First, we sorted out hematopoietic stem cells (HSCs) and hematopoietic stem and progenitor cells (HSPCs) from wildtype (WT) and Apc knockout (Apc KO, Apc^−/−) mice, which are Apc^fl/fl and Apc^fl/flMx1Cre respectively. The Apc deletion was induced by Poly(I:C) injection. Quantitative PCR (qPCR) demonstrated a significant decrease of Msi2 expression in the HSCs (lin⁻c-Kit⁺ Sca1⁺CD150⁺CD48⁻) and HSPCs (lin⁻c-Kit⁺) of Apc KO mice (Fig. 1B, C). To investigate the role of MSI2 in the WNT signaling pathway, MSI2 was re-expressed in Apc^−/− HSPCs by retrovirus infection, and western blot (WB) confirmed Msi2 was successfully overexpressed (Supplementary Fig. 1). The colony-forming assay showed that restoring MSI2 expression could partially rescue the diminished colony-forming ability observed in Apc^+/− and Apc^−/− cells (Fig. 1D, E). Next, we collected colony cells from the above colony-forming assay, and found that restoration of MSI2 expression can significantly reduce the high apoptosis rate induced by Apc KO (Fig. 1F and Supplementary Fig. 2).

To further investigate the relationship between MSI2 and WNT signaling pathway, we performed in vivo transplantation experiments following the restoration of MSI2 expression in Apc KO cells. Five days after the 5-FU injection, HSPCs from WT (Apc^fl/fl) or Apc^fl/fl Mx1Cre mice were infected with MigR1 vector or MSI2, and then were transplanted into CD45.1 recipient mice irradiated lethally (10 Gray). One month later, pIpC was injected intra-peritoneally at bi-daily intervals to induce the expression of Mx1Cre to knock out Apc. Seven days after the third pIpC injection, the mice were euthanized, and bone marrow cells were gathered for detection (Fig. 1G). The restoration of MSI2 expression considerably increased the populations of HSCs (Lin⁻c-Kit⁺Sca1⁺CD48⁻CD150⁺), LSK (Lin⁻c-Kit⁺Sca1⁺) and HPCs (Lin⁻c-Kit⁺Sca1⁻) compared to cells from Apc KO mice (Fig. 1H and Supplementary Fig. 3). Furthermore, the proportion of granulocyte-monocyte progenitors (GMP) and common myeloid progenitors (CMP) in the HPC population returned to normal. The megakaryocyte-erythroid progenitors (MEP) also changed, but not significantly (Fig. 1I and Supplementary Fig. 4). Apc deletion can induce a significant increase in apoptosis, which is one of the main reasons for the decline of HSPCs [7]. We found that the restoration of MSI2 expression can significantly inhibit the increase of apoptosis caused by Apc deletion (Fig. 1J and Supplementary Fig. 5A, B), suggesting that MSI2 can partially rescue the decline of HSPCs induced by Apc loss through inhibiting apoptosis. In addition to HSPCs, we also analyzed the changes in mature cells and found that the restoration of MSI2 expression significantly increased myeloid cells in both bone marrow and spleen (Fig. 1K and Supplementary Fig. 6A, B). Conversely, B cells in bone marrow (Supplementary Fig. 7A, B) and T cells in the thymus (Supplementary Fig. 8A, B) showed no significant alterations.

首先，我们从野生型 （WT） 和 Apc 敲除 （Apc KO， Apc ^−/- ） 小鼠中分选出造血干细胞 （HSC） 和造血干细胞和祖细胞 （HSPCs），它们分别是 Apc^fl/fl 和 Apc^fl/flMx1Cre。Apc 缺失由 Poly（I：C） 注射诱导。定量 PCR （qPCR） 显示 Apc KO 小鼠的 HSC （^{lin-c-Kit +} Sca1 ⁺ CD150 ⁺ CD48 ⁻） 和 HSPC （^{lin-c-Kit +} ） 中 Msi2 的表达显著降低（图 1B、C）。为了研究 MSI2 在 WNT 信号通路中的作用，通过逆转录病毒感染在 Apc^-/-HSPCs 中重表达 MSI2，Western blot （WB） 证实 Msi2 成功过表达（补充图 1）。集落形成测定表明，恢复 MSI2 表达可以部分挽救在 Apc ^{+ / -} 和 Apc ^{- /-} 细胞中观察到的降低的集落形成能力（图 1D、E）。接下来，我们从上述集落形成试验中收集集落细胞，发现 MSI2 表达的恢复可以显着降低 Apc KO 诱导的高细胞凋亡率（图 1F 和补充图 2）。

为了进一步研究 MSI2 与 WNT 信号通路之间的关系，我们在 Apc KO 细胞中恢复 MSI2 表达后进行了体内移植实验。注射 5-FU 后 5 天，用 MigR1 载体或 MSI2 感染来自 WT （Apc^fl/fl） 或 Apc^fl/fl Mx1Cre 小鼠的 HSPCs，然后移植到照射致死的 CD45.1 受体小鼠中 （10 Gray）。一个月后，每两天腹膜内注射 pIpC，诱导 Mx1Cre 的表达敲除 Apc。第三次 pIpC 注射后 7 天，对小鼠实施安乐死，并收集骨髓细胞进行检测（图 1G）。与 Apc KO 小鼠的细胞相比，MSI2 表达的恢复显着增加了 HSC （^{Lin-c-Kit +} Sca1 ⁺ CD48 ^- CD150 ⁺）、LSK （^{Lin-c-Kit +} Sca1 ⁺ ）和 HPC （^{Lin-c-Kit +} Sca1 ^- ）的数量（图 1H 和补充图 3）。此外，HPC 群体中粒细胞-单核细胞祖细胞 （GMP） 和共同髓系祖细胞 （CMP） 的比例恢复正常。巨核细胞-红系祖细胞 （MEP） 也发生了变化，但并不显着（图 1I 和补充图 4）。Apc 缺失可诱导细胞凋亡显著增加，这是 HSPCs 下降的主要原因之一 [7]。我们发现 MSI2 表达的恢复可以显着抑制 Apc 缺失引起的细胞凋亡增加 （图 1J 和补充图 5A、B），表明 MSI2 可以通过抑制细胞凋亡部分挽救 Apc 缺失诱导的 HSPCs 的下降。 除了 HSPCs，我们还分析了成熟细胞的变化，发现 MSI2 表达的恢复显着增加了骨髓和脾脏中的髓系细胞（图 1K 和补充图 6A、B）。相反，骨髓中的 B 细胞（补充图 7A、B）和胸腺中的 T 细胞（补充图 8A、B）没有显示显着改变。