Lynch Maureen R, Gasson Judith C, Paz Helicia
Department of Medicine, Hematology-Oncology, University of California, Los Angeles, USA.
J Vis Exp. 2011 Jun 7(52):2559. doi: 10.3791/2559.
The in vitro differentiation of ES cells towards a hematopoietic cell fate is useful when studying cell populations that are difficult to access in vivo and for characterizing the earliest genes involved in hematopoiesis, without having to deal with embryonic lethalities. The ES/OP9 co-culture system was originally designed to produce hematopoietic progeny, without the over production of macrophages, as the OP9 stromal cell line is derived from the calvaria of osteopetrosis mutant mice that lack functional M-CSF. The in vitro ES/OP9 co-culture system can be used in order to recapitulate early hematopoietic development. When cultured on OP9 stromal cells, ES cells differentiate into Flk-1+ hemangioblasts, hematopoietic progenitors, and finally mature, terminally differentiated lineages. The standard ES/OP9 co-culture protocol entails the placement of ES cells onto a confluent layer of OP9 cells; as well as, periodic replating steps in order to remove old, contaminating OP9 cells. Furthermore, current protocols involve evaluating only the hematopoietic cells found in suspension and are not optimized for evaluation of ES-derived progeny at each day of differentiation. However, with replating steps and the harvesting of only suspension cells one potentially misses a large portion of ES-derived progeny and developing hematopoietic cells. This issue becomes important to address when trying to characterize hematopoietic defects associated with knockout ES lines. Here we describe a modified ES/mStrawberry OP9 co-culture, which allows for the elimination of contaminating OP9 cells from downstream assays. This method allows for the complete evaluation of all ES-derived progeny at all days of co-culture, resulting in a hematopoietic differentiation pattern, which more directly corresponds to the hematopoietic differentiation pattern observed within the embryo.
在研究体内难以获取的细胞群体以及表征造血过程中最早涉及的基因时，胚胎干细胞（ES细胞）向造血细胞命运的体外分化很有用，且无需应对胚胎致死问题。ES/OP9共培养系统最初设计用于产生造血后代，而不会过度产生巨噬细胞，因为OP9基质细胞系源自缺乏功能性巨噬细胞集落刺激因子（M-CSF）的骨硬化突变小鼠的颅骨。体外ES/OP9共培养系统可用于重现早期造血发育过程。当在OP9基质细胞上培养时，ES细胞会分化为Flk-1+成血管细胞、造血祖细胞，最终分化为成熟的终末分化谱系。标准的ES/OP9共培养方案包括将ES细胞接种到汇合的OP9细胞层上；以及定期传代步骤，以去除老化的、污染的OP9细胞。此外，当前的方案仅涉及评估悬浮液中的造血细胞，并未针对分化各阶段ES来源后代的评估进行优化。然而，通过传代步骤且仅收集悬浮细胞，可能会遗漏很大一部分ES来源的后代和正在发育的造血细胞。在试图表征与基因敲除ES细胞系相关的造血缺陷时，解决这个问题变得很重要。在此，我们描述了一种改良的ES/mStrawberry OP9共培养方法，该方法可从下游分析中消除污染的OP9细胞。这种方法能够在共培养的所有天数对所有ES来源的后代进行全面评估，从而产生一种造血分化模式，该模式更直接地对应于胚胎内观察到的造血分化模式。
