Micromarrows-Three-Dimensional Coculture of Hematopoietic Stem Cells and Mesenchymal Stromal Cells
Micromarrows-Three-Dimensional Coculture of Hematopoietic Stem Cells and Mesenchymal Stromal Cells
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DOI：
10.1089/ten.tec.2011.0159
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发表时间：
2012-05-01
影响因子：
3
通讯作者：
Doran, Michael
中科院分区：
文献类型：
--
作者：
Cook, Matthew M.;Futrega, Kathryn;Doran, Michael
造血干细胞(HSC)移植是治疗某些血液系统恶性肿瘤的有效方法。然而，从一些供体组织获得足够的HSC供应可能会限制其应用和最终疗效。这一局限性可以通过在移植前扩大HSC数量来克服的理论已经激励了许多实验室开发体外扩增过程。这一领域的开创性工作利用基质细胞作为支持细胞与HSC共培养，以模拟HSC的生态位。我们假设，通过将这种经典的共培养系统转换为三维(3D)结构，我们可以更好地复制利基环境，进而促进HSC的扩张。在这里，我们描述了一种新的高通量3D共培养系统，其中小鼠来源的HSC可以与间充质干细胞/基质细胞(MSC)在3D微聚体中共培养--我们称之为“微骨髓”。使用表面修饰的微孔形成微骨髓，并将其支持HSC扩增的能力与经典的二维(2D)共培养进行比较。虽然2D和3D系统在最少补充的培养液中都只提供适度的总细胞扩增，但微骨髓系统支持大约两倍于2D对照的HSC候选细胞的扩增。组织学显示，在第7天，大多数结合的造血细胞位于集合体的外层。定量聚合酶链式反应表明，保持在3D聚集体中的MSC比其2D等价物表达显著更高水平的关键造血生态位因子。因此，我们认为，微骨髓平台是迈向高通量HSC 3D共培养系统的有希望的第一步，该系统可以在体外重现HSC生态位，并随后在体外进行广泛的HSC自我更新。
Hematopoietic stem cell (HSC) transplant is a well established curative therapy for some hematological malignancies. However, achieving adequate supply of HSC from some donor tissues can limit both its application and ultimate efficacy. The theory that this limitation could be overcome by expanding the HSC population before transplantation has motivated numerous laboratories to develop ex vivo expansion processes. Pioneering work in this field utilized stromal cells as support cells in cocultures with HSC to mimic the HSC niche. We hypothesized that through translation of this classic coculture system to a three-dimensional (3D) structure we could better replicate the niche environment and in turn enhance HSC expansion. Herein we describe a novel high-throughput 3D coculture system where murine-derived HSC can be cocultured with mesenchymal stem/stromal cells (MSC) in 3D microaggregates-which we term "micromarrows." Micromarrows were formed using surface modified microwells and their ability to support HSC expansion was compared to classic two-dimensional (2D) cocultures. While both 2D and 3D systems provide only a modest total cell expansion in the minimally supplemented medium, the micromarrow system supported the expansion of approximately twice as many HSC candidates as the 2D controls. Histology revealed that at day 7, the majority of bound hematopoietic cells reside in the outer layers of the aggregate. Quantitative polymerase chain reaction demonstrates that MSC maintained in 3D aggregates express significantly higher levels of key hematopoietic niche factors relative to their 2D equivalents. Thus, we propose that the micromarrow platform represents a promising first step toward a high-throughput HSC 3D coculture system that may enable in vitro HSC niche recapitulation and subsequent extensive in vitro HSC self-renewal.