Spheroid Coculture of Hematopoietic Stem/Progenitor Cells and Monolayer Expanded Mesenchymal Stem/Stromal Cells in Polydimethylsiloxane Microwells Modestly Improves In Vitro Hematopoietic Stem/Progenitor Cell Expansion.
Spheroid Coculture of Hematopoietic Stem/Progenitor Cells and Monolayer Expanded Mesenchymal Stem/Stromal Cells in Polydimethylsiloxane Microwells Modestly Improves In Vitro Hematopoietic Stem/Progenitor Cell Expansion.
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DOI：
10.1089/ten.tec.2016.0329
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发表时间：
2017-04
期刊：
影响因子：
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通讯作者：
Doran MR
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文献类型：
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作者：
Futrega K;Atkinson K;Lott WB;Doran MR
虽然已经显示间充质干细胞/基质细胞（MSC）的二维（2D）单层在体外增强造血干细胞/祖细胞（HSPC）扩增，但扩增的细胞不会长期植入人受体中。这一结果归因于2D培养未能重现骨髓（BM）龛信号环境。在此，我们评估了一种新型三维（3D）共培养系统支持HSPC体外扩增的能力。使用高通量聚二甲基硅氧烷（PDMS）微孔平台来制造数千个均匀的3D多细胞共培养球体。在3D球状体与2D贴壁BM来源的MSC培养物中的相对基因表达被表征并与文献报道进行比较。我们评估了共培养球体，每个球体含有25-400个MSC和10个脐带血（CB）衍生的CD 34+祖细胞。在低外源性细胞因子浓度下，2D和3D MSC共培养适度改善了整体造血细胞和CD 34+细胞扩增结果。相比之下，在PDMS微孔培养物中观察到CD 34 + CD 38 −细胞产量的大幅增加，无论是否存在MSC。这一结果表明，即使没有MSC共培养支持，单独使用微孔平台也可以提高CD 34 + CD 38 −细胞培养产量。我们发现，在PDMS微孔培养物中观察到的CD 34 + CD 38 −细胞产量的增加并没有转化为NOD/SCID γ（NSG）小鼠中的移植增强或移植小鼠中建立的相对人类造血谱系的修饰。总之，2D或3D共培养的CD 34+细胞产量没有统计学差异，MSC共培养支持在任一几何结构中仅提供适度的益处。虽然高通量3D微孔平台可以为研究共培养中的细胞提供有用的模型系统，但需要进一步优化以产生适用于临床应用的HSPC产量。
While two-dimensional (2D) monolayers of mesenchymal stem/stromal cells (MSCs) have been shown to enhance hematopoietic stem/progenitor cell (HSPC) expansion in vitro, expanded cells do not engraft long term in human recipients. This outcome is attributed to the failure of 2D culture to recapitulate the bone marrow (BM) niche signal milieu. Herein, we evaluated the capacity of a novel three-dimensional (3D) coculture system to support HSPC expansion in vitro. A high-throughput polydimethylsiloxane (PDMS) microwell platform was used to manufacture thousands of uniform 3D multicellular coculture spheroids. Relative gene expression in 3D spheroid versus 2D adherent BM-derived MSC cultures was characterized and compared with literature reports. We evaluated coculture spheroids, each containing 25–400 MSCs and 10 umbilical cord blood (CB)-derived CD34+ progenitor cells. At low exogenous cytokine concentrations, 2D and 3D MSC coculture modestly improved overall hematopoietic cell and CD34+ cell expansion outcomes. By contrast, a substantial increase in CD34+CD38− cell yield was observed in PDMS microwell cultures, regardless of the presence or absence of MSCs. This outcome indicated that CD34+CD38− cell culture yield could be increased using the microwell platform alone, even without MSC coculture support. We found that the increase in CD34+CD38− cell yield observed in PDMS microwell cultures did not translate to enhanced engraftment in NOD/SCID gamma (NSG) mice or a modification in the relative human hematopoietic lineages established in engrafted mice. In summary, there was no statistical difference in CD34+ cell yield from 2D or 3D cocultures, and MSC coculture support provided only modest benefit in either geometry. While the high-throughput 3D microwell platform may provide a useful model system for studying cells in coculture, further optimization will be required to generate HSPC yields suitable for use in clinical applications.