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. 2012 Mar 9;335(6073):1195-200.
doi: 10.1126/science.1216557. Epub 2012 Feb 16.

Lin28b reprograms adult bone marrow hematopoietic progenitors to mediate fetal-like lymphopoiesis

Joan Yuan  1 , Cuong K Nguyen, Xiuhuai Liu, Chrysi Kanellopoulou, Stefan A Muljo
Affiliations

Lin28b reprograms adult bone marrow hematopoietic progenitors to mediate fetal-like lymphopoiesis

Joan Yuan et al. Science. .

Abstract

The immune system develops in waves during ontogeny; it is initially populated by cells generated from fetal hematopoietic stem cells (HSCs) and later by cells derived from adult HSCs. Remarkably, the genetic programs that control these two distinct stem cell fates remain poorly understood. We report that Lin28b is specifically expressed in mouse and human fetal liver and thymus, but not in adult bone marrow or thymus. We demonstrate that ectopic expression of Lin28 reprograms hematopoietic stem/progenitor cells (HSPCs) from adult bone marrow, which endows them with the ability to mediate multilineage reconstitution that resembles fetal lymphopoiesis, including increased development of B-1a, marginal zone B, gamma/delta (γδ) T cells, and natural killer T (NKT) cells.

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Conflict of interest statement

The authors declare no competing financial interests.

Figures

Fig. 1
Fig. 1
Differential let-7 and Lin28b expression in fetal and adult lymphocyte progenitors. Mouse expression data represent RNA of FACS-sorted populations pooled from at least three adult or neonatal mice or 12 FLs. (A) Graph shows relative expression of mature let-7 isomiRs in FL and adult BM pro-B cells (B220+CD43+IgM−veCD19+CD24+) determined by NanoString global miRNA-expression profiling. (B) Graph shows qRT-PCR analysis of mature let-7g and let-7c expression in pre-pro-B cells (B220+CD43+IgM−veCD19−veCD24−ve) from FL and adult BM. (C) Graph shows qRT-PCR analysis of Lin28b mRNA expression in FACS-sorted B cell precursor subsets from the indicated organs. Pre-pro-B (B220+CD43+IgM−veCD19−veCD24−ve), pro-B (B220+CD43+IgMveCD19+CD24+), pre-B (B220+CD43−veIgM−veCD19+CD24+), immature B (B220+CD43veIgM+CD19+CD24+). (D) Graph shows qRT-PCR analysis of Lin28b mRNA in sorted FL and BM HSPC populations: LSK (Lin−veSca-1+c-Kit+), CLP (Lin−ve c-Kitint Sca-1int CD127+). (E) Graph shows qRT-PCR analysis of Lin28b mRNA expression in FACS-sorted thymocyte subsets from mice of the indicated age. DN2/3 (CD4−ve CD8veCD25+CD44int), DN4 (CD4−veCD8−veCD25−veCD44−ve), γδ-T (γδ-TCR+ CD3+), DP (CD4+CD8+CD3lo), CD4SP (CD4+CD8−veCD3+). All thymocyte subsets except for the γδ-T cells were also gated through a γδ-TCR−ve CD1dPBS57−ve gate. (F) Graph shows qRT-PCR analysis of human Lin28b mRNA expression in the indicated fetal and adult organs: FL, FT, FS, CD34+ CB, BM, thymus (Thy), lymph nodes (LN), spleen (SPL). Human samples contain commercially obtained RNA pooled from at least 3 donors each. For all panels, error bars represent standard error of triplicate experimental replicates. n.d. indicates not detectable or below background signal level.
Fig. 2
Fig. 2
Lin28-mediated depletion of let-7 and multi-lineage reconstitution in Lin28-RV BM chimeras. (A) FACS plot shows representative frequency of GFP+ cells among lineage-depleted adult BM cells enriched in HSPCs 24 hours post-transduction with Lin28-RV. (B) Absolute numbers of GFP+ (green) and GFP−ve (white) CD19+ B and CD4+ and CD8+ T-lymphocyte subsets in lymph nodes of three Lin28-RV BM chimeras 6–8 weeks post-adoptive transfer are plotted. Data are representative of >5 independent reconstitution experiments. (C) Upper panel shows Lin28 western blot of total thymocyte lysate from GFP-RV and Lin28-RV BM chimeras. Lower panel shows tubulin western blot as a loading control. (D) Results of NanoString global miRNA-expression profiling analysis of FACS-sorted GFP+ and GFP−ve DP thymocytes (CD4+CD8+CD3lo) are shown. Normalized counts of individual mature miRNAs in each population are plotted on x and y-axis respectively (log scale). RNA was pooled from FACS-sorted populations of 3 individual Lin28-RV BM chimeras. (E) Graph shows qRT-PCR validation of mature let-7a and let-7g expression levels in GFP−ve and GFP+ DP thymocytes from Lin28-RV BM chimera. Error bars indicate standard error of triplicate experimental replicates.
Fig. 3
Fig. 3. Ectopic Lin28 expression in adult BM HSPCs confers fetal-like B cell development
Plots depict flow cytometric analyses of (A) CD19+ peritoneal cavity (PerC) and (C) B220+ splenic (SPL) B-cell subsets in recipients of adult BM HSPCs transduced with the indicated retroviral particles. (B) Graph shows percentages of B-2 (CD19+ B220hi CD5−ve), B-1a (CD19+ B220lo CD5+) and B-1b (CD19+ B220lo CD5−ve) B-lymphocytes among the GFP+ and GFP−ve fractions of peritoneal cavity CD19+ B cells in eight independent BM chimeras. (D) Graph shows percentages of marginal zone (MZ) (B220+ CD1d+ CD23−ve) and follicular B-2 (FoB) cells (B220+ CD1d−ve CD23+) among GFP+ and GFP−ve splenic CD19+ B cells in six independent BM chimeras. (E) FACS analysis show the presence of B-cells in the peritoneal cavity and spleen of BM chimeras reconstituted with Lin28-RV transduced Il7rα−/−BM HSPCs. Data are representative of two independent BM chimeras. ***P value<0.0001 represent statistical significance calculated by t-test. n.s indicates not statistically significant.
Fig. 4
Fig. 4. Ectopic Lin28 expression in adult BM HSPCs promotes the development of Vγ1.1+Vδ6.3+ T cells
(A) Flow cytometric analysis to enumerate γδ-TCR+ CD3+ cells in thymi of GFP-RV and Lin28-RV BM chimeras. (B) Graph shows the percentages of γδ-TCR+ CD3+ thymocytes among GFP+ and GFP−ve cells in the thymi of five independent Lin28-RV BM chimeras. *P value<0.05 represent statistical significance calculated by t-test. (C) Flow cytometric analysis shows distribution of surface CD4 expression among GFP+ γδ-TCR+ CD3+ thymocytes in the Lin28-RV (red line) and GFP-RV BM (blue line) chimeras. (D) Flow cytometric clonotype analysis of γγ-TCR+ CD3+ thymocytes in the GFP+ and GFP−ve fractions of the Lin28-RV BM chimeras.
Fig. 5
Fig. 5. Ectopic Lin28 expression in adult BM HSPCs promotes the development of iNKT cells
(A) Plots depict flow cytometric analysis of thymic iNKT cells defined by by double staining of CD1d tetramer loaded with PBS57 (CD1dPBS57+) and anti-CD3 in GFP-RV and Lin28-RV BM chimeras. (B) Graphs show the percentages of iNKT cells among the GFP+ and GFP−ve cells in the indicated organs from Lin28-RV BM chimeras. *P<0.05 **P<0.01 ***P<0.005. Indicated P-values represent statistical significance calculated by t-test. (C) Plots depict flow cytometric analysis of developmental stages 1–3 within the iNKT cell compartment among GFP+CD1dPBS57+CD3+ thymocytes in GFP-RV (left) and Lin28-RV BM chimeras (right). Stage 1 (CD44−veNK1.1−ve), 2 (CD44+NK1.1−ve) and 3 (CD44+NK1.1+) (D) Plots depict flow cytometric analysis of developmental stages 1–3 iNKT cells in the thymi of intact wild-type C57BL/6 mice of the indicated age. (E) Precursor frequency analysis show iNKT cell potential as a function of age. Left y-axis (blue line) indicates the ratio of stage 1 to DP thymocytes in intact C57BL/6 mice of the indicated age (equation shown above graph). Right y-axis (grey line) indicates the percentage of DP thymocytes.
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