Epigenetic reprogramming induces the expansion of cord blood stem cells
Pratima Chaurasia, … , Sunita D’Souza, Ronald Hoffman
Pratima Chaurasia, … , Sunita D’Souza, Ronald Hoffman
Published April 24, 2014
Citation Information: J Clin Invest. 2014;124(6):2378-2395. https://doi.org/10.1172/JCI70313.
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Epigenetic reprogramming induces the expansion of cord blood stem cells

Abstract

Cord blood (CB) cells that express CD34 have extensive hematopoietic capacity and rapidly divide ex vivo in the presence of cytokine combinations; however, many of these CB CD34+ cells lose their marrow-repopulating potential. To overcome this decline in function, we treated dividing CB CD34+ cells ex vivo with several histone deacetylase inhibitors (HDACIs). Treatment of CB CD34+ cells with the most active HDACI, valproic acid (VPA), following an initial 16-hour cytokine priming, increased the number of multipotent cells (CD34+CD90+) generated; however, the degree of expansion was substantially greater in the presence of both VPA and cytokines for a full 7 days. Treated CD34+ cells were characterized based on the upregulation of pluripotency genes, increased aldehyde dehydrogenase activity, and enhanced expression of CD90, c-Kit (CD117), integrin α6 (CD49f), and CXCR4 (CD184). Furthermore, siRNA-mediated inhibition of pluripotency gene expression reduced the generation of CD34+CD90+ cells by 89%. Compared with CB CD34+ cells, VPA-treated CD34+ cells produced a greater number of SCID-repopulating cells and established multilineage hematopoiesis in primary and secondary immune–deficient recipient mice. These data indicate that dividing CB CD34+ cells can be epigenetically reprogrammed by treatment with VPA so as to generate greater numbers of functional CB stem cells for use as transplantation grafts.

Authors

Pratima Chaurasia, David C. Gajzer, Christoph Schaniel, Sunita D’Souza, Ronald Hoffman

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Figure 10

siRNA-mediated knock down of pluripotency genes.

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siRNA-mediated knock down of pluripotency genes. (A) PCs were treated wi...
(A) PCs were treated with VPA in SF cultures. After 3 days, cells were transfected with a pool of SOX2, OCT4, NANOG (SON), scrambled (negative control), and GAPDH siRNA (positive control) as described in Methods. SOX2, OCT4, NANOG, GAPDH, and ZIC3 transcripts were quantitated by SYBR Green qPCR and normalized to the level of CD34 transcripts *P < 0.05; **P ≤ 0.006; ***P = 0.0001 (mean ± SEM; ANOVA, P < 0.0001; n = 3). (B) Expression of SOX2, OCT4, NANOG, CD34, and GAPDH following siRNA-mediated knock down was analyzed by RT-PCR. M, DNA markers. ES cells were a positive control for SOX2, OCT4, NANOG, and ZIC3. Lanes were run on the same gel. (C) Pluripotency gene expression was analyzed by confocal microscopy. Upper panel: scrambled siRNA; lower panel: SON siRNA showing SOX2, OCT4, NANOG, and ZIC3 expression in VPA-treated cells. Images represent an optical section of confocal z-stack series. Scale bars: 10 μm (original magnification, ×40). Similar data were obtained in two additional experiments. (D) After 7 days of transfection, the percentage of cells that were CD34+ and CD34+CD90+ was analyzed using flow cytometry. Graph represents a comparative analysis of the percentage of CD34+ and CD34+CD90+ cells generated in VPA cultures after transfection with siRNA including scrambled and SON. *P ≤ 0.05 (mean ± SEM; ANOVA, P = 0.0005; n = 3). (E) Absolute numbers of CD34+ and CD34+CD90+ cells per CB collection generated in VPA-containing cultures following transfection with scrambled or SON siRNA were calculated. *P ≤ 0.05; ***P = 0.0001 (mean ± SEM; ANOVA, P = 0.0008; n = 3).