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L. Crisafulli et al.
Table 1. Analyzed and/or sorted cell populations. Cell
Acronyms
or abbreviation
LKS HSC
MPP
CLP
CMP GMP MEP Pre-MegE
Pre-E
MkP
From R1 stage to R41
PMN
ProB
PreB B imm B mat NK
Phenotype
Lin–/c-Kithi/Sca1hi
LKS/CD34–/Flk2– for miRNA
profiling; LKS/CD48–/CD150+/Flk2– for
analysis of transplanted mice; Sca1hi/CD48– CD150+-/EPCR+ for in vitro differentiation experiments
LKS/CD34+/Flk2- for miRNA profiling; LKS/CD127-/CD34+/Flk2int or Flk2high
for all other analysis Lin–/CD127+/Flk2high/c-Kitint/Sca1int Lin–/c-Kit+/Sca1-/CD34+/CD16/32- Lin–/c-Kit+/Sca1-/CD34+//CD16/32high Lin–/c-Kit+/Sca1-/CD34–/CD16/32– Lin–/c-Kit+/Sca1-/CD41–/CD16/32–/CD105–/CD150+
Lin–/c-Kit+/Sca1–/CD41–/CD16/32–/CD105+/ Lin–/c-Kit+/Sca1–/CD150+/CD41+/ Ter119–/CD44high (R1); Ter119+/CD44high
or CD44int or CD44– (R2 to R4) CD11b+/Ly6Ghigh
CD11b–/CD31+/Ly6C– CD11b–/CD31+/Ly6C+ CD11b+/CD31–/Ly6C+ NK1.1–/CD3–/CD43+/B220+ NK1.1–/CD3–/CD43–/B220+/IgM– NK1.1–/CD3–/CD43–/B220+/IgM+ NK1.1–/CD3–/CD43–/B220high /IgM+ NK1.1+/CD3–
CD3+/ NK1.1–
Stem and progenitor cells
Lineage restricted progenitors
Myeloid precursors and mature cells
Lymphoid precursors
and mature cells
population
Hematopoietic Stem
and Progenitor Cells Hematopoietic Stem Cells
Multi-Potent Progenitors
(at different stages of maturation)
Common Lymphoid Progenitors
Common Myeloid Progenitors Granulocyte/Macrophage Progenitors Megakaryocyte/Erythrocyte Progenitors Megakaryocyte and
Erythrocyte Precursors
Erythrocyte Precursors
(at different stages of maturation) Poly-Morpho-Nucleated Leukocytes Monoblasts
Pro-Monocyte
Monocyte
Pro-B
Pre-B
Immature B cells Mature B cells Natural Killer
T cells
1Modified from Liu et al..46 Monoclonal antibodies are listed in Online Supplementary Table S4.
Importantly, the expression of most of the DE miRNA changed more than 2-fold. Of note, the most up-regulated miRNA in the normal HSC-to-MPP transition is miR-221 (Online Supplementary Figure S1). This represents a posi- tive control of our analysis, since miR-221 has been pre- dicted to be an important regulator of HSC maturation due to its ability to repress cKit.15 We compared the lists of DE miRNA with those of DE transcripts obtained by microarray.4 None of the DE miRNA between mutant HSC or Flk2–MPP and corresponding controls are located within a transcript detected in the microarray, indicating that their different level of expression is not the result of the level of expression of a host gene.
Among the 23 over-lapping miRNA (Figure 1C), we applied stringent criteria to select a few candidates for subsequent studies. We considered only miRNA that: 1) were DE also by applying a different normalization method; 2) had a fold difference higher than five; 3) had anti-correlated predicted targets (PT) among previously described DE mRNA in Pbx1-deficient HSC;4 4) were DE also with independent real-time PCR in wild-type (wt) samples. Only three miRNA candidates fulfilled each
condition, all evolutionary conserved and all down-regu- lated (miR-127-3p, miR-411-5p, miR-34b-3p). Among these, the most DE both in Pbx1-deficient HSC and in the normal HSC-to-MPP transition is miR-127-3p (Online Supplementary Table S3). This miRNA was therefore cho- sen as a candidate HSC– self-renewal regulator.
miR-127-3p is an HSC-specific miRNA
As further proof that miR-127-3p downregulation cor- relates with loss of self-renewal, we confirmed its DE in phenotypically-defined HSC from a different previously described Pbx1-cKO mouse model (Tie2Cre+Pbx1-/f)4,5 (Figure 2A).
We then prospectively isolated HSC and Flk2-MPP from wt mice and compared the expression level of miR- 127-3p to that of other miRNA previously associated to HSC, such as miR-99b, miR-125, let-7, miR-221 and miR- 126.11,15-19 We found that miR-127-3p expression in HSC is not particularly high, being similar to that of other miRNA already reported to play a role in HSC biology (Figure 2B). We confirmed the recently reported down- regulation of miR-99b in Flk2–MPP20 (Figure 2C).
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