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A novel application for a 3-dimensional timelapse assay that distinguishes chemotactic from chemokinetic responses of hematopoietic CD133(+) stem/progenitor cells.

ABSTRACT: Efficient homing/mobilization of human hematopoietic stem/progenitor cells to/from bone marrow niches enhances their therapeutic efficacy. Additionally, homing is dependent on cell source and may be modulated by prior ex vivo cell expansion. Here, we describe a novel application of a 3-dimensional time-lapse method for assessing trafficking of individual human cord blood CD133(+) hematopoietic stem/progenitor cells in vitro, using the key chemokine CXCL12 as a paradigm. This new methodology allows distinction between chemotactic responses (displacement of center of mass and the forward migration index of the cells), and chemokinetic responses such as total cell path traveled in any direction (accumulated distance) and cell velocity in a 3-dimensional matrix. Other key advantages of this novel assay over existing assays include the ability to assess individual cell migration over times comparable to in vivo homing and rapid mobilization assays (18-24h) and to directly compare the strength or response of individual hematopoietic progenitor cells to different or competing stimuli and small molecule inhibitors in a single assay prior to analyses in vivo. Importantly, using this method, our results demonstrate definitively that CXCL12 regulates the chemotactic responses of human cord blood CD133(+) cells, but not their random migration or chemokinesis.

SUBMITTER: Pepperell EE

PROVIDER: S-EPMC3744817 | biostudies-literature |

REPOSITORIES: biostudies-literature

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CD133 expression in circulating hematopoietic progenitor cells.

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CD133 is a modifier of hematopoietic progenitor frequencies but is dispensable for the maintenance of mouse hematopoietic stem cells.

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CD133/CD140a-based isolation of distinct human multipotent neural progenitor cells and oligodendrocyte progenitor cells.

Project description:The mechanisms underlying the specification of oligodendrocyte fate from multipotent neural progenitor cells (NPCs) in developing human brain are unknown. In this study, we sought to identify antigens sufficient to distinguish NPCs free from oligodendrocyte progenitor cells (OPCs). We investigated the potential overlap of NPC and OPC antigens using multicolor fluorescence-activated cell sorting (FACS) for CD133/PROM1, A2B5, and CD140a/PDGFαR antigens. Surprisingly, we found that CD133, but not A2B5, was capable of enriching for OLIG2 expression, Sox10 enhancer activity, and oligodendrocyte potential. As a subpopulation of CD133-positive cells expressed CD140a, we asked whether CD133 enriched bone fide NPCs regardless of CD140a expression. We found that CD133(+)CD140a(-) cells were highly enriched for neurosphere initiating cells and were multipotent. Importantly, when analyzed immediately following isolation, CD133(+)CD140a(-) NPCs lacked the capacity to generate oligodendrocytes. In contrast, CD133(+)CD140a(+) cells were OLIG2-expressing OPCs capable of oligodendrocyte differentiation, but formed neurospheres with lower efficiency and were largely restricted to glial fate. Gene expression analysis further confirmed the stem cell nature of CD133(+)CD140a(-) cells. As human CD133(+) cells comprised both NPCs and OPCs, CD133 expression alone cannot be considered a specific marker of the stem cell phenotype, but rather comprises a heterogeneous mix of glial restricted as well as multipotent neural precursors. In contrast, CD133/CD140a-based FACS permits the separation of defined progenitor populations and the study of neural stem and oligodendrocyte fate specification in the human brain.

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