Effects of autologous stromal cells and cytokines on differentiation of equine bone marrow–derived progenitor cells

Ute E. Schwab Equine Immunology Laboratory, Department of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853.

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Rebecca L. Tallmadge Equine Immunology Laboratory, Department of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853.

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Mary Beth Matychak Equine Immunology Laboratory, Department of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853.

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M. Julia B. Felippe Equine Immunology Laboratory, Department of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853.

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DOI:
https://doi.org/10.2460/ajvr.78.10.1215
Volume/Issue:
Volume 78: Issue 10
Online Publication Date:
01 Oct 2017
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Abstract

OBJECTIVE To develop an in vitro system for differentiation of equine B cells from bone marrow hematopoietic progenitor cells on the basis of protocols for other species.

SAMPLE Bone marrow aspirates aseptically obtained from 12 research horses.

PROCEDURES Equine bone marrow CD34+ cells were sorted by use of magnetic beads and cultured in medium supplemented with cytokines (recombinant human interleukin-7, equine interleukin-7, stem cell factor, and Fms-like tyrosine kinase-3), murine OP9 stromal cell preconditioned medium, and equine fetal bone marrow mesenchymal stromal cell preconditioned medium. Cells in culture were characterized by use of flow cytometry, immunocytofluorescence microscopy, and quantitative reverse-transcriptase PCR assay.

RESULTS For these culture conditions, bone marrow–derived equine CD34+ cells differentiated into CD19+IgM+ B cells that expressed the signature transcription factors early B-cell factor and transcription factor 3. These conditions also supported the concomitant development of autologous stromal cells, and their presence was supportive of B-cell development.

CONCLUSIONS AND CLINICAL RELEVANCE Equine B cells were generated from bone marrow aspirates by use of supportive culture conditions. In vitro generation of equine autologous B cells should be of use in studies on regulation of cell differentiation and therapeutic transplantation.

Abstract

OBJECTIVE To develop an in vitro system for differentiation of equine B cells from bone marrow hematopoietic progenitor cells on the basis of protocols for other species.

SAMPLE Bone marrow aspirates aseptically obtained from 12 research horses.

PROCEDURES Equine bone marrow CD34+ cells were sorted by use of magnetic beads and cultured in medium supplemented with cytokines (recombinant human interleukin-7, equine interleukin-7, stem cell factor, and Fms-like tyrosine kinase-3), murine OP9 stromal cell preconditioned medium, and equine fetal bone marrow mesenchymal stromal cell preconditioned medium. Cells in culture were characterized by use of flow cytometry, immunocytofluorescence microscopy, and quantitative reverse-transcriptase PCR assay.

RESULTS For these culture conditions, bone marrow–derived equine CD34+ cells differentiated into CD19+IgM+ B cells that expressed the signature transcription factors early B-cell factor and transcription factor 3. These conditions also supported the concomitant development of autologous stromal cells, and their presence was supportive of B-cell development.

CONCLUSIONS AND CLINICAL RELEVANCE Equine B cells were generated from bone marrow aspirates by use of supportive culture conditions. In vitro generation of equine autologous B cells should be of use in studies on regulation of cell differentiation and therapeutic transplantation.

Contributor Notes

Address correspondence to Dr. Felippe (mbf6@cornell.edu).
Cover American Journal of Veterinary Research
American Journal of Veterinary Research
Publication Date:
01 Oct 2017

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