用于宫内移植的胎儿肝脏造血干细胞储存的生物学与伦理学问题

Mychaliska G B, Muench M O, Rice H E, Leavitt A D, Cruz J, Harrison M R

Department of Surgery, The Fetal Treatment Center, University of California, San Francisco 94143-0570, USA.

J Pediatr Surg. 1998 Feb;33(2):394-9. doi: 10.1016/s0022-3468(98)90470-5.

BACKGROUND/PURPOSE: Transplantation of fetal liver hematopoietic stem cells (HSCs) in utero has the potential to treat a variety of hematologic, immunologic, and metabolic diseases. One prerequisite for broad clinical application is the establishment of a bank of fetal liver HSC tissue. The authors describe their methods for processing fetal liver free of known human pathogens while maximizing HSC activity after cryopreservation.

METHODS

The authors developed a protocol that separates the abortion decision from the donation decision and preserves confidentiality between donor and recipient. Human fetal livers (12 to 14 weeks' gestation) were procured from aborted specimens and the light-density hematopoietic cells isolated by density centrifugation. Total viable cell count increased with gestational age and averaged from 4.36 x 10(7) cells for 12-week livers to 2.0 x 10(8) cells for 14-week livers.

RESULTS

Flow cytometric analysis demonstrated the presence of early progenitors in fresh and thawed specimens and a low number of T cells in each group. The functional capacity of fetal liver progenitors was assessed with colony-forming assays before and after cryopreservation. Thawed specimens showed an average 63% recovery rate for the high-proliferative potential colony-forming cells, a primitive subset of progenitors thought to include HSC. However, the more mature fraction of low-proliferative potential colony-forming cells had a recovery rate of only 35%. These data suggest that fetal liver HSC maybe more resistant to the detrimental effects of cryopreservation than mature progenitors. The fetal liver was screened for bacterial, fungal, and viral contaminates and the serum from donor mothers was screened for human immunodeficiency virus (HIV), hepatitis A, B, and C, human T-cell lymphoma virus (HTLV I/II), rapid plasma reagent (RPR), cytomegalovirus (CMV), and toxoplasmosis IgM. The bacterial contamination rate was 14% (n = 28). The maternal serum was positive for CMV in 78% of cases, and positive for hepatitis C in 0.7% of cases (n = 28). However, all fetal liver specimens were culture negative for CMV.

CONCLUSIONS

These findings demonstrate that human fetal liver HSCs can be procured ethically and processed to ensure a safe graft with a small number of T-cells, and a high yield of progenitors after cryopreservation. A bank of fetal liver HSC will prove useful in treating a variety of genetic diseases before birth by in utero HSC transplantation.

背景/目的：子宫内移植胎儿肝脏造血干细胞（HSCs）有潜力治疗多种血液学、免疫学和代谢性疾病。广泛临床应用的一个前提条件是建立胎儿肝脏HSC组织库。作者描述了他们在处理不含已知人类病原体的胎儿肝脏的方法，同时使冷冻保存后的HSC活性最大化。

方法

作者制定了一项方案，将流产决定与捐赠决定分开，并保持供体和受体之间的保密性。从流产标本中获取人类胎儿肝脏（妊娠12至14周），通过密度离心分离低密度造血细胞。总活细胞计数随胎龄增加，12周肝脏平均为4.36×10⁷个细胞，14周肝脏平均为2.0×10⁸个细胞。

结果

流式细胞术分析显示新鲜和解冻标本中存在早期祖细胞，每组中T细胞数量较少。在冷冻保存前后，用集落形成试验评估胎儿肝脏祖细胞的功能能力。解冻标本中高增殖潜能集落形成细胞（一种被认为包括HSC的祖细胞原始亚群）的平均回收率为63%。然而，低增殖潜能集落形成细胞的更成熟部分的回收率仅为35%。这些数据表明，胎儿肝脏HSC可能比成熟祖细胞对冷冻保存的有害影响更具抵抗力。对胎儿肝脏进行细菌、真菌和病毒污染筛查，对供体母亲的血清进行人类免疫缺陷病毒（HIV）、甲型、乙型和丙型肝炎、人类T细胞淋巴瘤病毒（HTLV I/II）、快速血浆反应素（RPR）、巨细胞病毒（CMV）和弓形虫IgM筛查。细菌污染率为14%（n = 28）。78%的病例中母亲血清CMV呈阳性，0.7%的病例中丙型肝炎呈阳性（n = 28）。然而，所有胎儿肝脏标本CMV培养均为阴性。