Isolation of CD133+ Liver Stem Cells for Clonal Expansion

Abstract

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 flowcytometry procedures. The specific goal of this process is to isolate a population of liver stem cells that can be clonally expanded in vitro. Video LinkThe video component of this article can be found at https://www.jove.com/video/3183/ ProtocolAll 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.

The role of CD133 in cancer: a concise review

“…Based on the evidence in the prostate alone, it appears to be clear that not all CD133 pos cells are stem cells and that CD133 neg cells may also possess stem-like properties. Several additional studies support this hypothesis by demonstrating that CD133 was expressed in differentiated epithelial cells in a variety of other organs including the pancreas [ 71 , 72 ], liver [ 73 , 74 ], colon [ 56 , 75 ], gastric contents [ 76 ], sweat glands [ 77 ], salivary and lacrimal glands [ 77 , 78 ], uterus [ 77 ], and kidneys [ 79 ]. Altogether, these studies indicate that the overall expression of human CD133 expands beyond stem cell populations and although it appears to negatively correlate with cell differentiation, it is likely not a regulator of stemness in most tissues [ 75 ].…”

Doxorubicin and 5-fluorouracil resistant hepatic cancer cells demonstrate stem-like properties

“…The existence of cancer stem cells (CSCs) in malignant tumors has been proposed for some decades, and much evidence supports this hypothesis. Hepatic cancer stem cells (HCSCs) have been identified and isolated from HCCs (Rountree et al 2011;Mikhail and He 2011;Cao et al 2011;Cheung et al 2011;Jia et al 2012;Yoon 2012;Shen and Cao 2012;Piao et al 2012;, and characterized by specific properties such as expression of CD133 (Suetsugu et al 2006;Yin et al 2007;Ma et al 2007), CD44, CD133 (Zhu et al 2010), CD90 (Yang et al 2008), EpCAM (Epithelial cell adhesion molecule) (Yamashita et al 2008(Yamashita et al , 2009), CD13 (Haraguchi et al 2010), OV6 (Yang et al 2008) and ALDH (Aldehyde hydrogenase) (Ma et al 2008).…”

Lack of Immunomodulatory Interleukin-27 Enhances Oncogenic Properties of Mutant p53 In Vivo

“…Femur, tibia, and lumbar vertebras from mice were cut aseptically in 1.5 mm small pieces, rinsed in PBS, and placed in digestion media consisting 5 mg collagenase, 5 mg pronase, and 1 mg DNAse for 45 minutes at 37° C (19). Following digestion, single cell suspension was passed through a 70 µm strainers twice.…”