Assessment of Ex Vivo Prostaglandin pathway activation in HSCs. Homo sapiens

Transplantation with low numbers of hematopoietic stem cells (HSCs), found in many of the publically accessible cryopreserved umbilical cord blood (UCB) units, leads to delayed time to engraftment, high graft failure rates, and early mortality in many patients. A chemical screen in zebrafish identified the prostaglandin compound, 16,16 dimethyl prostaglandin E2 (dmPGE2), to be a critical regulator of hematopoietic stem cell homeostasis. We hypothesized that an ex vivo modulation with dmPGE2 prior to transplantation would lead to enhanced engraftment by increasing the “effective” dose of hematopoietic stem cells (HSCs) in cord blood. A phase I trial of reduced-intensity double UCB transplantation was performed to evaluate safety, rates of engraftment and fractional chimerism of dmPGE2 enhanced UCB units. To explore potential causes of the lack of enhanced efficacy in the first cohort, we characterized HSCs to determine whether the prostaglandin pathway was being activated under the ex vivo incubation conditions (4°C, 10µM dmPGE2, 60 minutes). Incubation conditions were identified (37°C, 10µM dmPGE2, 120 minutes) that maximize the activation of the prostaglandin pathway by dmPGE2 in human CD34+ cells. Overall design: Isolated human CD34+ from umbilical cord blood were incubated ex vivo in Stem Span media with 10uM 16,16-dmPGE2 or DMSO. Two treatment conditions were evaluated (4 deg C for 1 hour, 37 deg C for 2 hours) with either 3 or 7 biological replicates at each condition. Total RNA was isolated post incubation and analyzed on Affymetrix microarrays for pathway activation.

低剂量造血干细胞（hematopoietic stem cells, HSCs）移植常见于众多公开可获取的冷冻保存脐带血（umbilical cord blood, UCB）单元中，该类移植会导致多数患者出现植入延迟、移植失败率升高以及早期死亡等问题。本研究通过斑马鱼化学筛选实验发现，前列腺素类化合物16,16-二甲基前列腺素E2（16,16 dimethyl prostaglandin E2, dmPGE2）是造血干细胞稳态的关键调控因子。我们提出假说：移植前采用dmPGE2进行体外调控，可通过提升脐带血中造血干细胞（HSCs）的“有效”剂量，增强移植植入效果。为此本研究开展了一项减低强度预处理的双份脐带血移植I期临床试验，以评估dmPGE2处理后的脐带血单元的安全性、植入率及嵌合率。为探究首批队列未出现疗效提升的潜在原因，我们对造血干细胞进行了表征分析，以明确在原体外孵育条件（4℃、10μM dmPGE2、60分钟）下，前列腺素通路是否被激活。最终我们确定了可使dmPGE2在人CD34+细胞中最大化激活前列腺素通路的孵育条件：37℃、10μM dmPGE2、120分钟。实验整体设计：从脐带血中分离得到的人CD34+细胞，在含10μM 16,16-二甲基前列腺素E2（16,16-dmPGE2）或二甲基亚砜（DMSO）的Stem Span培养基中进行体外孵育。本研究评估了两种处理条件：4℃孵育1小时、37℃孵育2小时，每个条件设置3或7次生物学重复。孵育结束后分离总RNA，通过Affymetrix微阵列分析以检测通路激活情况。