Fetal Liver CD34 Research Articles

Enhancement of HIV Infection of Human Fetal Liver and Cord Blood CD34+ CD4- Cells Is Mediated by Upregulation of Chemokine Receptor Expression on These Cells

Enhancement of HIV Infection of Human Fetal Liver and Cord Blood CD34+ CD4- Cells Is Mediated by Upregulation of Chemokine Receptor Expression on These Cells

Gene transfer into fetal baboon hematopoietic progenitor cells.

We studied hematopoietic progenitors from fetal baboon blood, marrow, and liver at four time points (125, 140, 160, and 175 days) during the third trimester (gestation approximately 180 days) to determine if fetal baboons might be an appropriate model for in utero gene therapy of hematopoietic stem cells (HSCs). Cells were studied for expression of CD34, CD33, CD38, and HLA-DR, for progenitor content in colony-forming cell assays, and for susceptibility of CD34+ progenitors to retrovirus-mediated gene transfer. Throughout the third trimester, the frequency of CD34+ progenitors in blood and marrow appears to remain unchanged at approximately 0.6 and 5.0%, respectively. In liver, progenitors progressively decrease to undetectable levels by day 175. The proportion of fetal baboon bone marrow and liver CD34+ cells expressing CD38 and HLA-DR appears to increase with increasing fetal age, similar to changes reported for human cord blood CD34+ cells. In fetal baboon blood the proportion of CD34+ cells expressing CD33 appears to decrease with increasing gestational age, also similar to changes reported for human cord blood cells. Progenitors from human cord blood and baboon fetal tissues were similarly susceptible to transduction by the gibbon ape leukemia pseudotyped retroviral vector LAPSN(PG13) containing the genes for human placental alkaline phosphatase (AP) and the bacterial neomycin phosphotransferase (neo). Fetal baboon and human hematopoietic progenitor cells undergo similar phenotypic changes during the third trimester of fetal development and are similarly susceptible to retrovirus-mediated gene transfer. The fetal baboon may be a model in which approaches to mobilization and gene transfer into fetal HSCs can be studied.

Expression and function of cell adhesion molecules on fetal liver, cord blood and bone marrow hematopoietic progenitors: Implications for anatomical localization and developmental stage specific regulation of hematopoiesis

The mechanism of localization, migration, and regulation of hematopoiesis at different stages of ontogeny is not well understood, but may relate to the specific adhesive interactions between hematopoietic stem cells and their microenvironment at different ontogenic stages. We studied the expression of cell adhesion molecules (CAM) on fetal liver (FL), umbilical cord blood (UCB) and adult bone marrow (ABM) CD34 + cells, and the adhesion of committed progenitors (CFC) from all three sources to ABM stromal layers and purified extracellular matrix proteins. Compared to ABM CFC, significantly more UCB CFC and fewer FL CFC adhered to ABM stroma. Adhesion of FL CFC to fibronectin (FN), the 75 kD RGD containing FN fragment and the 33–66 kD COOH-terminal heparin binding FN fragment was also significantly less than that of ABM CFC. Like ABM CFC, the adhesion of FL CFC was mediated through α4β1 and α5β1 integrins. Of note, more FL CD34 + cells expressed α5 integrins and the number of α4, α5 and β1 integins per cell (mean channel frequency) was similar or higher for FL CD34 + cells than ABM CD34 + cells. Further, treatment of FL CFC with a β1 integrin activating antibody (8A2), increased adhesion of FL CFC to FN to the same level as that of 8A2 treated ABM CFC. This suggests that the α4β1 and α5β1 integrins on FL CD34 + cells may be present in a low avidity/affinity state. We also show that unlike ABM, FL CD34 + cells expressed α2 and that approximately 20% FL CFC adhered to collagen IV. Further, α2β1 integrin on FL CFC was functional since their engagement, either by adhesion to collagen IV or through blocking α2 antibodies, transmitted proliferation inhibitory signals. In contrast to α4b and α5β1 integrin dependent adhesion, α2β1 dependent adhesion of FL CFC to collagen IV was not enhanced after treatment with 8A2. The reason for this is not clear but suggests that α2 integrins on FL CFC are maximally activated. This novel adhesive interaction with collagen IV, reminiscent of that described for CML progenitors, may have a role in the extramedullary localization of FL hematopoiesis or its developmental stage-specific regulation by its microenvironment. Studies to evaluate these possibilities are underway.

In Vitro Intrathymic Differentiation Kinetics of Human Fetal Liver CD34+CD38− Progenitors Reveals a Phenotypically Defined Dendritic/T-NK Precursor Split

Human CD34+CD38- hematopoietic precursor cells from fetal liver are able to develop into T, NK, and dendritic cells in a hybrid human/mouse fetal thymic organ culture (FTOC). In this report, we pay particular attention to the early events in differentiation of these precursor cells. We show that the CD34+CD38- precursor cells, which are CD4-CD7-cyCD3-HLA-DR-/++ (cy, cytoplasmatic), differentiate into a CD4+ population that remained CD7-cyCD3-HLA-DR++ and a CD4- population that expressed CD7 and cyCD3. The CD4+CD7-cyCD3- cells differentiate into phenotypically and functionally mature dendritic cells, but do not differentiate into T or NK cells. The CD4-CD7+cyCD3+ population later differentiates into a CD4+CD7+cyCD3+HLA-DR- population, which has no potential to differentiate into dendritic cells but is able to differentiate into NK cells and gammadelta and alphabeta T lymphocytes. These findings support the notion that the T/NK split occurs downstream of the NK/dendritic split.

The expression and differentiation pattern of cell antigens and adhesion molecules on the nonadherent cell population in canine long‐term marrow culture: a biphasic development of myeloid and lymphoid cells

During maturation of normal hematopoietic progenitors, there appears to be differentiation-dependent expression of adhesion receptors, which may contribute to the homing and lodging of stem progenitor cells into the marrow and for the eventual release of mature effector cells from the marrow cavity. Using a model of long-term marrow culture, we studied the expression pattern of different lineage cell antigens and cell adhesion molecules on the nonadherent cells in canine long-term marrow culture. CD4+ cells became a major proportion of both the small and large cell subsets by day 8 of culture. Small CD4+ cells, the majority of which are negative for other T-cell antigens during the first 2 weeks of culture, express low levels of CD4 (CD4lo) and coexpress granulocytic and/or monocytic markers. These CD4lo cells have progenitor potential as measured by the long-term culture-initiating cell assay and differentiate into myeloid (first wave) and lymphoid (second wave) cells. The T cells, which appear in 2-week-old long-term marrow culture, respond to Con A but not to alloantigen. At the same time, most of the large cells are CD14+, CD11b+, DLA-DR+ and CD4lo+, while granulocytes are not observed. This phenotypic pattern closely resembles that found on myelomonocytic cells developing from fetal thymic and fetal liver CD34+ precursors in a model of human fetal thymic organ culture. The cell adhesion molecules--CD44, CD18, L-selectin, CD11a-c as well as VLAalpha4, dramatically increase from the first week of long-term marrow culture, while ICAM-1 and a new beta2 cell adhesion molecule, alpha dbeta2, increased slightly from the third week on. In the large ("monocytic") cell population, alpha dbeta2 was exclusively expressed by CD4+ cells. The differentiation pattern of T-cell antigens and adhesion molecules seen in the canine long-term marrow culture appear to mimic those required for developmental interactions between leukocytes and endothelial cells; that is, an early expression of L-selectin and CD44, followed by the integrins, and later on by ICAM-1 and alpha dbeta2. Our data support the view that in a model of canine long-term marrow culture hematopoietic precursors retain their intrinsic developmental potential.

Differential infection of CD34+ cell-derived dendritic cells and monocytes with lymphocyte-tropic and monocyte-tropic HIV-1 strains.

Monocytes and dendritic cells are infected by HIV-1 and subsequently produce virions that initiate further rounds of infection. Current methods for the isolation and study of dendritic cells are hampered by the low frequency of these cells and contamination with other cell types. A two-step culture method was devised to generate large numbers of either dendritic cells or monocytes from fetal liver CD34+ progenitors. CD34+ cells were first expanded with the growth factors granulocyte-macrophage CSF and stem cell factor to generate a population of intermediate progenitor cells with a relatively immature phenotype. To induce specific differentiation to dendritic cells, the cultures were switched to serum-free medium with the growth factors granulocyte-macrophage CSF, stem cell factor, TNF-alpha, and IL-4. The cells became highly positive for HLA class II Ags and the dendritic cell marker CD1a. Culture of the intermediate progenitors in serum-containing medium with macrophage CSF resulted in differentiation to adherent monocytes expressing high levels of CD14 with low CD1a expression. The intermediate progenitors were permissive for HIV infection by both monocyte- and lymphocyte-tropic strains. In contrast, differentiation to monocytes or dendritic cells resulted in restricted viral tropism. Dendritic cells efficiently replicated the lymphocyte-tropic virus HIV-1MN, but not the monocyte-tropic virus HIV-1ADA. As expected, monocytes only supported replication of HIV-1ADA. This two-step culture method allows for the production of large numbers of monocytes or dendritic cells from a common precursor pool for studying the development of tropism-associated events.

Lymphoid and myeloid differentiation of fetal liver CD34+lineage- cells in human thymic organ culture.

In this article, we report that the human fetal thymus contains CD34bright cells (< 0.01% of total thymocytes) with a phenotype that resembles that of multipotent hematopoietic progenitors in the fetal bone marrow. CD34bright thymocytes were CD33-/dull and were negative for CD38, CD2, and CD5 as well as for the lineage markers CD3, CD4, and CD8 (T cells), CD19 and CD20 (B cells), CD56 (NK cells), glycophorin (erythrocytes), and CD14 (monocytes). In addition, total CD34+ lineage negative (lin-) thymocytes contained a low number of primitive myeloid progenitor cells, thus suggesting that the different hematopoietic lineages present in the thymus may be derived from primitive hematopoietic progenitor cells seeding the thymus. To investigate whether the thymus is permissive for the development of non-T cells, human fetal organ culture (FTOC) assays were performed by microinjecting sorted CD34+lin- fetal liver cells into fragments of HLA-mismatched fetal thymus. Sequential phenotypic analysis of the FTOC-derived progeny of CD34+lin- cells indicated that the differentiation into T cells was preceded by a wave of myeloid differentiation into CD14+CD11b+CD4dull cells. Donor-derived B cells (CD19+CD20+) were also generated, which produced immunoglobulins (IgG and IgM) when cultured under appropriate conditions, as well as functional CD56+CD3- NK cells, which efficiently killed K562 target cells in cytotoxicity assays. These results demonstrate that the microinjection of fetal liver hematopoietic progenitors into fetal thymic organ fragments results in multilineage differentiation in vitro.