Endometrial Derivation in a Human and Macaque Chimeric Bone Marrow Model

为了表征骨髓对子宫的贡献，我们研究了人类和灵长类外周血干细胞移植（PBSCT）模型的子宫内膜。 在移植自体绿色荧光蛋白（GFP）标记的CD 34+细胞后的恒河猴长期稳定的混合骨髓嵌合体模型中，进行子宫切开术以获得子宫内膜组织。 在贴壁基质培养物中观察到罕见的GFP+细胞（表明转导的造血干细胞或HSC衍生）;然而，没有一个细胞对间充质干细胞标记物CD 146呈双阳性。 类似地，当从该自体恒河猴模型检查骨髓时，在培养物中观察到罕见的GFP+细胞（指示转导的HSC衍生）。然而，GFP+细胞不是CD 146双阳性的，表明供体细胞仅植入骨髓的造血区室，而不是基质区室。 一只移植动物用于免疫组织化学（IHC）。 PDGFr β（CD 140 b），一种子宫内膜干细胞标记物，在子宫内膜中不与供体来源的（GFP）细胞共定位。 子宫内膜内的供体来源细胞不是CD 45+，这与先前的发现一致。 两只动物用于新鲜消化和FACS分析;尝试原代细胞培养，但GFP+分选的细胞都不能建立原代细胞培养物，而GFP-分选的级分容易建立原代培养物。 在人类中，评估了14名PBSCT接受者。 十个子宫内膜样本中有九个能够产生贴壁基质细胞培养。 子宫内膜和基质细胞表现出高水平的间充质干细胞表面标志物CD 146和PDGFr β。 由于新鲜的子宫内膜和骨髓样本有血液污染（已知是供体来源的），对这些样本进行微嵌合体检测将导致供体细胞的假阳性结果。 为了验证培养来自子宫内膜和骨髓培养物的基质细胞具有低水平的供体血细胞污染，我们通过FACS分析确定了对白色血细胞（WBC）标志物CD 45呈阳性的细胞的百分比。 在确认培养样品纯化了具有低水平WBC污染的感兴趣的基质区室之后，使用PCR分析样品的供体微嵌合体以检测供体和受体STR序列的百分比。 来自子宫内膜样本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<0.001）。 类似地，在骨髓组群中，与来自外周血细胞（阳性对照）样品的97.3%微嵌合体相比，来自骨髓样品的基质细胞的供体微嵌合体为0%（p<0.001）。 总之，PBSCT没有导致供体来源的子宫内膜基质细胞的植入。 我们的研究结果提示骨髓对子宫内膜干细胞龛没有贡献。

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.