人脐带血来源的间充质干细胞特性及对卵巢癌细胞趋化作用的研究

目的为人脐带血（HUCD）可成为间充质干细胞（MSCs）重要新来源提供依据,着重研究对人卵巢癌细胞的趋化作用,为临床靶向治疗卵巢癌提供新的载体。方法征求足月健康的自然产孕妇同意,获得脐带血,从中提取MSCs。获得稳定增殖传代的MSCs后,鉴定其生物特性和抗原表型,将其与人卵巢癌HO-8910细胞共培养,探讨其对卵巢癌细胞的趋化作用。结果成功从HUCD中提取MSCs,筛选出稳定传代的细胞系。其生物学特性与骨髓来源的MSCs一样具有多向细胞分化潜能,可诱导分化成脂肪细胞、成骨细胞;同时还具有相同的免疫表型,CD29、CD44、CD105阳性表达,CD13、CD14、CD34、CD45阴性表达,并且其免疫表型不随着细胞传代而改变。HUCD来源的MSCs与HO-8910细胞共培养时发现,MSCs对其有趋化作用。结论证实HUCD可以作为MSCs的新的重要来源。实验发现MSCs对人卵巢癌细胞株HO-8910有明显趋化作用,这一发现可能为卵巢肿瘤靶向治疗提供了新的有效载体。     

Thiol-ene Michael-type formation of gelatin/poly(ethylene glycol) biomatrices for three-dimensional mesenchymal stromal/stem cell administration to cutaneous wounds

Mesenchymal stromal/stem cells (MSCs) are considered promising cellular therapeutics in the fields of tissue engineering and regenerative medicine. MSCs secrete high concentrations of immunomodulatory cytokines and growth factors, which exert paracrine effects on infiltrating immune and resident cells in the wound microenvironment that could favorably promote healing after acute injury. However, better spatial delivery and improved retention at the site of injury are two factors that could improve the clinical application of MSCs. In this study, we utilized thiol-ene Michael-type addition for rapid encapsulation of MSCs within a gelatin/poly(ethylene glycol) biomatrix. This biomatrix was also applied as a provisional dressing to full thickness wounds in Sprague–Dawley rats. The three-way interaction of MSCs, gelatin/poly(ethylene glycol) biomatrices, and host immune cells and adjacent resident cells in the wound microenvironment favorably modulated wound progression and host response. In this model we observed attenuated immune cell infiltration, lack of foreign giant cell (FBGC) formation, accelerated wound closure and re-epithelialization, as well as enhanced neovascularization and granulation tissue formation by 7 days. The MSC entrapped in the gelatin/poly(ethylene glycol) biomatrix localized cell presentation adjacent to the wound microenvironment and thus mediated the early resolution of inflammatory events and facilitated the proliferative phases in wound healing.     

Ultrastructural characteristics of human mesenchymal stromal (stem) cells derived from bone marrow and term placenta

Human mesenchymal stromal (stem) cells (hMSCs) isolated from adult bone marrow (BM-hMSCs) as well as amnion (AM-hMSCs) and chorion (CM-hMSCs) term placenta leaves were studied by transmission electron microscopy (TEM) to investigate their ultrastructural basic phenotype. At flow cytometry, the isolated cells showed a homogeneous expression of markers commonly used to identify hMSCs, i.e., CD105, CD44, CD90, CD166, HLA-ABC positivities, and CD45, AC133, and HLA-DR negativities. However, TEM revealed subtle yet significant differences. BM-hMSCs had mesenchymal features with dilated cisternae of rough endoplasmic reticulum (rER) and peripheral collections of multiloculated clear blisters; this latter finding mostly representing complex foldings of the plasma membrane could be revelatory of the in situ cell arrangement in the niche microenvironment. Unlike BM-hMSCs, CM-hMSCs were more primitive and metabolically quiescent, their major features being the presence of rER stacks and large peripheral collections of unbound glycogen. AM-hMSCs showed a hybrid epithelial-mesenchymal ultrastructural phenotype; epithelial characters included non-intestinal-type surface microvilli, intracytoplasmic lumina lined with microvilli, and intercellular junctions; mesenchymal features included rER profiles, lipid droplets, and well-developed foci of contractile filaments with dense bodies. These features are consistent with the view that AM-hMSCs have a pluripotent potential. In conclusion, this study documents that ultrastructural differences exist among phenotypically similar hMSCs derived from human bone marrow and term placenta leaves; such differences could be revelatory of the hMSCs in vitro differentiation potential and may provide useful clues to attempt their in situ identification.     

Human IgA‐secreting cells induced by intestinal,but not systemic,immunization respond to CCL25 (TECK) and CCL28 (MEC)

Organ‐specific homing of lymphoid cells depends on the expression of tissue‐specific adhesion molecules and production of specific chemokines. CCL25 (TECK) and CCL28 (MEC) have been reported to direct circulating memory/effector B cells to mucosal tissues. Here, we examined if differential responsiveness to mucosal and systemic chemokines could explain the differential migration pattern of circulating human antibody‐secreting cells (ASC), induced by mucosal and systemic immunization. There was a robust migration of specific IgA‐ and IgM‐ASC induced by Salmonella vaccination toward the mucosal chemokines CCL25 and CCL28. In contrast, tetanus‐specific ASC migrated to the systemic chemokine CXCL12 (SDF‐1α) and showed no response to CCL25 or CCL28, not even tetanus‐specific IgA‐ASC. Cell sorting experiments demonstrated that Salmonella‐specific ASC co‐expressed CCR9 and CCR10. Our results show that induction site, rather than isotype commitment, determines the chemokine responsiveness and migration pattern of human effector B cells.     

LIF对人胎脑神经干细胞体外增殖和分化的影响

目的:观察白血病抑制因子(LIF)对体外培养的人胎脑神经干细胞增殖和分化的影响。方法:用添加表皮生长因子(EGF)和碱性成纤维细胞生长因子(FGF2)的N2培养基培养人神经干细胞(hNSC)。实验分添加LIF(LIF+)组和无LIF(LIF-)组,接种12天后计数细胞集落(球)的形成率。传代培养观察120天,定期进行活细胞计数,绘制生长速率曲线。取第6代细胞球进行分化诱导,免疫荧光技术鉴别神经细胞的特异性抗原标志,并计算各细胞类型间的比例。结果:两组集落形成百分比分别为:LIF+为0.50%-0.91%;LIF-为0.49%-0.94%。两组间的差异并无显著意义(P>0.05)。在相同培养条件下,各例胎脑来源的NSC扩增速率的相差并无显著性意义(P>0.05),但LIF对NSC扩增有重要作用,刺激细胞扩增了约4000-8400倍,无分化发生;LIF-组仅为43-97倍,培养两个月后可观察到分化现象。在培养过程中观察到:LIF的作用主要表现在细胞接种传代约50-60天以后。用免疫细胞化学荧光进行分化细胞类型鉴定,计数神经元和星形胶细胞数,并计算其中神经元所占的百分比。LIF+培养为12%-83%,明显高于LIF-组的8%-23%(P<0.005),来源于海马的NSC分化为神经元的比例要高于来源于纹状体的NSC。结论:LIF能阻抑人胎脑NSC的分化,促进其体外长期增殖,其效应主要表现在接种传代培养的50-60天以后。LIF还影响NSC的分化,可显著提高分化细胞中神经元的百分比,海马源性hNSC对LIF更为敏感。     

Non‐inferiority of microencapsulated mesenchymal stem cells to free cells in cardiac repair after myocardial infarction: A rationale for using paracrine factor(s) instead of cells

A major translational barrier to the use of stem cell (SC)‐based therapy in patients with myocardial infarction (MI) is the lack of a clear understanding of the mechanism(s) underlying the cardioprotective effect of SCs. Numerous paracrine factors from SCs may account for reduction in infarct size, but myocardial salvage associated with transdifferentiation of SCs into vascular cells as well as cardiomyocyte‐like cells may be involved too. In this study, bone marrow‐derived rat mesenchymal SC (MSCs) were microencapsulated in alginate preventing viable cell release while supporting their secretory phenotype. The hypothesis on the key role of paracrine factors from MSCs in their cardioprotective activity was tested by comparison of the effect of encapsulated vs free MSCs in the rat model of MI. Intramyocardial administration of both free and encapsulated MSCs after MI caused reduction in scar size (12.1 ± 6.83 and 14.7 ± 4.26%, respectively, vs 21.7 ± 6.88% in controls, P = 0.015 and P = 0.03 respectively). Scar size was not different in animals treated with free and encapsulated MSC (P = 0.637). These data provide evidence that MSC‐derived growth factors and cytokines are crucial for cardioprotection elicited by MSC. Administration of either free or encapsulated MSCs was not arrhythmogenic in non‐infarcted rats. The consistency of our data with the results of other studies on the major role of MSC secretome components in cardiac protection further support the theory that the use of live, though encapsulated, cells for MI therapy may be replaced with heart‐targeted‐sustained delivery of growth factors/cytokines.     

Long‐term human immune system reconstitution in non‐obese diabetic (NOD)‐Rag (–)‐γ chain (–) (NRG) mice is similar but not identical to the original stem cell donor

The murine immune system is not necessarily identical to it human counterpart, which has led to the construction of humanized mice. The current study analysed whether or not a human immune system contained within the non‐obese diabetic (NOD)‐Rag1^null‐γ chain^null (NRG) mouse model was an accurate representation of the original stem cell donor and if multiple mice constructed from the same donor were similar to one another. To that end, lightly irradiated NRG mice were injected intrahepatically on day 1 of life with purified cord blood‐derived CD34⁺ stem and progenitor cells. Multiple mice were constructed from each cord blood donor. Mice were analysed quarterly for changes in the immune system, and followed for periods up to 12 months post‐transplant. Mice from the same donor were compared directly with each other as well as with the original donor. Analyses were performed for immune reconstitution, including flow cytometry, T cell receptor (TCR) and B cell receptor (BCR) spectratyping. It was observed that NRG mice could be ‘humanized’ long‐term using cord blood stem cells, and that animals constructed from the same cord blood donor were nearly identical to one another, but quite different from the original stem cell donor immune system.     

Adhesion to fibronectin via alpha4 integrin (CD49d) protects B cells from apoptosis induced by serum deprivation but not via IgM or Fas/Apo-1 receptors

Apoptosis is a regulated event crucial to the development and proliferation of normal and malignant B cells. We have studied the role of signals delivered via alpha4 integrin on apoptosis triggered by three different pathways on these cells. For apoptosis induced by serum deprivation, culturing B cells on the recombinant fibronectin fragment H89, a known ligand for alpha4beta1 integrin, resulted in statistically significant (P < 0.005) higher viability values (68%, 65% and 67%) for Ramos, Nalm-6 and EHEB cells, respectively, than culturing cells on poly lysine (42%, 42% and 48%). An antialpha4 MoAb reverted the protecting effect, thus confirming that it was due specifically to alpha4 engagement. Similarly, cells cultured on FN-III4-5, a recently identified fibronectin region which binds activated alpha4 integrin, also showed statistically significant higher viability than poly lysine cultures. Alpha4 engagement however, did not prevent apoptosis induced on Ramos cells via surface IgM. Adhesion of IM-9 cells, a myeloma cell line carrying functional Fas receptors, to the H89 fragment neither increased cell viability upon triggering apoptosis via Fas when compared to poly lysine. These results indicate that alpha4 signalling may overcome B cell apoptosis induced by the lack of growth factors but does not seem to affect the IgM or Fas apoptotic pathways, thus suggesting different intracellular mechanisms for these processes.