Introduction The most commonly-used method to assess progenitors and hematopoietic stem cells (HSC) present in a graft is the enumeration of phenotypically-defined CD34þ cells. A number of manuscripts, editorials and letters published recently in the Journal of Hematotherapy have addressed standardization methods for CD34þ cell determination [1– 7]. Even though higher numbers of CD34þ cells in the graft correlate with earlier white blood cell (WBC) engraftment and platelet engraftment [8,9] the CD34þ cell population is still extremely heterogeneous and contains a wide range of progenitors, from HSC to single-lineage committed progenitors. There is mounting interest in ex vivo manipulation of CD34þ cells, from ex vivo expansion of primitive progenitors and production of precursors, to the introduction of novel genetic material in HSC [10–14]. A number of culture systems have been described in which progenitors can be expanded and in which retroviral vectors can be introduced in CD34þ cells. However, transplantation of ex vivo manipulated progenitors in humans, as well as xenogeneic immunodeficient animals, has taught us that, although phenotypically-defined CD34þ cells or phenotypicallydefined primitive subsets of the CD34þ cell population can be maintained/expanded for several weeks, ex vivo manipulation for more than 4–7 days results in a dramatic loss of stem-cell capacity [15]. Although there is in vitro evidence that retroviruses can be introduced in > 50% of CD34þ cells, transplantation of such retrovirally-marked CD34þ cells in humans indicates that few, if any, vector-containing cells are detected after transplantation [16]. Therefore, the phenotypic characterization of primitive cells as CD34þ is far from sufficient and assays that can more accurately assess the fraction of progenitors with stem-cell characteristics within the CD34þ population are needed. A number of assays that evaluate CD34þ subpopulations which have more immature characteristics than the majority of the CD34þ population, are currently being used in research laboratories. In this overview, we will describe a number of assays developed in individual investigators’ laboratories that assess primitive human progenitors and, possibly, HSC. We have purposefully chosen to keep this review descriptive and not to recommend one or more assays to be adopted in the clinical setting for the following reasons: n First, assessment of the absolute frequency of primitive progenitors in a given cell population using similar assays in individual laboratories differs considerably. This may, in part, be due to minor differences in culture conditions and, in part, to differences in the interpretation of colony growth. Unless these culture assays are standardized, as has been done for the phenotypic determination of CD34þ cell number, comparison between different laboratories will remain difficult. n Second, it still remains to be determined which assays currently available accurately assess the number of repopulating HSC in a graft. n Third, the inability of the majority of the assays to provide a ‘real time’ answer to the question as to whether HSC are present or preserved, detracts from their clinical usefulness. However, comparisons between ‘stem cell content’ in a given cell population obtained in the research setting and clinical-transplantation results will eventually lead to improved clinical therapies.

中文翻译：

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ISHAGE 科学委员会报告测定血液或骨髓移植物中的造血干细胞含量

介绍 评估移植物中存在的祖细胞和造血干细胞 (HSC) 的最常用方法是枚举表型定义的 CD34+ 细胞。最近发表在《血液疗法杂志》上的许多手稿、社论和信件都涉及 CD34+ 细胞测定的标准化方法 [1-7]。尽管移植物中更多的 CD34+ 细胞与早期的白细胞 (WBC) 植入和血小板植入相关 [8,9]，但 CD34+ 细胞群仍然极其异质，并包含从 HSC 到单谱系的广泛祖细胞承诺的祖先。人们对 CD34+ 细胞的离体操作越来越感兴趣，从原始祖细胞的离体扩增和前体的产生，到在 HSC 中引入新的遗传物质 [10-14]。已经描述了许多培养系统，其中可以扩展祖细胞，并且可以在 CD34+ 细胞中引入逆转录病毒载体。然而，在人类以及异种免疫缺陷动物中移植体外操作的祖细胞，告诉我们，虽然表型定义的 CD34+ 细胞或 CD34+ 细胞群的表型定义的原始子集可以维持/扩展数周，体外操作超过 4-7 天会导致干细胞容量急剧下降 [15]。尽管有体外证据表明逆转录病毒可被引入 > 50% 的 CD34+ 细胞，但将这种带有逆转录病毒标记的 CD34+ 细胞移植到人体中表明，在移植后检测到的含有载体的细胞很少（如果有的话）[16]。所以，原始细胞的表型表征为 CD34þ 还远远不够，需要能够更准确地评估 CD34þ 群体中具有干细胞特征的祖细胞比例的分析。目前，研究实验室正在使用许多评估 CD34þ 亚群的分析，这些亚群比大多数 CD34þ 群体具有更多的不成熟特征。在本概述中，我们将描述在个别研究人员的实验室中开发的许多检测方法，这些检测方法评估原始人类祖细胞，可能还有 HSC。我们有意选择保持本综述的描述性，不推荐在临床环境中采用一种或多种检测方法，原因如下： n 首先，在各个实验室中使用类似测定对给定细胞群中原始祖细胞的绝对频率的评估有很大差异。这可能部分是由于培养条件的微小差异，部分是由于对菌落生长的解释不同。除非这些培养检测是标准化的，如 CD34+ 细胞数的表型测定那样，否则不同实验室之间的比较仍然很困难。n 其次，仍有待确定目前可用的哪些检测准确评估移植物中重新填充的 HSC 数量。n 第三，大多数检测无法对 HSC 是否存在或保留的问题提供“实时”答案，这有损于它们的临床实用性。然而，