt19, confirming bi-potential status of CD133+ cells.
Figure 5: CD133+ liver stem cells form tumors in nude mice.Arrows indicate tumors growing 4 weeks after 1x106 CD133+ cells injected from MAT1a-/- model. CD133+ cells from toxin induced chronic liver injury, such as CCl4 or 0.1% DDC diet, do not form tumors. Tumor formation is used to identify malignant potential, or cancer stem cell phenotype within the stem cell population.
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.
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).
|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|
|Collagenase D||Roche Group||1088874|
|70 micron mesh strainer||Fisher Scientific||352350|
|Omniscript RT||Qiagen||205111||50 reactions|
|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|