Hematopoietic Engraftment of Amniotic Fluid Stem Cells Following in Utero Transplantation

Introduction

Amniotic fluid stem cells (AFSC) are an appealing cell source for prenatal treatment of congenital blood disorders. They can be obtained safely during amniocentesis, are autologous to the fetus, and have shown hematopoietic potential when transplanted postnatally. Ex-vivo expansion to obtain adequate cell numbers is required for the clinical use of AFSC for in utero transplantation (IUT). The aim of this study was to assess long-term hematopoietic engraftment of freshly-isolated and cultured AFSC post IUT in mice, and compare it to that achieved following IUT of adult, bone marrow-derived hematopoietic stem cells (BM-HSC).

Methods

Lineage-depleted/c-Kit+ AFSC were isolated at embryonic day 13 (E13) from C57BL/6TgN(act-EGFP)OsbY01 (GFP) dams. AFSC were cultured for 7 days on mitotically-inactivated mouse embryonic fibroblasts with mouse embryonic stem cell media, and were sorted by flow cytometry (GFP+/c-Kit+) prior to IUT. BM-HSC (lineage-depleted/c-Kit+/Sca-1+) were isolated from 6-week old GFP mice. 10⁴ AFSC (fresh or cultured) or BM-HSC (fresh) were injected intravenously into E14 C57BL/6J fetuses representing a congenic (autologous-like) IUT. Donor cell engraftment was assessed in the peripheral blood (PB) at 4 and 12 weeks of age by flow cytometry (% GFP+ cells within CD45+ population). Lineage characterization of engrafted cells was performed at both time points using antibodies against lymphoid (CD3, B220), and myeloid (CD11b, Gr-1) cell surface markers. Results are expressed as mean±SEM, and statistical analysis was performed using 1- or 2-way ANOVA with Bonferroni post-hoc tests. Experimental protocols were approved by the Institutional Animal Care and Use Committee at The Children’s Hospital of Philadelphia.

Results

Freshly-isolated AFSC expressed pluripotency (Oct-4, c-Myc, Klf-4) and hematopoietic markers (Sca-1+: 34.1±8.3%, CD34+: 10.6±3.1%, CD45+: 97.9±2.7%). Cultured AFSC had a doubling time of 3.2±0.2 days, and 80.7±1.6% of cells maintained c-Kit expression after 7 days of culture. c-Kit+ cultured AFSC expressed pluripotency and hematopoietic markers at similar levels as fresh AFSC. Array analysis of hematopoietic genes demonstrated significant under-expression of hematopoiesis-related transcription factors and regulators (including Gata1, Gata2, and Lmo2) in fresh and cultured AFSC compared to BM-HSC. Fetal survival rate was comparable following IUT with fresh AFSC (14/23; 60.9%), cultured AFSC (13/21; 61.9%) and BM-HSC (16/26; 61.5%) (p=0.9). Successful hematopoietic engraftment was seen at 4 weeks of age in pups that underwent fresh and cultured AFSC IUT, with comparable PB levels between groups (fresh AFSC: 21.9±1.0% vs. cultured AFSC: 20.4±1.4%; p=0.9). In contrast, IUT of BM-HSC resulted in hematopoietic micro-chimerism (engraftment: 0.04±0.02%; p<0.0001 vs. fresh and cultured AFSC). Hematopoietic engraftment was maintained in fresh and cultured AFSC groups at 12 weeks of age (fresh AFSC: 21.6±1.1% vs. cultured AFSC: 19.4±2.4%; p=0.9), and donor cell characterization demonstrated multi-lineage hematopoietic differentiation, with distribution similar to that of host cells (p=0.8).

Conclusions

Congenic (autologous-like) IUT of AFSC results in successful multi-lineage hematopoietic engraftment in immune-competent mice, which is superior to that achieved with BM-HSC. AFSC can be expanded in vitro without differentiation, and the hematopoietic potential of cultured cells post IUT is similar to that achieved with freshly-isolated AFSC. Combined with gene therapy, IUT of autologous AFSC could be a promising approach for prenatal treatment of congenital blood disorders.