Identifying States along the Hematopoietic Stem Cell Differentiation Hierarchy with Single Cell Specificity via Raman Spectroscopy.
Identifying States along the Hematopoietic Stem Cell Differentiation Hierarchy with Single Cell Specificity via Raman Spectroscopy.
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DOI：
10.1021/acs.analchem.5b02537
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发表时间：
2015-11-17
影响因子：
7.4
通讯作者：
Kraft ML
中科院分区：
文献类型：
--
作者：
Ilin Y;Choi JS;Harley BA;Kraft ML
体外扩增特定类型的造血细胞用于治疗血细胞病理的主要挑战是确定细胞和基质线索的组合，这些线索可指导造血干细胞（HSC）在体外自我更新或分化为细胞群。微型筛选平台能够最大限度地减少筛选对HSC命运决定的众多线索的影响所需的罕见HSC数量。这些平台创造了对无标签方法的强烈需求，这些方法可以准确地识别平台上特定位置的单个造血细胞的命运决定。我们展示了通过拉曼光谱的多变量分析，沿HSC分化层次识别离散细胞的能力。值得注意的是，细胞状态鉴定对单个细胞是准确的，并且独立于培养这些细胞的功能化聚丙烯酰胺凝胶的生物物理特性。我们报告单细胞拉曼光谱的偏最小二乘判别分析（PLS-DA）模型能够在HSC谱系规范中识别四种不同的造血细胞群。成功区分了长期再生的造血干细胞（lt - hsc）和其分化程度更高的后代，包括密切相关的短期再生的造血干细胞（st - hsc），以及完全分化的淋巴细胞（B细胞）和骨髓细胞（粒细胞）。这四个亚群细胞的谱系特异性分化状态被准确地识别出来，与底层生物材料底物的硬度无关，表明区分这些群体的细微光谱变化并未被培养底物的特征所掩盖。这种方法能够识别造血细胞在不同组成的生物材料基质上的谱系特异性分化阶段，并可能促进将造血细胞命运决定与引发它们的外部线索联系起来。
A major challenge for expanding specific types of hematopoietic cells ex vivo for the treatment of blood cell pathologies is identifying the combinations of cellular and matrix cues that direct hematopoietic stem cells (HSC) to self-renew or differentiate into cell populations ex vivo. Microscale screening platforms enable minimizing the number of rare HSCs required to screen the effects of numerous cues on HSC fate decisions. These platforms create a strong demand for label-free methods that accurately identify the fate decisions of individual hematopoietic cells at specific locations on the platform. We demonstrate the capacity to identify discrete cells along the HSC differentiation hierarchy via multivariate analysis of Raman spectra. Notably, cell state identification is accurate for individual cells and independent of the biophysical properties of the functionalized polyacrylamide gels upon which these cells are cultured. We report partial least-squares discriminant analysis (PLS-DA) models of single cell Raman spectra enable identifying four dissimilar hematopoietic cell populations across the HSC lineage specification. Successful discrimination was obtained for a population enriched for long-term repopulating HSCs (LT-HSCs) versus their more differentiated progeny, including closely-related short-term repopulating HSCs (ST-HSCs), and fully differentiated lymphoid (B cells) and myeloid (granulocytes) cells. The lineage-specific differentiation states of cells from these four sub-populations were accurately identified independent of the stiffness of the underlying biomaterial substrate, indicating subtle spectral variations that discriminated these populations were not masked by features from the culture substrate. This approach enables identifying the lineage-specific differentiation stages of hematopoietic cells on biomaterial substrates of differing composition, and may facilitate correlating hematopoietic cell fate decisions with the extrinsic cues that elicited them.