在脐血培养物中添加脂肪来源的干细胞可刺激其多能分化。

Addition of adipose-derived stem cells in cord blood cultures stimulates their pluripotent differentiation.

作者信息

Tsagias N, Kouzi-Koliakos K, Koliakos I, Kostidou E, Karagiannis V, Daniilidis A, Koliakos G

机构信息

3rd University Obstetrics and Gynaecology Clinic, Ippokration General Hospital, Medical School, Aristotle University Thessaloniki, Thessaloniki 54124, Greece.

出版信息

Transplant Proc. 2009 Dec;41(10):4340-4. doi: 10.1016/j.transproceed.2009.09.053.

DOI:10.1016/j.transproceed.2009.09.053

PMID:20005395

Abstract

INTRODUCTION

Adipose tissue is recognized as an important source of postnatal mesenchymal stem cells for generative medicine applications. Moreover, cord blood stem cells have been shown to contain pluripotent stem cells called unrestricted somatic stem cells (USSCs). However, this population is rare and cannot be generated from every cord blood sample. In this study, we have presented a new method of co-culture of adipose-derived stem cells (ADPCs) and cord blood stem cells that results in pluripotent differentiation.

MATERIALS AND METHODS

ADPCs were obtained from a piece of adipose tissue after treatment with 0.075% collagenase, which was subsequently inactivated with DMEM/10% FBS. The cellular pellet of centrifugation was plated at 5-7 x 10(6) cells/mL in T25 culture flasks in a low-glycose DMEM with 30% FCS. Cord blood stem cells were obtained by centrifugation following double-processing in the presence of 2% HES 200/0.5 and plated at 5-7 x 10(6) cells/mL in the same medium. To investigate the crucial role of ADPCs in pluripotent cord blood differentiation, we added a ADPCS as (1 x 10(4) cells/mL) to the cord blood cultures and analyzed the contribution of ADPCs using a microscope as well as with flow cytometry.

RESULTS

After only 3 days, adherent cells (USSC colonies) of fibroblastic morphology were detected in all co-cultured samples, whereas this was observed later or not at all in the non-co-cultured samples. The greater density of colonies in the co-coltured samples was another point. Hematopoietic CD45 cells were no longer detected after the first passage. Pluripotent stem cells were obtained from all co-cultured samples that contained stem cells positive for CD29, CD44, CD49e, CD90, CD105, CD51 Stro, and C-kit antibodies but negative for CD34, CD45, CD133, and glycophorin A.

CONCLUSION

Addition of ADPCs was crucial to generate pluripotent-derived stem cells from cord blood samples. This double culture may be a useful tool for a universal allogeneic stem cell source for tissue repair or regeneration.

摘要

引言

脂肪组织被认为是用于再生医学应用的产后间充质干细胞的重要来源。此外，已证明脐带血干细胞含有称为无限制体细胞干细胞（USSCs）的多能干细胞。然而，这一群体很罕见，并非每个脐带血样本都能产生。在本研究中，我们提出了一种脂肪来源干细胞（ADPCs）与脐带血干细胞共培养的新方法，该方法可导致多能分化。

材料与方法

用0.075%胶原酶处理一块脂肪组织后获得ADPCs，随后用含10%胎牛血清的DMEM使其失活。将离心后的细胞沉淀以5 - 7×10(6)个细胞/毫升的密度接种于T25培养瓶中，培养基为含30%胎牛血清的低糖DMEM。在2%羟乙基淀粉200/0.5存在下进行双重处理后，通过离心获得脐带血干细胞，并以5 - 7×10(6)个细胞/毫升的密度接种于相同培养基中。为了研究ADPCs在脐带血多能分化中的关键作用，我们向脐带血培养物中添加ADPCS（1×10(4)个细胞/毫升），并使用显微镜以及流式细胞术分析ADPCs的作用。

结果

仅3天后，在所有共培养样本中均检测到成纤维细胞形态的贴壁细胞（USSC集落），而在未共培养的样本中，这种情况出现较晚或根本未出现。共培养样本中集落密度更高是另一个特点。首次传代后不再检测到造血CD45细胞。从所有共培养样本中获得了多能干细胞，这些样本中的干细胞对CD29、CD44、CD49e、CD90、CD105、CD51 Stro和C - kit抗体呈阳性，但对CD34、CD45、CD133和血型糖蛋白A呈阴性。