Fetal hematopoietic cells in early gestation : aspects in view of fetal transplantation

Abstract

The fetus as a patient is a challenge. There are opportunities for early, sometimes life-saving procedures or there may be advantages in prenatal rather than postnatal intervention. This is the case in fetal hematopoietic cell (FHC) transplantation where conditions caused by a defect in blood cell formation may be life-threatening to the fetus. In other diseases the dysfunction may be slower. Nonetheless, an early treatment may improve prognosis.

The aim of the studies on which this thesis is based was to lay the foundation of a program for human fetus-to-fetus transplantation with FHC. As the logistics of such transplantation demand a tissue bank of cells from elective abortions, the following items were studied: methods for retrieval of fetal cells, cryopreservation of FHC, deterrnination of immunological capacity, and different modalities for improving hematological capacity. In addition, the safety of FHC with regard to bacterial contamination, virus infection and cytogenetic abnormality was evaluated. Finally, different routes for the administration of FHC to human fetuses were compared.

FHC from the fetal liver (FL) were analyzed with conventional immunological techniques. Monoclonal antibodies were used, and it was found that common surface antigens were present on FL cells, but to a lesser extent than in bone marrow (BM). As compared to BM, FHC from FL were found to manifest more Ms positive cells (monocytes/macrophages) and the same amount of CD34 positive (stemlearly progenitor) cells. The number of CD34+ cells did not change after cryopreservation, nor was cellular function altered, as measured by colony formation.

We studied the immunological capacity of FHC before the 12th week, as those are the cells that will be used for transplantation. This was done in various assays: mitogen stimulation, polyclonal B-cells activation (PBA), mixed Iymphocyte culture (MLC), HLA surface markers, HLA-typing with anti-sera and genomic HLA determination. Nowhere was any significant immunological activity found, although it was possible to demonstrate that FHC did activate adult peripheral blood leukocytes (PBL) in MLC, but not the opposite. Genomic HLA determination was possible, which was expected.

Engraftment of transplanted cells may be enhanced. As the recipient is a developing fetus, manipulations are restricted to the graft. Different methods, namely cytokine stimulation and CD34+ enrichment of FHC, were studied. The effects of these manipulations were evaluated in terms of colony formation (BFU-E, CFU-GM and -GEMM). It was possible to demonstrate effects of each single cytokine, and of cytokines used simultaneously. CD34+ purification was also beneficial for cellular expansion, and a combination of cytokines and purification seemed to be most advantageous for colony formation. However, crude cells may be used in transplantation instead of CD34+ FHC, as the CD34 negative FHC also could form colonies.

All FHC retrieved after elective abortions were screened for bacterial contamination, virus infection and chromosomal abnormality. With these screening precautions as routine procedure, the use of FHC from elective abortions seems safe and practicable.

The administration of cells to fetuses in utero is possible with an ultrasound-guided technique. Theintravascular(iv) and the intraperitoneal (ip) routes in fetuses at 13-17 weeks of gestation were compared. Of the two the iv route yielded significantly higher cell dose of radiolabeled FHC in hematopoiesis-associated organs, such as liver, spleen and thymus, in the recipient fetuses. There was a higher level of activity after injection of FHC than after injection of maternal plasma, suggesting an active homing of FHC in these organs.