Safety Evaluation of Thrombin-pretreated Human Umbilical Cord Blood-derived Mesenchymal Stem Cells: Biodistribution, Subchronic Toxicity and Tumorigenicity

다국어 초록 (Multilingual Abstract)

Cellular therapy is a treatment that injects living cells into a patient to restore the functions of cells and damaged tissues. Stem cell therapeutic agent is an regenerative medicine using undifferentiated cells, and it is necessary to secure the safety of stem cell therapeutics by evaluating nonclinical studies prior to clinical trials in humans because it has the risk of tumorigenicity or potential toxicity owing to its pluripotent and multipotent properties. The primary goal of this study was to evaluate the nonclinical safety assessment of thrombin-pretreated human umbilical cord blood-derived mesenchymal stem cells (th-UCB-MSCs) targeting hypoxic-ischemic encephalopathy (HIE), a fatal brain injury in newborns, for clinical trial. We investigated the in vivo biodistribution, subchronic general toxicity and in vivo/in vitro tumorigenicity of th-UCB-MSCs in compliance with Good Laboratory Practice. The MSCs manufactured under Good Manufacturing Practice were administered once at 1×105 cells/10 µL/head as the maximum feasible dose, or at lower doses into the brain ventricle. To support the clinical use of th-UCB-MSCs for brain injury in newborns, neonatal Sprague-Dawley (SD) rats were used in the in vivo biodistribution, subchronic general toxicity studies. Addtionally, growth process evaluations including physical development, behavioral function, sexual maturation, memory and learning were included to investigate the impact of th-UCB-MSCs on the growth of human neonates.
As the results, intracerebroventricular (ICV) injection of th-UCB-MSCs for HIE therapy induced no toxicologically relevant changes in clinical sign, body weight, food consumption, clinical pathology, gross findings, organ weights and histopathology. In addition, there was no effect immunotoxicity and growth process by administration of th-UCB-MSCs. Evidence of tumorigenesis related to th-UCB-MSCs was not observed in in vivo/in vitro tumorigenicity, and it was confirmed that th-UCB-MSCs remain for up to 7 days in the brain. These results are expected to play an important role in providing information on the thrombin-pretreatment of cell therapies and clinical use of th-UCB-MSCs.

국문 초록 (Abstract)

세포치료제는 살아있는 세포를 환자에게 주입하여 세포와 손상된 조직의 기능을 회복시키는 치료법이다. 그중 줄기세포치료제는 미분화 세포를 이용한 재생의약으로, 다능성(pluripotent) 및 다분화능(multipotent)이라는 세포 자체의 특성으로 인해 종양발생 또는 잠재적 독성의 위험성이 존재한다. 따라서 인체에 대한 임상시험을 실시하기 전 동물에 대한 비임상시험을 실시하여 줄기세포치료제의 안전성을 확보하는 것이 필요하다.
본 연구에서는 주산기 가사 유발 저산소성-허혈성 뇌병증(hypoxic-ischemic encephalopathy, HIE) 치료를 목적으로 개발 중인 트롬빈 전처리 인간제대혈 유래 중간엽줄기세포 치료제(thrombin-pretreated human umbilical cord blood-derived mesenchymal stem cells, th-UCB-MSCs)의 임상적용 전 비임상 안전성평가를 진행하였다. 본 연구는 줄기세포의 주된 치료 효과를 향상시킨 트롬빈 전처리된 th-UCB-MSCs에 대해 생체내 분포분석(in vivo biodistribution), 일반독성(subchronic general toxicity), 생체내 종양원성(in vivo tumorigenicity) 및 생체외 종양원성(in vitro tumorigenicity)을 평가하였으며, 비임상시험관리기준(Good Laboratory Practice, GLP)에 따라 수행하였다. 최대 투여가능농도인 1×105 cells/10 µL/head를 고용량으로 설정하여 마우스 및 랫드의 뇌실(brain ventricle)에 단회 투여하였으며, 신생아 손상 및 투여시기를 고려하여 분포분석 및 일반독성평가에서도 랫드 7일령의 신생자를 사용하였다. 또한 신생아의 성장과정에서의 영향을 확인하기 위해 랫드 신생자를 이용하여 피모발생, 절치 붕출 및 안검개열 완료일을 확인하는 성장시험(physical development), 부주지성, 견인력, 청각반사, 동공반사, 회전봉시험 및 운동성 검사를 실시하는 행동시험(behavioral function), 수미로시험을 통한 학습기능시험(learning and memory)과 질개구 및 포피개열 완료일을 확인하는 성성숙 측정(sexual maturation)을 평가하였다.
본 연구 결과를 통해 th-UCB-MSCs를 마우스 및 랫드 신생자에게 단회 뇌실내 투여하였을 때 일반증상, 체중 및 사료섭취량 측정, 임상병리학적 검사, 장기중량, 육안적 소견 및 병리조직학적 검사에서 독성 변화가 관찰되지 않는 것을 확인하였다. 또한 th-UCB-MSCs 투여로 인한 면역독성 및 성장과정에 미치는 영향은 없는 것으로 확인되었다. 면역부전 마우스를 이용한 생체내/생체외 종양원성 평가에서도 th-UCB-MSCs와 관련된 종양형성이 관찰되지 않았으며, 분포분석을 통해 th-UCB-MSCs가 투여 후 7일까지 뇌에 검출되고 이후 각 장기에서 검출되지 않는 것이 확인되었다. 결론적으로, 이 연구를 통해 HIE 치료를 위한 th-UCB-MSCs의 임상적 사용과 트롬빈으로 전처리된 세포치료제의 비임상 안전성평가에 대한 중요한 정보를 제공할 것으로 기대된다.

목차 (Table of Contents)

• Abstract 1
• General Introduction 4
• Chapter I. Subchronic general toxicity and in vivo biodistribution assessment of thrombin pretreated human umbilical cord blood-derived mesenchymal stem cells 9
• I. Materials and Methods 10
• 1.1. Preparation of th-UCB-MSCs 10
• 1.2. Dose selection 11
• 1.3. Animal housing and care 12
• 1.4. Surgical procedure and intracerebroventricular injection 12
• 1.5. Subchronic general toxicity study in newborn SD rats 13
• 1.6. In vivo biodistribution test in neonatal disease model 16
• 1.7. Data and statistical analysis 20
• II. Results 21
• 2.1. Subchronic general toxicity study in newborn SD rats 21
• 2.1.1. Mortality, clinical observation, body weight, food consumption, macro/microscopic observation 21
• 2.1.2. Clinical pathology and lymphocyte analysis 26
• 2.1.3. Physical development, behavioral function, memory and learning, and sexual maturation 31
• 2.2. In vivo biodistribution test in neonatal disease model 38
• III. Discussion and Conclusion 40
• Chapter II. In vivo and in vitro tumorigenicity assessment of thrombin-pretreated human umbilical cord blood–derived mesenchymal stem cells 46
• I. Materials and Methods 47
• 1.1. Preparation of th-UCB-MSCs 47
• 1.2. Dose selection 48
• 1.3. Animal housing and care (In vivo study) 49
• 1.4. Surgical procedure and intracerebroventricular injection (In vivo study) 49
• 1.5. In vivo tumorigenicity study in nude mice 50
• 1.6. In vitro tumorigenicity study (Soft agar colony formation assay) 52
• 1.7. Data and statistical analysis 53
• II. Results 55
• 2.1. In vivo tumorigenicity study in nude mice 55
• 2.1.1. Mortality, clinical observation, body weight, hematology 55
• 2.1.2. Macroscopic and microscopic observation 58
• 2.2. In vitro tumorigenicity study (Soft agar colony formation assay) 61
• III. Discussion and Conclusion 64
• References 68
• Abstract (Korean) 79