PMID- 23644405
OWN - NLM
STAT- MEDLINE
DCOM- 20130927
LR  - 20240610
IS  - 1528-0012 (Electronic)
IS  - 0016-5085 (Print)
IS  - 0016-5085 (Linking)
VI  - 145
IP  - 2
DP  - 2013 Aug
TI  - Isolation and characterization of intestinal stem cells based on surface marker 
      combinations and colony-formation assay.
PG  - 383-95.e1-21
LID - S0016-5085(13)00701-4 [pii]
LID - 10.1053/j.gastro.2013.04.050 [doi]
AB  - BACKGROUND & AIMS: Identification of intestinal stem cells (ISCs) has relied 
      heavily on the use of transgenic reporters in mice, but this approach is limited 
      by mosaic expression patterns and difficult to directly apply to human tissues. 
      We sought to identify reliable surface markers of ISCs and establish a robust 
      functional assay to characterize ISCs from mouse and human tissues. METHODS: We 
      used immunohistochemistry, real-time reverse-transcription polymerase chain 
      reaction, and fluorescence-activated cell sorting (FACS) to analyze intestinal 
      epithelial cells isolated from mouse and human intestinal tissues. We compared 
      different combinations of surface markers among ISCs isolated based on expression 
      of Lgr5-green fluorescent protein. We developed a culture protocol to facilitate 
      the identification of functional ISCs from mice and then tested the assay with 
      human intestinal crypts and putative ISCs. RESULTS: CD44(+)CD24(lo)CD166(+) 
      cells, isolated by FACS from mouse small intestine and colon, expressed high 
      levels of stem cell-associated genes. Transit-amplifying cells and progenitor 
      cells were then excluded based on expression of GRP78 or c-Kit. 
      CD44(+)CD24(lo)CD166(+) GRP78(lo/-) putative stem cells from mouse small 
      intestine included Lgr5-GFP(hi) and Lgr5-GFP(med/lo) cells. Incubation of these 
      cells with the GSK inhibitor CHIR99021 and the E-cadherin stabilizer Thiazovivin 
      resulted in colony formation by 25% to 30% of single-sorted ISCs. CONCLUSIONS: We 
      developed a culture protocol to identify putative ISCs from mouse and human 
      tissues based on cell surface markers. CD44(+)CD24(lo)CD166(+), GRP78(lo/-), and 
      c-Kit(-) facilitated identification of putative stem cells from the mouse small 
      intestine and colon, respectively. CD44(+)CD24(-/lo)CD166(+) also identified 
      putative human ISCs. These findings will facilitate functional studies of mouse 
      and human ISCs.
CI  - Copyright (c) 2013 AGA Institute. Published by Elsevier Inc. All rights reserved.
FAU - Wang, Fengchao
AU  - Wang F
AD  - Stowers Institute for Medical Research, Kansas City, Missouri 64110, USA.
FAU - Scoville, David
AU  - Scoville D
FAU - He, Xi C
AU  - He XC
FAU - Mahe, Maxime M
AU  - Mahe MM
FAU - Box, Andrew
AU  - Box A
FAU - Perry, John M
AU  - Perry JM
FAU - Smith, Nicholas R
AU  - Smith NR
FAU - Lei, Nan Ye
AU  - Lei NY
FAU - Davies, Paige S
AU  - Davies PS
FAU - Fuller, Megan K
AU  - Fuller MK
FAU - Haug, Jeffrey S
AU  - Haug JS
FAU - McClain, Melainia
AU  - McClain M
FAU - Gracz, Adam D
AU  - Gracz AD
FAU - Ding, Sheng
AU  - Ding S
FAU - Stelzner, Matthias
AU  - Stelzner M
FAU - Dunn, James C Y
AU  - Dunn JC
FAU - Magness, Scott T
AU  - Magness ST
FAU - Wong, Melissa H
AU  - Wong MH
FAU - Martin, Martin G
AU  - Martin MG
FAU - Helmrath, Michael
AU  - Helmrath M
FAU - Li, Linheng
AU  - Li L
LA  - eng
GR  - U01 DK085535/DK/NIDDK NIH HHS/United States
GR  - R01 DK091427/DK/NIDDK NIH HHS/United States
GR  - U01DK85532/DK/NIDDK NIH HHS/United States
GR  - U01 DK085507/DK/NIDDK NIH HHS/United States
GR  - U24 DK085532/DK/NIDDK NIH HHS/United States
GR  - U01DK085507/DK/NIDDK NIH HHS/United States
GR  - U01 DK085547/DK/NIDDK NIH HHS/United States
GR  - U01 DK085532/DK/NIDDK NIH HHS/United States
GR  - U01DK085547/DK/NIDDK NIH HHS/United States
GR  - U01DK85535/DK/NIDDK NIH HHS/United States
GR  - U01 DK085525/DK/NIDDK NIH HHS/United States
GR  - U01DK85525/DK/NIDDK NIH HHS/United States
PT  - Journal Article
PT  - Research Support, N.I.H., Extramural
PT  - Research Support, Non-U.S. Gov't
DEP - 20130502
PL  - United States
TA  - Gastroenterology
JT  - Gastroenterology
JID - 0374630
RN  - 0 (Activated-Leukocyte Cell Adhesion Molecule)
RN  - 0 (Antigens, Surface)
RN  - 0 (CD24 Antigen)
RN  - 0 (Endoplasmic Reticulum Chaperone BiP)
RN  - 0 (HSPA5 protein, human)
RN  - 0 (Heat-Shock Proteins)
RN  - 0 (Hspa5 protein, mouse)
RN  - 0 (Hyaluronan Receptors)
RN  - EC 2.7.10.1 (Proto-Oncogene Proteins c-kit)
SB  - IM
MH  - Activated-Leukocyte Cell Adhesion Molecule/metabolism
MH  - Adult Stem Cells/*metabolism
MH  - Animals
MH  - Antigens, Surface/*metabolism
MH  - CD24 Antigen/metabolism
MH  - Cell Culture Techniques
MH  - Colon/cytology
MH  - Colony-Forming Units Assay
MH  - Endoplasmic Reticulum Chaperone BiP
MH  - Flow Cytometry
MH  - Heat-Shock Proteins/genetics
MH  - Humans
MH  - Hyaluronan Receptors/metabolism
MH  - Intestinal Mucosa/*cytology
MH  - Intestine, Small/cytology
MH  - Mice
MH  - Proto-Oncogene Proteins c-kit/metabolism
PMC - PMC3781924
MID - NIHMS499368
OTO - NOTNLM
OT  - CBC
OT  - CFE
OT  - CoSC
OT  - Differentiation
OT  - FACS
OT  - Flow Cytometry Analysis
OT  - GRP
OT  - IEC
OT  - IHC
OT  - ISC
OT  - PC
OT  - Paneth cell
OT  - Single-Cell Sorting
OT  - Stemness
OT  - colonic stem cell
OT  - colony-forming efficiency
OT  - crypt base columnar
OT  - fluorescence-activated cell sorting
OT  - green fluorescent protein
OT  - immunohistochemistry
OT  - intestinal epithelial cell
OT  - intestinal stem cell
OT  - qRT-PCR
OT  - quantitative reverse-transcription polymerase chain reaction
EDAT- 2013/05/07 06:00
MHDA- 2013/09/28 06:00
PMCR- 2014/08/01
CRDT- 2013/05/07 06:00
PHST- 2012/11/11 00:00 [received]
PHST- 2013/04/10 00:00 [revised]
PHST- 2013/04/19 00:00 [accepted]
PHST- 2013/05/07 06:00 [entrez]
PHST- 2013/05/07 06:00 [pubmed]
PHST- 2013/09/28 06:00 [medline]
PHST- 2014/08/01 00:00 [pmc-release]
AID - S0016-5085(13)00701-4 [pii]
AID - 10.1053/j.gastro.2013.04.050 [doi]
PST - ppublish
SO  - Gastroenterology. 2013 Aug;145(2):383-95.e1-21. doi: 
      10.1053/j.gastro.2013.04.050. Epub 2013 May 2.