Baumert B, Grymuła K, Pietruszka D, Kotowski M, Mielczarek M, Dziedziejko V, Hałasa M, Czerny B, Walczak M, Machaliński B
Department of General Pathology, Pomeranian Medical University, Szczecin, Poland.
Folia Histochem Cytobiol. 2008;46(3):299-305. doi: 10.2478/v10042-008-0045-0.
The transplantation of hematopoietic stem and progenitor cells (HSPC) is an established lifesaving therapy. Bone marrow (BM), harvested from heparinized cadaveric organ donors, peripheral blood (PB) and cord blood (CB), are important sources of hematopoietic stem cells. HSPCs, which are used for transplantation purposes, are routinely evaluated in terms of number of mononuclear cells (MNCs), CD34+ MNCs count and viability. The efficacy of grafting is determined additionally in clonogenic tests in vitro. These tests deliver important information about the number of HSPCs and their proliferative potential. Unfortunately, they do not give a possibility to evaluate the functional HSPC chemotactic reactivity in the SDF-1 gradient, which is probably the key phenomenon for HSPC homing after transplantation procedure. Thus, the aim of our study was to optimize HSPC isolation according to their chemotactic reactivity in SDF-1 gradient. Using multiparameter cell sorter (FACS Aria, BD) we examined the HSPCs attracted by SDF-1 on a single cell level. The population of cells which participated in the chemotactic process was highly enriched in CXCR4+lin-AC133+CD45+ cells (referred as hematopoietic stem cells) and to our surprise in CXCR4+lin-AC133+CD45- cells (referred as pluripotent stem cells) in quantitative amounts. Since reactivity of HSPCs may depend on various factors involved in the protocol of their isolation and short-term storage, we tested the most commonly used anticoagulants (ACD, CPDA-1, EDTA and Heparin) and culture media (DME, IMDM, RPMI). HSPCs, harvested from CB, PB and BM, were subsequently investigated for clonogenic growth of CFU-GM in methylcellulose cultures and for the level of apoptosis by employing annexin V staining. Evaluating clonogenic potential, ability of chemotactic reactivity in SDF-1 gradient and intensification of apoptosis of HSPC as the most safe anticoagulant and medium were selected. This study has proved that chemotactic reactivity of HSPCs is a new but very important parameter which should be included in the procedure of their isolation.
造血干细胞和祖细胞（HSPC）移植是一种成熟的挽救生命的治疗方法。从肝素化尸体器官供体采集的骨髓（BM）、外周血（PB）和脐带血（CB）是造血干细胞的重要来源。用于移植的HSPC通常根据单核细胞（MNC）数量、CD34 + MNC计数和活力进行评估。此外，通过体外克隆形成试验来确定移植的疗效。这些试验提供了有关HSPC数量及其增殖潜力的重要信息。不幸的是，它们无法评估HSPC在SDF-1梯度中的功能性趋化反应性，而这可能是移植后HSPC归巢的关键现象。因此，我们研究的目的是根据HSPC在SDF-1梯度中的趋化反应性来优化HSPC的分离。使用多参数细胞分选仪（FACS Aria，BD），我们在单细胞水平上检测了被SDF-1吸引的HSPC。参与趋化过程的细胞群体在CXCR4 + lin-AC133 + CD45 +细胞（称为造血干细胞）中高度富集，令我们惊讶的是，在CXCR4 + lin-AC133 + CD45-细胞（称为多能干细胞）中也有大量富集。由于HSPC的反应性可能取决于其分离和短期储存方案中涉及的各种因素，我们测试了最常用的抗凝剂（ACD、CPDA-1、EDTA和肝素）和培养基（DME、IMDM、RPMI）。随后，对从CB、PB和BM中采集的HSPC进行了甲基纤维素培养中CFU-GM的克隆生长研究，并通过膜联蛋白V染色检测了凋亡水平。通过评估克隆形成潜力、在SDF-1梯度中的趋化反应能力以及HSPC凋亡的增强情况，选择了最安全的抗凝剂和培养基。这项研究证明，HSPC的趋化反应性是一个新的但非常重要的参数，应纳入其分离过程中。
