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. 2013 Aug 8;122(6):1034-41.
doi: 10.1182/blood-2012-12-472308. Epub 2013 Jun 24.

LIN28B-mediated expression of fetal hemoglobin and production of fetal-like erythrocytes from adult human erythroblasts ex vivo

Y Terry Lee  1 , Jaira F de Vasconcellos, Joan Yuan, Colleen Byrnes, Seung-Jae Noh, Emily R Meier, Ki Soon Kim, Antoinette Rabel, Megha Kaushal, Stefan A Muljo, Jeffery L Miller
Affiliations

LIN28B-mediated expression of fetal hemoglobin and production of fetal-like erythrocytes from adult human erythroblasts ex vivo

Y Terry Lee et al. Blood. .

Abstract

Reactivation of fetal hemoglobin (HbF) holds therapeutic potential for sickle cell disease and β-thalassemias. In human erythroid cells and hematopoietic organs, LIN28B and its targeted let-7 microRNA family, demonstrate regulated expression during the fetal-to-adult developmental transition. To explore the effects of LIN28B in human erythroid cell development, lentiviral transduction was used to knockdown LIN28B expression in erythroblasts cultured from human umbilical cord CD34+ cells. The subsequent reduction in LIN28B expression caused increased expression of let-7 and significantly reduced HbF expression. Conversely, LIN28B overexpression in cultured adult erythroblasts reduced the expression of let-7 and significantly increased HbF expression. Cellular maturation was maintained including enucleation. LIN28B expression in adult erythroblasts increased the expression of γ-globin, and the HbF content of the cells rose to levels >30% of their hemoglobin. Expression of carbonic anhydrase I, glucosaminyl (N-acetyl) transferase 2, and miR-96 (three additional genes marking the transition from fetal-to-adult erythropoiesis) were reduced by LIN28B expression. The transcription factor BCL11A, a well-characterized repressor of γ-globin expression, was significantly down-regulated. Independent of LIN28B, experimental suppression of let-7 also reduced BCL11A expression and significantly increased HbF expression. LIN28B expression regulates HbF levels and causes adult human erythroblasts to differentiate with a more fetal-like phenotype.

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Figures

Figure 1
Figure 1
LIN28B regulates HbF levels in cultured human cord blood erythroblasts. (A) Detection of primary let-7d miRNA in erythroblasts cultured from human CD34+ cord blood cells after RNA IP with antibodies against LIN28B or control (rabbit IgG) with DNA ladder in the right lane shown for comparison. (B) LIN28B knockdown (LIN28B-KD) confirmation by qRT-PCR quantitation of copy number per nanogram complementary DNA (cDNA) (copies/ng cDNA). LIN28B-KD and control samples were evaluated. (C) The relative expression levels of the let-7 family of miRNAs were determined by qRT-PCR. Open bars represent control samples and black bars represent LIN28B-KD. Standard deviation bars are shown. *P < .05 in triplicate experiments. Hemoglobin profiles demonstrated by HPLC analysis are shown for (D) control and (E) LIN28B-KD cultures. HbF and HbA peaks are labeled on each graph (y-axis: mVolts; x-axis: elution time in minutes). C, empty vector control; KD, LIN28B knockdown; Pri-Let7d, primary let-7d miRNA.
Figure 2
Figure 2
LIN28B overexpression in adult human erythroblasts. LIN28B-OE was confirmed by (A) qRT-PCR quantitation of copy number per nanogram complementary DNA (cDNA) (copies/ng cDNA); (B) Western blot analysis and (C-F) confocal images of control and LIN28B-OE cells were stained with DAPI (4′,6-diamidino-2-phenylindole) (blue) and LIN28B (green). Cells were transfected with empty vector (C-D), and LIN28B (E-F). Analyses were performed at culture day 14. Mean value ± standard deviation of 3 independent donors for each condition. C, empty vector control; OE, LIN28B overexpression.
Figure 3
Figure 3
LIN28B overexpression enhances HbF levels in adult human erythroblasts. Flow cytometry analyses of (A) control and (B) LIN28B-OE at culture day 14, and (C) control, and (D) LIN28B-OE at culture day 21 stained with anti-transferrin receptor (CD71) and anti-glycophorin A (GPA) antibodies. Enucleation is represented by staining of culture day 21 cells with thiazole orange for (E) control and (F) LIN28B-OE. The enucleated cells were imaged in panels (G) control and (H) LIN28B-OE. HPLC analysis of hemoglobin from (I) control and (J) LIN28B-OE, and (K) control and (L) LIN28A-OE samples performed at culture day 21 shown for comparison with LIN28B-OE. HbF and HbA peaks are labeled on each graph (y-axis: mVolts; x-axis: elution time in minutes). Data are representative of more than 3 independent experiments. LIN28B-OE, LIN28B overexpression; LIN28A-OE, LIN28A overexpression.
Figure 4
Figure 4
LIN28B expression regulates γ-globin and erythroid-related fetal genes in adult erythroblasts. (A) α, μ, θ, and ζ globins, (B) β, δ, γ, and ε globins, (C) CA1, and (D) GCNT2 mRNA expression analysis of LIN28B-OE compared with control samples. Open bars represent control and black bars represent LIN28B-OE. qRT-PCR analyses were performed at culture day 14. Mean value ± standard deviation of 3 independent donors for each condition. P values were calculated using two-tailed Student t test. C, empty vector control; OE, LIN28B overexpression. *P < .05.
Figure 5
Figure 5
LIN28B modulation of adult CD34+ cells toward a fetal-like phenotype involves the let-7 family of miRNAs, miR-96, and BCL11A. LIN28B-OE compared with control samples in (A) the relative expression levels of the let-7 family of miRNAs (open bars represent control and black bars represent LIN28B-OE), the mRNA expression levels of (B) HMGA2 and (C) IGF2, (D) the relative expression levels of miR-96, miR-29c, miR-451, miR-144, and miR-142, and the mRNA expression of the transcription factors (E) BCL11A, (F) GATA1, (G) KLF1, and (H) SOX6. The qRT-PCR analyses were performed at culture day 14. The miRNAs relative expression levels (y-axis) in the control cells were defined as a level of one for comparison. Error bars denote ± standard deviation of 3 independent donors for each condition. P values were calculated using two-tailed Student t test. C, empty vector control; OE, LIN28B overexpression. *P < .05.
Figure 6
Figure 6
LIN28B is an upstream regulator of BCL11A. Western blot analyses with protein extracts from empty vector control (C) versus LIN28B-OE (OE) cells (A-B), or BCL11A knockdown (KD) cells (C-D). The blots were probed with anti-LIN28B, and anti-BCL11A as labeled at the left of each band pair. An anti-β-actin probe was used as a loading control.
Figure 7
Figure 7
Retroviral suppression of the let-7 family of miRNAs regulates BCL11A and HbF. (A) Relative expression levels of the let-7 family of miRNAs (open bars represent control, black bars represent LIN28B-OE, and hatch-marked bars represent let-7 sponge) after transduction of the let-7 sponge or LIN28B-OE encoding retrovirus. The qRT-PCR analyses were performed at culture day 14, and compared with control transductions. The miRNAs relative expression levels (y-axis) in the control cells were defined as a level of one for comparison. Error bars denote ± standard deviation of 3 independent donors for each condition. (B) Western blot analyses with protein extracts from the empty vector control (C) versus LIN28B-OE (OE) and let-7 sponge (SP) cells. The membranes were probed with anti-BCL11A. Anti-β-actin probe was used as a loading control. HPLC analyses of hemoglobin from (C) control, (D) LIN28B-OE, and (E) let-7 sponge samples were performed at culture day 21. HbF and HbA peaks are labeled on each graph (y-axis: mVolts; x-axis: elution time in minutes). Data are representative of 3 independent experiments. P values were calculated using two-tailed Student t test. C, empty vector control; OE, LIN28B overexpression; SP, let-7 sponge. *P < .05.
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