Human Umbilical Cord Matrix Stem Cells Maintain Multilineage Differentiation Abilities and Do Not Transform during Long-Term Culture

Umbilical cord matrix stem cells (UCMSC) have generated great interest in various therapeutic approaches, including liver regeneration. This article aims to analyze the specific characteristics and the potential occurrence of premalignant alterations of UCMSC during long-term expansion, which are important issues for clinical applications. UCMSC were isolated from the umbilical cord of 14 full-term newborns and expanded in vitro until senescence. We examined the long-term growth potential, senescence characteristics, immunophenotype and multilineage differentiation capacity of these cells. In addition, their genetic stability was assessed through karyotyping, telomerase maintenance mechanisms and analysis of expression and functionality of cell cycle regulation genes. The tumorigenic potential was also studied in immunocompromised mice. In vitro, UCMSC reached up to 33.7±2.1 cumulative population doublings before entering replicative senescence. Their immunophenotype and differentiation potential, notably into hepatocyte-like cells, remained stable over time. Cytogenetic analyses did not reveal any chromosomal abnormality and the expression of oncogenes was not induced. Telomere maintenance mechanisms were not activated. Just as UCMSC lacked transformed features in vitro, they could not give rise to tumors in vivo. UCMSC could be expanded in long-term cultures while maintaining stable genetic features and endodermal differentiation potential. UCMSC therefore represent safe candidates for liver regenerative medicine.     

体外培养脐血单个核细胞与CD34+富集细胞

对比MNC和CD34 +富集细胞在SCF +IL 3+IL 6 +FL +Tpo细胞因子组合下的体外扩增特性 ,发现 :CD34 +富集细胞具有很高的扩增潜力 ,在本实验条件下其总细胞持续扩增了 8周 ,扩增倍数达 312 70 9± 86 40 5倍 ;而MNC在培养至第 4周扩增就已呈现下降趋势 ,最大仅扩增了 5 3 3± 6 2倍。对比集落和CD34 +细胞的扩增发现 ,MNC的集落密度和CD34 +细胞含量由第 0天至第 7天有一个上升的过程 ,而CD34 +富集细胞在培养过程中 ,集落密度和CD34 +细胞含量却始终呈下降趋势。在体外培养过程中 ,CD34 +富集细胞的CFU GM和CD34 +细胞最大分别扩增了 185 7± 14 1和 191 7± 188 8倍 ,明显高于MNC的 12 4± 3 2和 5 0 6± 33 2倍 ;而CD34 +富集细胞和MNC的BFU E则只实现了少量扩增 ,分别为 7 2± 5 2和 10 1± 3 4倍。结果显示 ,从CD34 +富集细胞出发扩增造血干 祖细胞 ,可以得到更多的CD34 +细胞和CFU GM集落形成细胞     

Magnetic Resonance Detection of CD34+ Cells from Umbilical Cord Blood Using a 19F Label

Impaired homing and delayed recovery upon hematopoietic stem cell transplantation (HSCT) with hematopoietic stem cells (HSC) derived from umbilical cord blood (UCB) is a major problem. Tracking transplanted cells in vivo will be helpful to detect impaired homing at an early stage and allows early interventions to improve engraftment and outcome after transplantation. In this study, we show sufficient intracellular labeling of UCB-derived CD34⁺ cells, with ¹⁹F-containing PLGA nanoparticles which were detectable with both flow cytometry and magnetic resonance spectroscopy (MRS). In addition, labeled CD34⁺ cells maintain their capacity to proliferate and differentiate, which is pivotal for successful engraftment after transplantation in vivo. These results set the stage for in vivo tracking experiments, through which the homing efficiency of transplanted cells can be studied.     

High Log-Scale Expansion of Functional Human Natural Killer Cells from Umbilical Cord Blood CD34-Positive Cells for Adoptive Cancer Immunotherapy

Immunotherapy based on natural killer (NK) cell infusions is a potential adjuvant treatment for many cancers. Such therapeutic application in humans requires large numbers of functional NK cells that have been selected and expanded using clinical grade protocols. We established an extremely efficient cytokine-based culture system for ex vivo expansion of NK cells from hematopoietic stem and progenitor cells from umbilical cord blood (UCB). Systematic refinement of this two-step system using a novel clinical grade medium resulted in a therapeutically applicable cell culture protocol. CD56⁺CD3⁻ NK cell products could be routinely generated from freshly selected CD34⁺ UCB cells with a mean expansion of >15,000 fold and a nearly 100% purity. Moreover, our protocol has the capacity to produce more than 3-log NK cell expansion from frozen CD34⁺ UCB cells. These ex vivo-generated cell products contain NK cell subsets differentially expressing NKG2A and killer immunoglobulin-like receptors. Furthermore, UCB-derived CD56⁺ NK cells generated by our protocol uniformly express high levels of activating NKG2D and natural cytotoxicity receptors. Functional analysis showed that these ex vivo-generated NK cells efficiently target myeloid leukemia and melanoma tumor cell lines, and mediate cytolysis of primary leukemia cells at low NK-target ratios. Our culture system exemplifies a major breakthrough in producing pure NK cell products from limited numbers of CD34⁺ cells for cancer immunotherapy.     

人脐血CD34+细胞在NOD/SCID小鼠免疫重建及其抗HBV作用的初步探讨

目的：探讨NOD／SCID（nonobese diabetic／severe combined immunodeficient）小鼠移植人脐带血（human umbilical cord blood,HUCB）CD34＋细胞后免疫重建的特性。建立hu—NOD／SCID人鼠嵌合模型并观察其人源化免疫细胞在小鼠体内的生长分化特性、存活时间及其对HBV感染的清除作用。方法：1．NOD／SCID小鼠于C0603．5Gy照射后24h内尾静脉输注HUCBCD34＋细胞;2．以流式细胞技术鉴定小鼠外周血中CD45＋,CD3＋,CD19＋,CD56＋等人源化细胞的比例;3．NOD／SCID小鼠于移植后第4wk注射HBV感染者血清并以未移植的NOD／SCID小鼠作为对照,注射同等量的患者血清;4．于感染后1、7、10、15天采血,免疫荧光定量PCR方法分别检测其HBV—DNA含量。结果：1．HUCBCD34＋细胞移植后第2wk,在小鼠外周血中检测出的CD3＋CD8＋T细胞、CD3＋CD4＋T细胞、CD19＋B细胞、CD56＋NK细胞的比例分别为18．6％、16．1％、13．1％和27．8％。各细胞比例随小鼠周龄而变化。所有移植小鼠存活时间均达9wk;2．移植后小鼠感染HBV血清后,病毒仅在感染后第一天检出,随后消失;未移植CD34＋细胞的小鼠外周血HBV—DNA一直维持在103水平：结论：1．NOD／SCID小鼠经射线照射后移植HUCBCD34＋细胞,在不加任何刺激因子的情况下小鼠可以长时间存活并重建免疫;2．hu—NOD／SCID人鼠嵌合模型小鼠免疫成功重建后,对HBV感染有快速的清除作用。     

The Notch Signaling Regulates CD105 Expression,Osteogenic Differentiation and Immunomodulation of Human Umbilical Cord Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are a group of multipotent cells with key properties of multi-lineage differentiation, expressing a set of relatively specific surface markers and unique immunomodulatory functions. IDO1, a catabolic enzyme of tryptophan, represents a critical molecule mediating immunomodulatory functions of MSCs. However, the signaling pathways involved in regulating these key properties still remain elusive. To investigate the involvement of Notch signaling as well as other potential signaling pathway(s) in regulating these critical properties of MSCs, we treated human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) with γ-secreatase inhibitor I (GSI-I), which inhibits both Notch signaling and ubiquitin-proteasome activities. It was shown that the GSI-I treatment resulted in apoptosis, reduced expression of surface markers CD73, CD90 and CD105, reduced osteogenic differentiation, and reduction of the hUC-MSCs-mediated suppression of Th1 lymphocyte proliferation and the IFN-γ-induced IDO1 expression. Through distinguishing the effects of GSI-I between Notch inhibition and proteasome inhibition, it was further observed that, whereas both Notch inhibition and proteasome inhibition were attributable to the reduced CD105 expression and osteogenic differentiation, but not to the induced apoptosis. However, Notch inhibition, but not proteasome inhibition, only contributed to the significant effect of GSI-I on Th1 proliferation probably through reducing IDO1 promoter activity. In conclusion, the Notch signaling may represent a very important cell signaling capable of regulating multiple critical properties, especially the immunomodulatory functions of MSCs.     

逆病毒载体介导双耐药基因在脐血CD34+细胞中的表达和抗性研究

为探讨转染醛脱氢酶基因(ALDHl)和多药耐药基因(MDRl)的人脐血CD34+细胞能否同时增强对活性环磷酰胺(4-HC)和MDRl基因靶药的抗性,构建了同时含ALDHl和MDRl双耐药基因的逆转录病毒表达质粒GlNa-ALDHl-IRES-MDRl,经LipofectAMINE介导转染GP+E86和PA317包装细胞,采用含长春新碱(VCR)和4-HC的培养基克隆选择后收集重组病毒上清于单向型GP+E86与双嗜型PA317包装细胞行乒乓交互感染,获得PA317重组病毒生产细胞(最高滴度达5.6×105CFU／ml),将含ALDHl和MDRl双耐药基因重组病毒的上清在细胞生长因子刺激下重复感染人脐血CD34+细胞,用PCR、RT-PCR、Southernblot、Northernblot、FACS和MTT等方法检测外源ALDHl与MDRl基因在CD34+细胞中的转移和表达。结果显示逆转录病毒载体介导的双耐药基因已经整合人转染靶细胞基因组并获得有效表达,同时传递不同的耐药表型。经双耐药基因修饰的脐血CD34+细胞对4-HC和VCR药物同时产生抗性,其IC50值分别比未转染细胞高4倍和7.2倍,本研究为开展肿瘤基因治疗的临床研究奠定了实验基础。     

Development of a Xeno-Free Feeder-Layer System from Human Umbilical Cord Mesenchymal Stem Cells for Prolonged Expansion of Human Induced Pluripotent Stem Cells in Culture

Various feeder layers have been extensively applied to support the prolonged growth of human pluripotent stem cells (hPSCs) for in vitro cultures. Among them, mouse embryonic fibroblast (MEF) and mouse fibroblast cell line (SNL) are most commonly used feeder cells for hPSCs culture. However, these feeder layers from animal usually cause immunogenic contaminations, which compromises the potential of hPSCs in clinical applications. In the present study, we tested human umbilical cord mesenchymal stem cells (hUC-MSCs) as a potent xeno-free feeder system for maintaining human induced pluripotent stem cells (hiPSCs). The hUC-MSCs showed characteristics of MSCs in xeno-free culture condition. On the mitomycin-treated hUC-MSCs feeder, hiPSCs maintained the features of undifferentiated human embryonic stem cells (hESCs), such as low efficiency of spontaneous differentiation, stable expression of stemness markers, maintenance of normal karyotypes, in vitro pluripotency and in vivo ability to form teratomas, even after a prolonged culture of more than 30 passages. Our study indicates that the xeno-free culture system may be a good candidate for growth and expansion of hiPSCs as the stepping stone for stem cell research to further develop better and safer stem cells.     

Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model

The field of human trophoblast research aids in understanding the complex environment established during placentation. Due to the nature of these studies, human in vivo experimentation is impossible. A combination of primary cultures, explant cultures and trophoblast cell lines¹ support our understanding of invasion of the uterine wall² and remodeling of uterine spiral arteries^3,4 by extravillous trophoblast cells (EVTs), which is required for successful establishment of pregnancy. Despite the wealth of knowledge gleaned from such models, it is accepted that in vitro cell culture models using EVT-like cell lines display altered cellular properties when compared to their in vivo counterparts^5,6. Cells cultured in the rotating cell culture system (RCCS) display morphological, phenotypic, and functional properties of EVT-like cell lines that more closely mimic differentiating in utero EVTs, with increased expression of genes mediating invasion (e.g. matrix metalloproteinases (MMPs)) and trophoblast differentiation^7,8,9. The Saint Georges Hospital Placental cell Line-4 (SGHPL-4) (kindly donated by Dr. Guy Whitley and Dr. Judith Cartwright) is an EVT-like cell line that was used for testing in the RCCS.The design of the RCCS culture vessel is based on the principle that organs and tissues function in a three-dimensional (3-D) environment. Due to the dynamic culture conditions in the vessel, including conditions of physiologically relevant shear, cells grown in three dimensions form aggregates based on natural cellular affinities and differentiate into organotypic tissue-like assemblies^10,11,12 . The maintenance of a fluid orbit provides a low-shear, low-turbulence environment similar to conditions found in vivo. Sedimentation of the cultured cells is countered by adjusting the rotation speed of the RCCS to ensure a constant free-fall of cells. Gas exchange occurs through a permeable hydrophobic membrane located on the back of the bioreactor. Like their parental tissue in vivo, RCCS-grown cells are able to respond to chemical and molecular gradients in three dimensions (i.e. at their apical, basal, and lateral surfaces) because they are cultured on the surface of porous microcarrier beads. When grown as two-dimensional monolayers on impermeable surfaces like plastic, cells are deprived of this important communication at their basal surface. Consequently, the spatial constraints imposed by the environment profoundly affect how cells sense and decode signals from the surrounding microenvironment, thus implying an important role for the 3-D milieu¹³.We have used the RCCS to engineer biologically meaningful 3-D models of various human epithelial tissues^7,14,15,16. Indeed, many previous reports have demonstrated that cells cultured in the RCCS can assume physiologically relevant phenotypes that have not been possible with other models^10,17-21. In summary, culture in the RCCS represents an easy, reproducible, high-throughput platform that provides large numbers of differentiated cells that are amenable to a variety of experimental manipulations. In the following protocol, using EVTs as an example, we clearly describe the steps required to three-dimensionally culture adherent cells in the RCCS.     

Suppression of Cholangiocarcinoma Cell Growth by Human Umbilical Cord Mesenchymal Stem Cells: A Possible Role of Wnt and Akt Signaling

Emerging evidence indicates that human mesenchymal stem cells (hMSCs) can be recruited to tumor sites, and affect the growth of human malignancies. However, little is known about the underlying molecular mechanisms. Here, we observed the effects of hMSCs on the human cholangiocarcinoma cell line, HCCC-9810, using an animal transplantation model, and conditioned media from human umbilical cord-derived mesenchymal stem cells (hUC-MSCs). Animal studies showed that hUC-MSCs can inhibit the growth of cholangiocarcinoma xenograft tumors. In cell culture, conditioned media from hUC-MSCs inhibited proliferation and induced apoptosis of tumor cells in a dose- and time-dependent manner. The proliferation inhibition rate increased from 6.21% to 49.86%, whereas the apoptosis rate increased from 9.3% to 48.1% when HCCC-9810 cells were cultured with 50% hUC-MSC conditioned media for 24 h. Immunoblot analysis showed that the expression of phosphor-PDK1 (Ser241), phosphor-Akt (Ser 437 and Thr308), phosphorylated glycogen synthase kinase 3β (phospho-GSK-3β^Ser9), β-catenin, cyclin-D1, and c-myc were down-regulated. We further demonstrated that CHIR99021, a GSK-3β inhibitor reversed the suppressive effects of hUC-MSCs on HCCC-9810 cells and increased the expression of β-catenin. The GSK-3β activator, sodium nitroprusside dehydrate (SNP), augmented the anti-tumor effects of hUC-MSCs and decreased the expression of β-catenin. IGF-1 acted as an Akt activator, and also reversed the suppressive effects of hUC-MSCs on HCCC-9810 cells. All these results suggest that hUC-MSCs could inhibit the malignant phenotype of HCCC-9810 human cholangiocarcinoma cell line. The cross-talk role of Wnt/β-catenin and PI3K/Akt signaling pathway, with GSK-3β as the key enzyme bridging these pathways, may contribute to the inhibition of cholangiocarcinoma cells by hUC-MSCs.     

Microvesicles Derived from Human Umbilical Cord Mesenchymal Stem Cells Facilitate Tubular Epithelial Cell Dedifferentiation and Growth via Hepatocyte Growth Factor Induction

During acute kidney injury (AKI), tubular cell dedifferentiation initiates cell regeneration; hepatocyte growth factor (HGF) is involved in modulating cell dedifferentiation. Mesenchymal stem cell (MSC)-derived microvesicles (MVs) deliver RNA into injured tubular cells and alter their gene expression, thus regenerating these cells. We boldly speculated that MVs might induce HGF synthesis via RNA transfer, thereby facilitating tubular cell dedifferentiation and regeneration. In a rat model of unilateral AKI, the administration of MVs promoted kidney recovery. One of the mechanisms of action is the acceleration of tubular cell dedifferentiation and growth. Both in vivo and in vitro, rat HGF expression in damaged rat tubular cells was greatly enhanced by MV treatment. In addition, human HGF mRNA present in MVs was delivered into rat tubular cells and translated into the HGF protein as another mechanism of HGF induction. RNase treatment abrogated all MV effects. In the in vitro experimental setting, the conditioned medium of MV-treated injured tubular cells, which contains a higher concentration of HGF, strongly stimulated cell dedifferentiation and growth, as well as Erk1/2 signaling activation. Intriguingly, these effects were completely abrogated by either c-Met inhibitor or MEK inhibitor, suggesting that HGF induction is a crucial contributor to the acceleration of cell dedifferentiation and growth. All these findings indicate that MV-induced HGF synthesis in damaged tubular cells via RNA transfer facilitates cell dedifferentiation and growth, which are important regenerative mechanisms.     

Purified Human Synovium Mesenchymal Stem Cells as a Good Resource for Cartilage Regeneration

Mesenchymal stem cells (MSCs) have the ability to differentiate into a variety of lineages and to renew themselves without malignant changes, and thus hold potential for many clinical applications. However, it has not been well characterized how different the properties of MSCs are depending on the tissue source in which they resided. We previously reported a novel technique for the prospective MSC isolation from bone marrow, and revealed that a combination of cell surface markers (LNGFR and THY-1) allows the isolation of highly enriched MSC populations. In this study, we isolated LNGFR⁺ THY-1 ⁺ MSCs from synovium using flow cytometry. The results show that the synovium tissue contained a significantly larger percentage of LNGFR ⁺ THY-1 ⁺ MSCs. We examined the colony formation and differentiation abilities of bone marrow-derived MSCs (BM-MSCs) and synovium-derived MSCs (SYN-MSCs) isolated from the same patients. Both types of MSCs exhibited a marked propensity to differentiate into specific lineages. BM-MSCs were preferentially differentiated into bone, while in the SYN-MSC culture, enhanced adipogenic and chondrogenic differentiation was observed. These data suggest that the tissue from which MSCs are isolated should be tailored according to their intended clinical therapeutic application.     

EB病毒对脐带血细胞产生免疫球蛋白的影响

探讨EB病毒是否使培养的脐带血B细胞、CD5+B细胞产生免疫球蛋白及具有天然自身抗体性质的免疫球蛋白。无菌采集新生儿脐带血,RosetteSepTM法分离全B细胞,免疫磁珠分离CD5+B细胞,将分离得到的全B,CD5+B,CD5-B三组细胞分别用EB病毒、紫外线照射灭活的EB病毒(UVEBV)、TPA刺激,于培养的第10～30d,每间隔4d分别留取上清,ELISA法检测培养上清中的IgG,IgM及抗角蛋白自身抗体(AKautoAb)IgG,IgM,阳性对照为健康成人血清,阴性对照为未加刺激的培养细胞上清。EB病毒感染的3组细胞于培养的第7d发生转化,14d以后的培养上清中IgM,AKautoAbIgMOD值较对照组显著升高(p<0.01);UVEBV刺激细胞存活15d,其第14d的CD5+B细胞培养上清中IgMOD值高于对照组(p<0.05);TPA及未刺激的细胞存活7d,培养上清中未检测到免疫球蛋白。可见,EB病毒能使培养的脐带血B细胞、CD5+B细胞产生可能具有天然自身抗体性质的免疫球蛋白。     

Assessment of Glial Scar,Tissue Sparing,Behavioral Recovery and Axonal Regeneration following Acute Transplantation of Genetically Modified Human Umbilical Cord Blood Cells in a Rat Model of Spinal Cord Contusion

Objective and Methods

This study investigated the potential for protective effects of human umbilical cord blood mononuclear cells (UCB-MCs) genetically modified with the VEGF and GNDF genes on contusion spinal cord injury (SCI) in rats. An adenoviral vector was constructed for targeted delivery of VEGF and GDNF to UCB-MCs. Using a rat contusion SCI model we examined the efficacy of the construct on tissue sparing, glial scar severity, the extent of axonal regeneration, recovery of motor function, and analyzed the expression of the recombinant genes VEGF and GNDF in vitro and in vivo.

Results

Transplantation of UCB-MCs transduced with adenoviral vectors expressing VEGF and GDNF at the site of SCI induced tissue sparing, behavioral recovery and axonal regeneration comparing to the other constructs tested. The adenovirus encoding VEGF and GDNF for transduction of UCB-MCs was shown to be an effective and stable vehicle for these cells in vivo following the transplantation into the contused spinal cord.

Conclusion

Our results show that a gene delivery using UCB-MCs-expressing VEGF and GNDF genes improved both structural and functional parameters after SCI. Further histological and behavioral studies, especially at later time points, in animals with SCI after transplantation of genetically modified UCB-MCs (overexpressing VEGF and GDNF genes) will provide additional insight into therapeutic potential of such cells.     

The Protective Effect of Human Umbilical Cord Blood CD34+ Cells and Estradiol against Focal Cerebral Ischemia in Female Ovariectomized Rat: Cerebral MR Imaging and Immunohistochemical Study

Human umbilical cord blood derived CD34⁺ stem cells are reported to mediate therapeutic effects in stroke animal models. Estrogen was known to protect against ischemic injury. The present study wished to investigate whether the protective effect of CD34⁺ cells against ischemic injury can be reinforced with complemental estradiol treatment in female ovariectomized rat and its possible mechanism. Experiment 1 was to determine the best optimal timing of CD34⁺ cell treatment for the neuroprotective effect after 60-min middle cerebral artery occlusion (MCAO). Experiment 2 was to evaluate the adjuvant effect of 17β-estradiol on CD34⁺ cell neuroprotection after MCAO. Experiment 1 showed intravenous infusion with CD34⁺ cells before MCAO (pre-treatment) caused less infarction size than those infused after MCAO (post-treatment) on 7T magnetic resonance T2-weighted images. Experiment 2 revealed infarction size was most significantly reduced after CD34⁺ + estradiol pre-treatment. When compared with no treatment group, CD34⁺ + estradiol pre-treatment showed significantly less ADC reduction at 2 h and 2 d, less CBF reduction at 2 h and less hyperperfusion at 2 d. The immunoreactivity of c-Fos, c-Jun and GFAP was attenuated, and BDNF showed significant recovery from 2 h to 2 d after MCAO, especially after CD34⁺ + estradiol pre-treatment. The present study suggests pre-treatment with CD34⁺ cells with complemental estradiol can be most protective against ischemic injury, which may act through stabilization of cerebral hemodynamics and normalization of the expressions of immediate early genes and BDNF.     

Phenotypic and Functional Properties of Human Steady State CD14+ and CD1a+ Antigen Presenting Cells and Epidermal Langerhans Cells

Cutaneous antigen presenting cells (APCs) are critical for the induction and regulation of skin immune responses. The human skin contains phenotypically and functionally distinct APCs subsets that are present at two separated locations. While CD1a^high LCs form a dense network in the epidermis, the CD14⁺ and CD1a⁺ APCs reside in the dermal compartment. A better understanding of the biology of human skin APC subsets is necessary for the improvement of vaccine strategies that use the skin as administration route. In particular, progress in the characterization of uptake and activatory receptors will certainly improve APC-targeting strategies in vaccination. Here we performed a detailed analysis of the expression and function of glycan-binding and pattern-recognition receptors in skin APC subsets. The results demonstrate that under steady state conditions human CD1a⁺ dermal dendritic cells (DCs) were phenotypically most mature as measured by the expression of CD83 and CD86, whereas the CD14⁺ cells showed a higher expression of the CLRs DC-SIGN, mannose receptor and DCIR and had potent antigen uptake capacity. Furthermore, steady state LCs showed superior antigen cross-presentation as compared to the dermal APC subsets. Our results also demonstrate that the TLR3 ligand polyribosinic-polyribocytidylic acid (pI:C) was the most potent stimulator of cytokine production by both LCs and dDCs. These studies warrant further exploration of human CD1a⁺ dDCs and LCs as target cells for cancer vaccination to induce anti-tumor immune responses.     

Imatinib and Nilotinib Inhibit Hematopoietic Progenitor Cell Growth,but Do Not Prevent Adhesion,Migration and Engraftment of Human Cord Blood CD34+ Cells

Background

The availability of tyrosine kinase inhibitors (TKIs) has considerably changed the management of Philadelphia chromosome positive leukemia. The BCR-ABL inhibitor imatinib is also known to inhibit the tyrosine kinase of the stem cell factor receptor, c-Kit. Nilotinib is 30 times more potent than imatinib towards BCR-ABL in vitro. Studies in healthy volunteers and patients with chronic myelogenous leukemia or gastrointestinal stromal tumors have shown that therapeutic doses of nilotinib deliver drug levels similar to those of imatinib. The aim of this study was to compare the inhibitory effects of imatinib and nilotinib on proliferation, differentiation, adhesion, migration and engraftment capacities of human cord blood CD34⁺ cells.

Design and Methods

After a 48-hour cell culture with or without TKIs, CFC, LTC-IC, migration, adhesion and cell cycle analysis were performed. In a second time, the impact of these TKIs on engraftment was assessed in a xenotransplantation model using NOD/SCID/IL-2Rγ (null) mice.

Results

TKIs did not affect LTC-IC frequencies despite in vitro inhibition of CFC formation due to inhibition of CD34⁺ cell cycle entry. Adhesion of CD34⁺ cells to retronectin was reduced in the presence of either imatinib or nilotinib but only at high concentrations. Migration through a SDF-1α gradient was not changed by cell culture in the presence of TKIs. Finally, bone marrow cellularity and human chimerism were not affected by daily doses of imatinib and nilotinib in a xenogenic transplantation model. No significant difference was seen between TKIs given the equivalent affinity of imatinib and nilotinib for KIT.

Conclusions

These data suggest that combining non-myeloablative conditioning regimen with TKIs starting the day of the transplantation could be safe.