There are some different protocols for isolation of epithelial (keratinocyte) stem cells locating at the bulge region of hair follicles, I want to know which one is more efficient and less time consuming?
First, it depends on which specie you have been working. It's obvious that human is different of mouse. As you are speaking of bulge, and not bulge region, i will suppose that's mouse. Second, it also depends on the technics you can use (FACS, culture, ...) For mouse, there is currently no good methods with culture because rodent keratinocytes really fast immortalize and culture conditions for mouse keratinocytes are really complicated (low calcium, 32°C, ....). Most of the people use FACs and select according to CD34, CD49f, eventually CD71 (read Villani et al., 2013). Of course, there are also transcription factors, but they are not usefull for selection...
I hope it helps
S.
Thanks a lot Stephanie. Actually I am going to work on human hair follicle bulge cells but I forgot to mention it in my question! So what about human? What are the positive markers for human?
I'd like to know an effective method for intraoperative isolation of mesenchymal stem cells for tissue rigeneration. In italy, national health system requires intraoperative isolation without expansion for stem cells transplant and tissue rigeneration. cells expansion is forbidden at today. can you help me to find the best isolation protocol? can you, altrenatively suggest me a device with which isolate stem cells? thank a lot dear collegues!
here is an article which can help you. So if you follow this, with FACS, you will enrich...
J Clin Invest. 2006 January 4; 116(1): 249–260.
Characterization and isolation of stem cell–enriched human hair follicle bulge cells
Manabu Ohyama,1 Atsushi Terunuma,1 Christine L. Tock,1 Michael F. Radonovich,2 Cynthia A. Pise-Masison,2 Steven B. Hopping,3 John N. Brady,2 Mark C. Udey,1 and Jonathan C. Vogel1
Abstract
The human hair follicle bulge is an important niche for keratinocyte stem cells (KSCs). Elucidation of human bulge cell biology could be facilitated by analysis of global gene expression profiles and identification of unique cell-surface markers. The lack of distinctive bulge morphology in human hair follicles has hampered studies of bulge cells and KSCs. In this study, we determined the distribution of label-retaining cells to define the human anagen bulge. Using navigated laser capture microdissection, bulge cells and outer root sheath cells from other follicle regions were obtained and analyzed with cDNA microarrays. Gene transcripts encoding inhibitors of WNT and activin/bone morphogenic protein signaling were overrepresented in the bulge, while genes responsible for cell proliferation were underrepresented, consistent with the existence of quiescent noncycling KSCs in anagen follicles. Positive markers for bulge cells included CD200, PHLDA1, follistatin, and frizzled homolog 1, while CD24, CD34, CD71, and CD146 were preferentially expressed by non-bulge keratinocytes. Importantly, CD200+ cells (CD200hiCD24loCD34loCD71loCD146lo) obtained from hair follicle suspensions demonstrated high colony-forming efficiency in clonogenic assays, indicating successful enrichment of living human bulge stem cells. The stem cell behavior of enriched bulge cells and their utility for gene therapy and hair regeneration will need to be assessed in in vivo assays.