Efficient retroviral transduction of human B-lymphoid and myeloid progenitors: marked inhibition of their growth by the Pax5 transgene - PubMed (original) (raw)

Efficient retroviral transduction of human B-lymphoid and myeloid progenitors: marked inhibition of their growth by the Pax5 transgene

Rieko Sekine et al. Int J Hematol. 2008 May.

Abstract

We applied a coculture system for the genetic manipulation of human B-lymphoid and myeloid progenitor cells using murine bone marrow stromal cell support, and investigated the effects of forced Pax5 expression in both cell types. Cytokine-stimulated cord blood CD34+ cells could be transduced at 85% efficiency and 95% cell viability by a single 24-h infection with RD114-pseudotyped retroviral vectors, produced by the packaging cell line Plat-F and bicistronic vector plasmids pMXs-Ig, pMYs-Ig, or pMCs-Ig, encoding EGFP. Infected CD34+ cells were seeded onto HESS-5 cells in the presence of stem cell factor and granulocyte colony-stimulating factor, allowing the extensive production of B progenitors and granulocytic cells. We examined the cell number and CD34, CD33, CD19, and CD20 lambda and kappa expressions by flow cytometry. Ectopic expression of Pax5 in CD34+ cells resulted in small myeloid progenitors coexpressing CD33 and CD19 and inhibited myeloid differentiation. After 6 weeks, the number of Pax5-transduced CD19+ cells was 40-fold lower than that of control cells. However, the expression of CD20 and the kappa/lambda chain on Pax5-transduced CD19+ cells suggests that the Pax5 transgene may not interfere with their differentiation. This report is the first to describe the effects of forced Pax5 expression in human hematopoietic progenitors.

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Figures

Fig. 1

Fig. 1

a Sequential phenotypic analysis of in vitro B-cell differentiation examined by flow cytometry of CD34, CD19, CD20, and light chain (kappa or lambda) expression. The quadrate gate of two-color analysis was determined by each isotype control. One representative result of four repeat experiments is shown. b Kinetic analysis of B-lymphopoiesis-associated gene expression by QR-PCR for the transcription factors E2A, EBF, Pax5, and RAG1 proteins. The results are the mean ± SD of duplicate experiments

Fig. 2

Fig. 2

Structure of pMXs-Ig, pMYs-Ig, and pMCs-Ig retrovirus vectors. ψ packaging signal, Δ gag truncated gag sequence, LTR long terminal repeat, MCS multi-cloning site, IRES internal ribosomal entry site, EGFP enhanced green fluorescent protein, white box Mo-MLV LTR, gray box PCMV LTR, hatched box MPSV LTR

Fig. 3

Fig. 3

a The relationship between EGFP expression and the surface marker CD34 at the end of infection. Cells were stained with PE-conjugated anti-CD34 and analyzed by two-color FCM. b After a 4-day coculture, EGFP expression levels in CD33+ cells were compared between the three vectors by FCM, and cytospin preparations revealed an immature myeloid cell morphology. The photograph of pMCs-Ig cells shown is representative (×600). c Coculture at 28-days. Constitutive EGFP expression by pMXs, pMYs, and pMCs in mature myeloid cells (upper panels), and B-lymphoid cells (lower panels). The photograph of pMCs-Ig cells is representative (×600)

Fig. 4

Fig. 4

a Transduction rates evaluated by the percentage of EGFP+ cells and percentage of CD34+ cells after 4× repeat infection. The mean transduction rate and SD were 80.4 ± 6%, the mean percentage of CD34+ cells decreased to 75.8 ± 2.7%, and the percentage of EGFP+ cells in the CD34-positive gate was 86.3 ± 4.6% (n = 3). The data shown are from one representative experiment of three. b The stability of EGFP expression in pMCs-Ig-transduced cells was determined after 1, 6, and 9 weeks of coculture. Non-adherent cells in the culture medium on day 7 (upper). Almost all cells were developing into CD33+ myeloid cells. All of the cocultured cells were trypsinized on day 42 (middle) and day 63 (lower), developing into CD19+ B-lymphoid cells. In each case, the percentage of EGFP+ cells determined by FACS was above 98%

Fig. 5

Fig. 5

a White box total number of control colonies, black box total number of Pax5 colonies, slanted box BFU-E colony plus mixed colony numbers, gray box CFU-GM colony number. Three hundred control and Pax5 cells were seeded, and the colony number was counted 14 days later (the data shown are representative of four independent experiments). b The total weekly number of non-adherent cells, control cells (open circles) versus Pax5 cells (filled circles). The number of Pax5 cells was consistently lower than that of control cells. c Surface marker and morphology of myeloid cells after 1 week of coculture. EGFP+ myeloid cells were stained with PE-CD33 and APC-CD19 and examined by three-color FCM. A minor but significant fraction of non-adherent cells could be detected in the lymphoid gate of the cell population transduced with Pax5 vector, but not the control vector. M-G-stained cytospin preparations revealed the myelogenetic maturation arrest of Pax5 cells (×600). One representative result of three repeat experiments is shown. d Image capture with an inverted Nikon TMD300 microscope after 1 and 6 weeks of coculture (×400). The cobblestone area generated by Pax5 cells was markedly reduced. e Total cell number of control and Pax5 cells on day 42 (n = 3). f Living EGFP+ cells from the Pax5-transduced CD34+ cells expressed CD20 at a higher level than the control. A small number of EGFP+ cells also expressed the kappa light chain (these data are representative of three independent experiments)

Fig. 6

Fig. 6

a Cell cycle analysis using 70% ethanol fixation and the apoptosis assay, comparing Pax5 cells with control cells on days 7, 14, and 21. b Cell cycle analysis of EGFP-positive cells using formaldehyde fixation and the apoptosis assay, comparing Pax5 cells with control cells on day 28

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