Endometrial Derivation in a Human and Macaque Chimeric Bone Marrow Model

为了确定骨髓对子宫的贡献，我们研究了人和灵长类外周血干细胞移植(PBSCT)模型中的子宫内膜。 在恒河猴长期稳定的混合骨髓嵌合模型中，用绿色荧光蛋白(GFP)标记的自体CD34+细胞移植后，剖宫产获取子宫内膜组织。在贴壁基质培养中观察到罕见的GFP+细胞(表明转导的造血干细胞或HSC来源)；然而，没有一例间充质干细胞标记CD146双重阳性。同样，当从这个自体恒河猴模型中检查骨髓时，在培养中观察到罕见的GFP+细胞(表明转导的HSC来源)。但GFP+细胞未见CD146双阳性，提示供者细胞仅植入造血室，而不植入骨髓基质室。 其中一只移植动物用于免疫组织化学(IHC)。子宫内膜干细胞标记物PDGFrbeta(CD140b)不与供体来源的(GFP)细胞在子宫内膜共定位。位于子宫内膜内的供体来源细胞不是CD45+，这与以前的发现一致。 两只动物用于新鲜消化和FACS分析；尝试了原代细胞培养，但没有一个GFP+分选的细胞能够建立原代细胞培养，而GFP分选的部分很容易建立原代培养。 在人类中，对14名接受PBSCT的人进行了评估。10个子宫内膜样本中有9个能够产生贴壁基质细胞培养。子宫内膜和间质细胞显示高水平的间充质干细胞表面标志CD146和PDGFrbeta。由于新鲜的子宫内膜和骨髓样本有血液污染(已知来自供体)，对这些样本进行微嵌合检测将导致供体细胞的假阳性结果。为了验证从子宫内膜和骨髓培养的基质细胞是否有较低的供体血细胞污染水平，我们通过FACS分析测定了白细胞(WBC)标记CD45阳性的细胞的百分比。 在确认培养的样本净化了感兴趣的间质间隔室，白细胞污染水平较低后，使用PCR分析供体微嵌合体，以检测供体和受体STR序列的百分比。来自子宫内膜的基质细胞的供体嵌合率为0%，而来自外周血细胞的嵌合率为91.2%(p&lt；0.001)。同样，在骨髓队列中，来自骨髓样本的基质细胞的供者微嵌合率为0%，而来自外周血细胞(阳性对照)样本的微嵌合率为97.3%(p&lt；0.001)。 综上所述，PBSCT没有导致供体来源的子宫内膜间质细胞植入。我们的研究结果表明，骨髓对子宫内膜干细胞的生态位没有贡献。

To characterize bone marrow contribution to the uterus, we studied the endometrium from human and primate peripheral blood stem cell transplant (PBSCT) models. In a Rhesus macaque long-term stable mixed bone marrow chimerism model after transplantation with autologous CD34+ cells labeled with green fluorescent protein (GFP), a hysterotomy was performed to obtain endometrial tissue. Rare GFP+ cells (indicating transduced hematopoeitic stem cell, or HSC,-derivation) were observed in adherent stromal culture; however, none were double positive for the mesenchymal stem cell marker CD146. Similarly, when bone marrow was examined from this autologous rhesus model, rare GFP+ cells (indicating transduced HSC-derivation) were observed in culture. However, GFP+ cells were not CD146 double positive indicating that donor cells only engrafted in the hematopoietic compartment, but not the stromal compartment of the bone marrow. One transplanted animal was used for immunohistochemistry (IHC). PDGFrbeta (CD140b), an endometrial stem cell marker, did not co-localize with donor-derived (GFP) cells in the endometrium. Donor derived cells residing within the endometrium were not CD45+, which is consistent with previous findings. Two animals were used for fresh digestion and FACS analysis; primary cell cultures were attempted, but none of the GFP+ sorted cells were able to establish a primary cell culture, while GFP- sorted fraction readily established primary cultures. In humans, 14 PBSCT recipients were assessed. Nine of ten endometrial samples were able to generate adherent stromal cells cultures. Endometrial and stromal cell exhibited high levels of mesenchymal stem cell surface markers CD146 and PDGFrbeta. As fresh endometrial and bone marrow samples have blood contamination (which is known to be donor derived), microchimerism testing on these samples would result in a false positive result for donor cells. To verify that culturing stromal cells from endometrium and bone marrow cultures had low levels of donor blood cell contamination, we determined the percentage of cells that were positive for the white blood cell (WBC) marker CD45 by FACS analysis. After confirming that culturing the samples purified the stromal compartment of interest, with low levels of WBC contamination, samples were analyzed for donor microchimerism using PCR to detect percentage of donor and recipient STR sequences. There was 0% donor chimerism of the stromal cells from the endometrial samples as compared to 91.2% chimerism from the peripheral blood cells (p<0.001). Similarly, in the bone marrow cohort, there was 0% donor microchimerism of the stromal cells from the bone marrow samples as compared to 97.3% microchimerism from the peripheral blood cell (positive control) samples (p<0.001). In summary, PBSCT did not result in the engraftment of donor derived endometrial stromal cells. Our results here suggest that the bone marrow does not contribute to the endometrial stem cell niche.