NIDCR Contribution to NIH Bone Marrow Stromal Cell Transplantation Center

在过去的一年里，BMSCTC开展了许多活动。使用中心批准的骨髓采集方案(10-CC-0053-收集健康志愿者和患者的骨髓用于生产临床骨髓基质细胞(BMSC)产品，Stroncek，CC，PI)，已经建立了供异基因使用的冷冻BMSCs库。骨髓基质细胞(BMSCs)已被用于治疗异基因造血干细胞移植(SCT)后的急性移植物抗宿主病(GVHD)和其他并发症。一项I期试验(12-H-0010-A期骨髓基质细胞输注治疗异基因干细胞移植后急性移植物抗宿主病、骨髓衰竭和组织损伤，Battiwalla和Barrett，NHLBI，PI)在SCT后使用第三方早期传代的BMSCs治疗激素难治性GVHD、组织损伤或骨髓衰竭患者，以研究其安全性和有效性。为了确定骨髓间充质干细胞免疫调节和组织修复的机制，对患者进行了血浆GVHD生物标志物、细胞因子和淋巴细胞表型的连续监测。10名受试者每周静脉注射固定剂量的骨髓间充质干细胞2×10(6)/kg，共3次。没有与治疗相关的毒性(主要终点)。8名受试者在最后一次输液后4周可评估反应。7例激素耐药的急性移植物抗宿主病患者中5例完全缓解，2例组织损伤(纵隔/气胸)患者完全缓解，2例延迟性骨髓衰竭患者无反应。炎性细胞因子迅速降低。临床反应与移植物抗宿主病或组织损伤相关的生物标志物(REG3、CK18和Elafin)下降有关。GVHD完全应答者存活时间显著延长，并具有更高的基线绝对淋巴细胞和中央记忆CD4和CD8计数。细胞因子的变化也与生存分离。这些结果证实了骨髓间充质干细胞与移植物抗宿主病和组织损伤的快速临床和生物标志物反应有关。然而，对于淋巴细胞计数较低的GVHD延长患者，BMSCs效果不佳，提示BMSCs有效控制GVHD需要相对完整的免疫系统。 同种异体细胞还被用于治疗炎症性肠病的试验(13-I-0077-一项开放标签的第1阶段研究，以评估骨髓基质细胞输注治疗中到重度炎症性肠病的安全性和耐受性)，目前正在患者评估中。 在接受冠状动脉旁路移植和经心肌激光血管重建术的患者中，生成了直接使用(无需冷冻)的自体细胞(12-H-0078-心肌内直接注射自体骨髓基质细胞的初步评估，用于治疗左心功能低下的冠心病患者，Robey，NIDCR，PI和Horvath，NHLBI，LAI)。到目前为止，已有9名患者接受了治疗。 除了准备临床级别的BMSCs外，该中心还一直在开发技术，以更好地监测它们在体外扩增过程中的生物学活性。使用骨髓基质干细胞的临床试验的结果是不同的；临床生产过程中BMSCs的扩张程度可能会导致这种差异，因为细胞的扩张受到衰老的限制。取健康人BMSCs进行连续传代培养，直至细胞停止生长。两组BMSCs的表型和功能检测包括衰老相关β-半乳糖苷酶染色和集落形成效率在第6、7代时由早衰变为衰老期。对来自5个受试者的10个早传代和15个晚期传代的BMSC进行转录组分析，发现2122个差异表达基因，这些基因与免疫应答、发育和细胞增殖途径有关。对来自7个捐献者的57个系列BMSC样本的分析表明，从早期到衰老的变化在受试者中是不同的，并且发生在表型变化之前。以最大群体倍增(PD)百分比表示的BMSC年龄是早期或衰老转录特征的良好指标，但这种BMSC寿命的测量只能在BMSCs扩增到衰老后才能计算出来。为了找到更有用的骨髓间充质干细胞年龄的替代指标，我们使用计算生物学的方法来识别一组基因，这些基因在每一代中的表达可以预测骨髓间充质干细胞的年龄。共有155个基因与BMSC年龄高度相关。最小角度回归算法确定了一组24个BMSC年龄预测基因。与衰老相关的分子变化的开始是不同的，并且先于骨髓间充质干细胞质量和衰老的其他指标的变化。24个骨髓间充质干细胞年龄预测基因将有助于评估临床骨髓间充质干细胞产品的质量。

There have been a number of activities in the BMSCTC during the past year. Using the Centers approved bone marrow acquisition protocol (10-CC-0053 - Collection of Bone Marrow from Healthy Volunteers and Patients for the Production of Clinical Bone Marrow Stromal Cell (BMSC) Products, Stroncek, CC, PI), a bank of frozen BMSCs has been established for allogeneic use. Bone marrow stromal cells (BMSCs) have been used to treat acute graft-versus-host disease (GVHD) and other complications following allogeneic hematopoietic stem cell transplantation (SCT). a phase I trial (12-H-0010 - A Phase I Study of Bone Marrow Stromal Cell Infusions to Treat Acute Graft Versus Host Disease, Marrow Failure and Tissue Injury After Allogeneic Stem Cell Transplantation, Battiwalla and Barrett, NHLBI, PIs) using third party, early passage BMSCs for patients with steroid-refractory GVHD, tissue injury, or marrow failure following SCT to investigate safety and efficacy was completed. To identify mechanisms of BMSC immunomodulation and tissue repair, patients were serially monitored for plasma GVHD biomarkers, cytokines, and lymphocyte phenotype. Ten subjects were infused a fixed dose of 2 10(6) BMSCs/kg intravenously weekly for three doses. There was no treatment-related toxicity (primary endpoint). Eight subjects were evaluable for response at 4 weeks after the last infusion. Five of the seven patients with steroid-refractory acute GVHD achieved a complete response, two of two patients with tissue injury (pneumomediastinum/pneumothorax) achieved resolution but there was no response in two subjects with delayed marrow failure. Rapid reductions in inflammatory cytokines were observed. Clinical responses correlated with a fall in biomarkers (Reg 3α, CK18, and Elafin) relevant for the site of GVHD or tissue injury. The GVHD complete responders survived significantly longer and had higher baseline absolute lymphocyte and central memory CD4 and CD8 counts. Cytokine changes also segregated with survival. These results confirm that BMSCs are associated with rapid clinical and biomarker responses in GVHD and tissue injury. However, BMSCs were ineffective in patients with prolonged GVHD with lower lymphocyte counts, which suggest that effective GVHD control by BMSCs requires a relatively intact immune system. The allogeneic cells are also being used in a trial to treat inflammatory bowel disease (13-I-0077 - An Open-Label, Phase 1 Study to Assess the Safety and Tolerability of Bone Marrow Stromal Cell Infusion for the treatment of Moderate to Severe Inflammatory Bowel Disease, Fuss, NIAID, PI), currently in patient accrual. Autologous cells were generated for direct use (no freezing) in patients under going coronary artery bypass grafting and trans myocardial laser revascularization (12-H-0078 - Preliminary Assessment of Direct Intra-Myocardial Injection of Autologous Bone Marrow-derived Stromal Cells on Patients Undergoing Revascularization for CAD with Depressed Left Ventricular Function, Robey, NIDCR, PI and Horvath, NHLBI, LAI). Nine patients have been treated to date. In addition to preparing clinical grade BMSCs, the Center also has been developing techniques to better monitor their biological activity during ex vivo expansion. The outcomes of clinical trials using bone marrow stromal cell BMSC are variable; the degree of the expansion of BMSCs during clinical manufacturing may contribute to this variability since cell expansion is limited by senescence. Human BMSCs from aspirates of healthy subjects were subcultured serially until cell growth stopped. Phenotype and functional measurements of BMSCs from two subjects including senescence-associated beta-galactosidase staining and colony formation efficiency changed from an early to a senescence pattern at passage 6 or 7. Transcriptome analysis of 10 early and 15 late passage BMSC samples from 5 subjects revealed 2122 differentially expressed genes, which were associated with immune response, development, and cell proliferation pathways. Analysis of 57 serial BMSC samples from 7 donors revealed that the change from an early to senescent profile was variable among subjects and occurred prior to changes in phenotypes. BMSC age expressed as a percentage of maximum population doublings (PDs) was a good indicator for an early or senescence transcription signature but this measure of BMSC life span can only be calculated after expanding BMSCs to senescence. In order to find a more useful surrogate measure of BMSC age, we used a computational biology approach to identify a set of genes whose expression at each passage would predict elapsed age of BMSCs. A total of 155 genes were highly correlated with BMSC age. A least angle regression algorithm identified a set of 24 BMSC age-predictive genes. The onset of senescence-associated molecular changes was variable and preceded changes in other indicators of BMSC quality and senescence. The 24 BMSC age predictive genes will be useful in assessing the quality of clinical BMSC products.