Demystifying SP cell purification: viability, yield, and phenotype are defined by isolation parameters

Montanaro, Federica; Liadaki, Kalliopi; Schienda, Jaclyn; Flint, Alan; Gussoni, Emanuela; Kunkel, Louis M.

doi:10.1016/j.yexcr.2004.04.010

Cited by 144 publications

(161 citation statements)

References 25 publications

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“…We labeled somitic cells using the methods described above, harvested and dissociated mature limb muscles, and determined the appropriate concentrations of Hoechst 33342 to identify SP cells (see Methods and ref. 29). Consistent with previous studies (29), we found that SP cells constitute 0.05-2.07% of the total mononuclear cells derived from avian and mouse limb muscle and were enriched in Sca1 (Tables 2 and 3, which are published as supporting information on the PNAS web site).…”

Section: Somitic Cells Give Rise To Satellite Cells In Both Chick Andsupporting

confidence: 92%

“…One possible source is bone marrow, as suggested by the expression of CD45, a pan marker of nucleated hematopoietic stem cells, found by some in muscle SP cells (9,28). However, the findings of others that muscle SP cells are CD45Ϫ (10,29) and not derived from labeled bone marrow transplants (30) argue against a bone marrow origin of muscle SP cells. Alternatively, it is reasonable to hypothesize, but not yet tested, that muscle SP cells may be derived from somites.…”

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confidence: 99%

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Somitic origin of limb muscle satellite and side population cells

Schienda

Engleka

Jun

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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Repair of mature skeletal muscle is mediated by adult muscle progenitors. Satellite cells have long been recognized as playing a major role in muscle repair, whereas side population (SP) cells have more recently been identified as contributing to this process. The developmental source of these two progenitor populations has been considerably debated. We explicitly tested and quantified the contribution of embryonic somitic cells to these progenitor populations. Chick somitic cells were labeled by using replicationdefective retroviruses or quail͞chick chimeras, and mouse cells were labeled by crossing somite-specific, Pax3-derived Cre driver lines with a Cre-dependent reporter line. We show that the majority of, if not all, limb muscle satellite cells arise from cells expressing Pax3 specifically in the hypaxial somite and their migratory derivatives. We also find that a significant number of, but not all, limb muscle SP cells are derived from the hypaxial somite. Notably, the heterogeneity in the developmental origin of SP cells is reflected in their functional heterogeneity; somitically derived SP cells are intrinsically more myogenic than nonsomitically derived ones. Thus, we show that the somites, which supply embryonic and fetal myoblasts, are also an important source of highly myogenic adult muscle progenitors.regeneration ͉ satellite cells ͉ somite

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Section: Somitic Cells Give Rise To Satellite Cells In Both Chick Andsupporting

confidence: 92%

mentioning

confidence: 99%

Somitic origin of limb muscle satellite and side population cells

Schienda

Engleka

Jun

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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189

174

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“…In our experiments, the percentages of CD34 þ cells in the Lin À SSC Low SP and Lin À SSC Low ALDH Br fractions were 72.6% AE 16.2% and 91.2% AE 13.1%, respectively. This important difference in CD34 þ cell enrichment was likely a result of the lineage depletion step that we incorporated into our protocol prior to SP/ALDH selection and that is not generally performed by others [25,27].…”

Section: Discussionmentioning

confidence: 99%

“…Hoechst staining was performed according to Goodell et al [25,26] and others [2,12,27,28], with slight modifications. Briefly, 10 6 Lin À cells/ml were suspended in prewarmed Dulbecco's modified Eagle's medium (Gibco, Cergy-Pontoise, France, http:// www.invitrogen.com) supplemented with 5% fetal calf serum and 10 mM HEPES.…”

Section: Facs Analysis and Cell Sortingmentioning

confidence: 99%

Dual SP/ALDH Functionalities Refine the Human Hematopoietic Lin−CD34+CD38− Stem/Progenitor Cell Compartment

et al. 2009

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Identification of prevalent specific markers is crucial to stem/ progenitor cell purification. Determinants such as the surface antigens CD34 and CD38 are traditionally used to analyze and purify hematopoietic stem/progenitor cells (HSCs/HPCs). However, the variable expression of these membrane antigens poses some limitations to their use in HSC/HPC purification. Techniques based on drug/stain efflux through the ATP-binding cassette (ABC)G2 pump (side population [SP] phenotype) or on detection of aldehyde dehydrogenase (ALDH) activity have been independently developed and distinguish the SP and ALDH Bright (ALDH Br ) cell subsets for their phenotype and proliferative capability. In this study, we developed a multiparametric flow cytometric method associating both SP and ALDH activities on human lineage negative (Lin 2 ) bone marrow cells and sorted different cell fractions according to their SP/ALDH activity level. We find that Lin 2 CD34 1 CD38 Low/2 cells are found throughout the spectrum of ALDH expression and are enriched especially in ALDH Br cells when associated with SP functionality (SP/ALDH Br fraction). Furthermore, the SP marker identified G 0 cells in all ALDH fractions, allowing us to sort quiescent cells regardless of ALDH activity. Moreover, we show that, within the Lin 2 CD34 1 CD38 2 ALDH Br population, the SP marker identifies cells with higher primitive characteristics, in terms of stemness-related gene expression and in vitro and in vivo proliferative potential, than the Lin 2 CD34 1 CD38 2 ALDH Br main population cells. In conclusion, our study shows that the coexpression of SP and ALDH markers refines the Lin 2 CD34 1 CD38 2 hematopoietic compartment and identifies an SP/ALDH Br cell subset enriched in quiescent primitive HSCs/HPCs.

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“…SP cells); their abundance are indicated by the number in the figure. Despite the initial excitment about using SP to identify CSCs, the ABCG2-highly expressing SP and ABCG2-negative non-SP tumor cells have been reported to be similarly tumorigenic (Patrawala et al, 2005). It is believed that the SP fraction obtained is not a pure stem-cell population, which is greatly affected by the isolation method (Montanaro et al, 2004). There were also report demonstrating that SP cells do not identify stem cell (Triel et al, 2004).…”

Section: Detection and Identification Of Cscsmentioning

confidence: 99%