Optimized Flow Cytometric Analysis of Central Nervous System Tissue Reveals Novel Functional Relationships Among Cells Expressing CD133, CD15, and CD24

Author:

Panchision David M.1,Chen Hui-Ling1,Pistollato Francesca1,Papini Daniela2,Ni Hsiao-Tzu3,Hawley Teresa S.4

Affiliation:

1. Center for Neuroscience Research, Children's Research Institute, Children's National Medical Center, Washington, DC, USA

2. Stem Cell Core Facility, Children's Research Institute, Children's National Medical Center, Washington, DC, USA

3. R&D Systems Inc., Minneapolis, Minnesota, USA

4. Flow Cytometry Core Facility, George Washington University Medical Center, Washington, DC, USA

Abstract

Abstract Although flow cytometry is useful for studying neural lineage relationships, the method of dissociation can potentially bias cell analysis. We compared dissociation methods on viability and antigen recognition of mouse central nervous system (CNS) tissue and human CNS tumor tissue. Although nonenzymatic dissociation yielded poor viability, papain, purified trypsin replacement (TrypLE), and two purified collagenase/neutral protease cocktails (Liberase-1 or Accutase) each efficiently dissociated fetal tissue and postnatal tissue. Mouse cells dissociated with Liberase-1 were titrated with antibodies identifying distinct CNS precursor subtypes, including CD133, CD15, CD24, A2B5, and PSA-NCAM. Of the enzymes tested, papain most aggressively reduced antigenicity for mouse and human CD24. On human CNS tumor cells, CD133 expression remained highest after Liberase-1 and was lowest after papain or Accutase treatment; Liberase-1 digestion allowed magnetic sorting for CD133 without the need for an antigen re-expression recovery period. We conclude that Liberase-1 and TrypLE provide the best balance of dissociation efficiency, viability, and antigen retention. One implication of this comparison was confirmed by dissociating E13.5 mouse cortical cells and performing prospective isolation and clonal analysis on the basis of CD133/CD24 or CD15/CD24 expression. Highest fetal expression of CD133 or CD15 occurred in a CD24hi population that was enriched in neuronal progenitors. Multipotent cells expressed CD133 and CD15 at lower levels than did these neuronal progenitors. We conclude that CD133 and CD15 can be used similarly as selectable markers, but CD24 coexpression helps to distinguish fetal mouse multipotent stem cells from neuronal progenitors and postmitotic neurons. This particular discrimination is not possible after papain treatment. Disclosure of potential conflicts of interest is found at the end of this article.

Publisher

Oxford University Press (OUP)

Subject

Cell Biology,Developmental Biology,Molecular Medicine

Reference65 articles.

1. The control of neural stem cells by morphogenic signals;Panchision;Curr Opin Genet Dev,2002

2. Single factors direct the differentiation of stem cells from the fetal and adult central nervous system;Johe;Genes Dev,1996

3. BMPs signal alternately through a SMAD or FRAP-STAT pathway to regulate fate choice in CNS stem cells;Rajan;J Cell Biol,2003

4. The avian embryo as a model in developmental studies: Chimeras and in vitro clonal analysis;Dupin;Curr Top Dev Biol,1998

5. Stem cell therapy for human neurodegenerative disorders-how to make it work;Lindvall;Nat Med,2004

同舟云学术

1.学者识别学者识别

2.学术分析学术分析

3.人才评估人才评估

"同舟云学术"是以全球学者为主线,采集、加工和组织学术论文而形成的新型学术文献查询和分析系统,可以对全球学者进行文献检索和人才价值评估。用户可以通过关注某些学科领域的顶尖人物而持续追踪该领域的学科进展和研究前沿。经过近期的数据扩容,当前同舟云学术共收录了国内外主流学术期刊6万余种,收集的期刊论文及会议论文总量共计约1.5亿篇,并以每天添加12000余篇中外论文的速度递增。我们也可以为用户提供个性化、定制化的学者数据。欢迎来电咨询!咨询电话:010-8811{复制后删除}0370

www.globalauthorid.com

TOP

Copyright © 2019-2024 北京同舟云网络信息技术有限公司
京公网安备11010802033243号  京ICP备18003416号-3