Murine Sca-1(+)/Lin(-) cells and human KG1a cells exhibit multiple pseudopod morphologies during migration.

OBJECTIVE: The migration of primitive hematopoietic cells has been studied mostly via population-based assays while the actual mechanisms of cell motion have been defined by tracking individual mature cells. In this report, we examined individual immature hematopoietic cells to determine if any notable differences in migration mechanisms exist due to the primitive nature of the cells. MATERIALS AND METHODS: Murine cells of the Sca-1(+)/Lin(-) phenotype were isolated from C57BL/6 mice using Miltenyi bead purification and flow cytometric sorting. These cells were then observed for long periods of time with an environmentally controlled time-lapse microscope system in either multiwell plates or micropore transwell chambers. Experiments were also performed with the human KG1a immature hematopoietic cell line. RESULTS: Murine Sca-1(+)/Lin(-) immature hematopoietic cells and human KG1a cells were observed to exhibit a variety of mechanisms/morphologies during migration, which include the classic "hand mirror" shape; broad, flat lamellipodia; trailing uropodia; dynamic filopodia; and retraction fibers. Time-lapse observations of transmembrane assays revealed long, thin magnupodia passing through the pores, while other measurements show magnupods can generate forces capable of accelerating a cell to a velocity of 5 microns/minute. CONCLUSION: Many of these mechanisms have been reported separately for differentiated cells; however, we show that immature hematopoietic cells are capable of exhibiting all of these mechanisms of migration. These data provide insight into the loss of phenotypic functions as stem cells differentiate.

Alloreactivity following in utero transplantation of cytokine-stimulated hematopoietic stem cells: the role of recipient CD4(-) cells.

OBJECTIVE: We have previously reported immunity to donor antigens following in utero transplantation (IUT) of cytokine-stimulated allogeneic hematopoietic stem cells (sca(+)/lin(-)) (day 9 of gestation). Transplanted mice showed accelerated rejection of donor skin grafts and high anti-donor cytotoxic response, a finding not seen in the control mice. This was accompanied by an enhancement of Th1 over Th2 cytokine production and persistent donor microchimerism. In order to assess the role of the thymus in allograft rejection, prenatal transplants were performed under similar experimental conditions at a later gestational age, when the thymus is more developed (day 13). MATERIALS AND METHODS: Cytokine-stimulated stem cell factor (SCF) and granulocyte colony-stimulating factor (G-CSF)-purified sca(+)/lin(-) cells of C57BL/6 (H-2b, 1E(+)) background were injected into MHC-mismatched BALB/c (H-2d, 1E(-)) fetal mouse recipients at day 13 of gestation. Chimerism was determined by highly sensitive (0.001%) semiquantitative polymerase chain reaction (PCR). Mixed lymphocyte reaction (MLR) and cytotoxic T-cell assay (CTL) were used to evaluate tolerance vs immunity. Cytokine levels were quantified in MLR supernatants using ELISA assay. The percent of T cells was determined by flow cytometry (FACS) and CD4/CD8 ratio calculated. Postnatal boosts (transplants without conditioning) were performed at 6 months of age to enhance donor chimerism and test the degree of tolerance. RESULTS: When assayed at 4 months of age, donor-type cells were not detected in the spleen or in the peripheral blood of BALB/c mice inoculated with C57BL/6 sca(+)/lin(-) cells. Transplanted but not control animals demonstrated high anti-donor MLR but not CTL responses. The increase of MLR reactivity was correlated with high levels of IL-2. Furthermore, transplanted mice showed higher resistance to postnatal boosts with allogeneic bone marrow (BM) cells, when compared to the control mice. The later resistance was accompanied by the expansion of host-type CD4 cells. CONCLUSION: These data demonstrate that transplantation of cytokine-stimulated sca(+)/lin(-) allogeneic cells at 13 days of fetal development leads to the allosensitization, characterized by an enhancement of MLR alloreactivity and by the rejection of postnatal boosts (transplants without conditioning). Host-type CD4 cells might play a central role in this rejection. These findings indicate that the late injection of allogeneic cells may result in the development of allosensitization with subsequent donor graft rejection. Precise conditions for the development of tolerance must be established before prenatal transplants in humans with conditions other than SCID can be done.

Chimerism and tolerance post-in utero transplantation with embryonic stem cells.

BACKGROUND: Clinical application of in utero transplantation (IUT) in human fetuses with intact immune systems resulted in a very low level of donor chimerism. In this study, we examined whether the fetal immune system early in the second trimester of pregnancy (13.5 dpc) can initiate immune tolerance for major histocompatibility complex (MHC)-mismatched embryonic stem (ES) cells. We also examined whether immune tolerance mechanisms respond differently to ontogenetically different stem cells. METHODS: MHC-mismatched ES, fetal liver (FL), and bone-marrow (BM) cells (H-2kd) at 1 x 10(9) cells/kg fetal body weight were injected intraperitoneally into 13.5 dpc BALB/c fetuses (H-2Kd). Peripheral chimerism was determined in blood by flow cytometry (sensitivity< or =0.1%) at monthly intervals. Donor-specific immune responses were determined by cytotoxic lymphocyte (CTL) assay, mixed lymphocyte reaction, and T helper (Th)1 and Th2 cytokine assays. Chimeric mice at the age of 9 months received postnatal boosts (PB) with minimal conditioning of 200 cGy by intravenous injection of 1 x 10(9) of the corresponding cells/kg body weight. RESULTS: After IUT with ES, FL, or BM cells, the level of peripheral chimerism within the first 9 months of life was 0% to 0.4%. PB with 1 x 10(9)/kg of corresponding cells resulted in a decrease in the peripheral chimerism to 0% within 2 weeks of PB. CTL and cytokine assays before and after PB demonstrated a shift toward immunity. CONCLUSIONS: Immunologic tolerance was not achieved after IUT of murine fetuses at 13.5 dpc with MHC-mismatched ES cells, and only a low level chimerism was achieved.

Non-myeloablative transplants for congenital diseases.

The morbidity and mortality associated with postnatal HSCT, toxicity of HSCT conditioning regimens, lifelong immunosuppressive therapy, and lack of compatible donors discourages many patients and physicians from utilizing postnatal HSCT as a treatment for congenital disease. Non-myeloblative in utero HSCT is now being considered as an alternate treatment with the hope that it will be more therapeutic with less toxicity to a wider spectrum of patients with congenital disorders. Prenatal stem cell transfer may eliminate many of the risks and hazards associated with postnatal HSCT, as the fetus may be less reactive than an immunologically mature individual such that tolerance to donor cells could be developed. GVHD and rejection of postnatal therapeutic grafts may be minimized thus reducing or eliminating altogether the need for postnatal myeloablation and immunosuppression. Much work must be done both in animal studies as well as in clinical trials. By using well-designed murine models such as the beta-thalassemic mouse outlined above, we believe we can determine the optimal conditions for non-myeloablative postnatal transplants with allogeneic or haplocompatible HSC following prenatal tolerance induction with these cells. In addition, we may answer basic immunology questions regarding the development and regulation of immunity and tolerance in both mice and humans.