The AFT024 stromal cell line supports long-term ex vivo maintenance of engrafting multipotent human hematopoietic progenitors.

The immortalized murine stromal cell line AFT024 has been reported to maintain human hematopoietic progenitors in an undifferentiated state in vitro. In the current studies the beige/nude/xid (bnx) mouse in vivo xenograft model was used to examine the engraftment and multilineage generative potential of human hematopoietic progenitors after 2-3 weeks growth on AFT024 stroma, in comparison to primary stromal monolayers derived from post-natal human bone marrow. Eight to 12 months after transplantation of human CD34+CD38- cells from umbilical cord blood, cultured on AFT024 vs human stroma for 2-3 weeks, the murine bone marrow was harvested and analyzed for the presence of human myeloid and lymphoid cells. The mean percent engraftment of total human hematopoietic cells in the murine marrow was significantly higher after co-cultivation on AFT024 than on human stroma. Human myeloid and lymphoid lineage cells were detected in all mice. However, engraftment of myeloid lineage cells (CD33+), B lymphoid (CD19+), and T lymphoid cells (CD4+and CD8+) were significantly higher after co-cultivation of the human cells on AFT024 than on human stroma, prior to transplantation. Interestingly, the length of time in culture did not significantly affect the engraftment of the myeloid and T lymphoid lineage progenitors, but the percentage of B lymphoid lineage engraftment decreased significantly between 2 and 3 weeks of co-cultivation on both types of stroma. Cells with a primitive phenotype (CD45+/CD34-/CD38- and CD45+/CD34-/lin-) and cells with the capacity to generate secondary human CFU after recovery from the bnx bone marrow were maintained at significantly higher levels during culture on AFT024 stroma than on human stroma. The current studies demonstrate that the AFT024 murine stromal cell line supports the ex vivo survival and maintenance of human hematopoietic progenitors that are capable of long-term multilineage reconstitution for 2-3 weeks ex vivo, to levels superior to those that can be obtained using human stromal cells.

Evaluation of a new simple and rapid enzyme-linked immunosorbent assay kit for neopterin determination.

A new commercially available enzyme-linked immunosorbent assay (ELISA) kit has been evaluated for the measurement of neopterin concentrations in serum, plasma and urine. This competitive ELISA is technically simple, requires only small sample volume and is rapid to perform. The assay procedure consists of sequential 1.5 h and 10 min room temperature incubation steps. The ELISA is accurate, sensitive, specific, and precise. Linear regression analysis of neopterin concentrations measured with the new ELISA and with an established method yielded a highly significant correlation (r = 0.99). The new assay is applicable to ELISA workstations, thus enabling determination of neopterin in large series of samples. The neopterin ELISA kit has been used in routine laboratory testing of blood donations in a blood bank.

The number and generative capacity of human B lymphocyte progenitors, measured in vitro and in vivo, is higher in umbilical cord blood than in adult or pediatric bone marrow.

The lack of human B lymphocyte development in beige/nude/XID (bnx) mice is in sharp contrast to the robust development observed in another immune deficient strain, the NOD/SCID mouse. The ability to generate human B lymphocytes in the NOD/SCID, but not bnx mouse has been hypothesized to be caused by differences in the microenvironments or systemic cytokine concentrations. In the current studies we report that the differences in development can be primarily attributed to the source of the progenitors transplanted into the mice. The prior studies in bnx mice used cultured pediatric or adult bone marrow (BM) as the source of the CD34+ cells, whereas the NOD/SCID studies have predominantly used fresh or cultured umbilical cord blood (UCB). We have analyzed BM and UCB for the number of human CD34+/CD38- cells capable of in vitro B lymphocyte development, and have found a lower frequency of B lymphocyte generation in BM. The individual B lymphocyte clones that developed from bone marrow produced 100-fold fewer cells than the UCB-derived clones. In agreement with the in vitro studies, human B lymphocytes developed in bnx mice from both CD34+ and CD34+/CD38- cells isolated from human umbilical cord blood, but not from equivalent numbers of CD34+ and CD34+/CD38- progenitors from bone marrow. Therefore, the lower generative capacity, and frequency of B lymphocyte precursors in human marrow may be responsible for the previous results that showed a lack of B lymphocyte development in bnx mice.

The murine stromal cell line AFT024 acts specifically on human CD34+CD38- progenitors to maintain primitive function and immunophenotype in vitro.

A stromal cell line derived from murine fetal liver (AFT024) has been demonstrated to maintain long-term repopulating murine stem cells for up to 7 weeks in vitro. We evaluated the ability of AFT024 to maintain the immunophenotype and function of primitive human progenitors in vitro by comparing the cocultivation of CD34+CD38 cells on AFT024 with that on primary human stroma (HS). We have previously reported that within the CD34+CD38- population of bone marrow and cord blood, a highly primitive progenitor subpopulation can be identified functionally by its ability to generate colony forming unit-cells (CFU-Cs) in extended long-term culture (ELTC), that is, beyond 60 days of stromal cocultivation. Cocultivation of bone marrow and cord blood CD34+CD38-cells on AFT024 produced significantly greater cell expansion (p=0.0002) and CFU-C output (p=0.0007) during the ELTC period compared with culturing on HS. CFU-C production continued up to 9 weeks longer on AFT024 stroma. After 3 to 4 weeks of bulk culture on either AFT024 or HS, cells were replated in a limiting dilution to measure the number of cobblestone area-forming cells (CAFCs) maintained on each stroma. AFT024 maintained significantly more CAFCs than did HS (n=3, p=0.002). Fluorescence-activated cell sorter analysis of AFT024 and HS cocultures showed that both the frequency (p=0.018) and absolute number (p=0.027) of CD34+CD38- cells were significantly higher in cultures on AFT024 than in those on HS (n=9). The effects of AFT024 on preservation of primitive progenitors were not seen in transwell (noncontact) cultures. Thus, AFT024 acts by direct contact to maintain the phenotype and function of the most primitive and quiescent human progenitors currently identifiable by in vitro assays.

Timing of the expression of enamel gene products during mouse tooth development.

In order to understand the mechanisms involved in tooth development it is important to define the timing for tissue-specific gene expression. A consequence of ameloblast cell differentiation is the sequential expression of tissue-specific genes whose products form the enamel extracellular matrix. The ameloblast phenotype has been characterized as consisting of two major classes of proteins: amelogenins and non-amelogenin proteins such as anionic enamel proteins (enamelins, tuft proteins, tuftelin, sulfated proteins) and enamel proteases. The postulated functions for the anionic enamel proteins are as nucleators for hydroxyapatite crystal formation while amelogenins control the crystal size, growth and orientation. While the amelogenins have been well characterized, detailed knowledge for anionic enamel proteins has been sparse. In the present study, we designed experiments to characterize one of the anionic enamel proteins from mouse molars, tuftelin, and to determine the timing of expression of this protein during molar tooth development. Our results showed the initial detection of tuftelin transcripts within proliferating inner enamel epithelial cells at very early stages of tooth development (13 days of embryonic development equivalent to the bud stage of tooth development). These data provide direct evidence that invalidates previous dogmas that enamel proteins were synthesized by polarized, non-dividing, fully differentiated ameloblast cells. In addition, tuftelin was found to be synthesized also by dental papilla mesenchyme cells suggesting that this protein is not enamel-specific. These data taken together open the possibility that the tuftelin present in the dentino-enamel junction could be secreted by both, preodontoblast cells and preameloblast cells. It might also suggest a possible different role for tuftelin than nucleator of hydroxyapatite crystals.

Differentiation of human CD34+CD38- cord blood stem cells into B cell progenitors in vitro.

Umbilical cord blood CD34+CD38- cells are a rare, quiescent, primitive progenitor subpopulation lacking expression of lymphoid and myeloid lineage specific antigens. Although myeloid, erythroid, and megakaryocytic differentiation from these cells has been described, B lineage differentiation has not been demonstrated previously. We report here that highly enriched human B cell progenitors can be consistently generated from CD34+CD38- cord blood cells using long-term culture on the murine stromal line, S17, in the absence of added cytokines. After 6-8 weeks, cell numbers increased up to 160-fold, and cultures contained > 80-90% CD10+CD19+ B progenitors. Consistent with previous reports describing delayed myeloid cell differentiation from CD34+CD38- cells, production of B cell progenitors from CD34+CD38- cord blood cells was delayed 2-4 weeks relative to cultures initiated with either CD34+CD38bright or CD34+CD38dim progenitors. Addition of Flt3 ligand to long-term cultures resulted in a 2-3-fold greater increase in the number of CD19+ cells generated after 4-8 weeks. The selective outgrowth of B cell progenitors using this culture model will be useful for a range of in vitro studies using primitive hematopoietic progenitors.

Extended long-term culture reveals a highly quiescent and primitive human hematopoietic progenitor population.

Long-term culture-initiating cells (LTC-IC) are hematopoietic progenitors able to generate colony-forming unit-cells (CFU) after 5 to 8 weeks (35 to 60 days) of culture on bone marrow (BM) stroma and represent the most primitive progenitors currently detectable in vitro. We have recently reported that long-term cultures initiated with CD34+CD38- cells from BM or cord blood are able to continue generating CFU for at least 100 days, ie, beyond the standard LTC-IC period. In this report, single-cell cultures from cord blood and retroviral marking of cord blood and BM were used to study whether the subpopulation of CD34+CD38- cells able to generate CFU beyond 60 days ("extended long-term culture-initiating cells" or ELTC-IC) are functionally distinct from LTC-IC in terms of timing of initial clonal proliferation and generative capacity. All cord blood LTC-IC formed clones of greater than 50 cells by day 30. In contrast, cord blood ELTC-IC proliferated later in culture, 50% forming clones after day 30. Although efficient retroviral marking of LTC-IC was seen (25% to 45%), marking of ELTC-IC was inefficient (< 1%), consistent with a more quiescent progenitor population. There was a positive correlation between time of clonal proliferation and generative capacity. ELTC-IC generated threefold to fourfold more progeny than did LTC-IC (P < .002). These studies show that there is a functional hierarchy of progenitors in long-term culture which correlates with their level of quiescence. By extending the LTC-IC assay, a more primitive progenitor may be studied that may be functionally closer to the human long-term repopulation stem cell in vivo.

A functional comparison of CD34 + CD38- cells in cord blood and bone marrow.

We present cell cycling and functional evidence that the CD34+CD38- immunophenotype can be used to define a rare and primitive subpopulation of progenitor cells in umbilical cord blood. CD34+CD38- cells comprise 0.05% +/- 0.08% of the mononuclear cells present in cord blood. Cell cycle analysis with the fluorescent DNA stain 7-aminoactinomycin D showed that the percentage of CD34+ cells in cycle directly correlated with increasing CD38 expression. CD34+CD38- cord blood cells were enriched for long-term culture-initiating cells (LTCIC; cells able to generate colony-forming unit-cells [CFU-C] after 35 to 60 days of coculture with bone marrow stroma) relative to CD34+CD38- cells. In an extended LTCIC assay, CD34+CD38- cells were able to generate CFU-C between days 60 and 100, clearly distinguishing them from CD34+CD38+ cells that did not generate CFU-C beyond day 40. When plated as single cells, onset of clonal proliferation was markedly delayed in a subpopulation of CD34+CD38- cells; clones (defined as > 100 cells) appeared after 60 days of culture in 2.9% of CD34+CD38- cells. In contrast, 100% of CD34+CD38+ cells formed clones by day 21. Although the CD34+CD38- immunophenotype defines highly primitive populations in both bone marrow and cord blood, important functional differences exist between the two sources. CD34+CD38- cord blood cells have a higher cloning efficiency, proliferate more rapidly in response to cytokine stimulation, and generate approximately sevenfold more progeny than do their counterparts in bone marrow.

Characterization of protein kinases involved in dentinogenesis.

Protein phosphorylation and dephosphorylation control many different cell functions as well as responses to internal and external signals. It has also been shown that highly phosphorylated acidic proteins have an important role in matrix mediated biomineralization, perhaps functioning as nucleators for crystal formation. Dentine phosphoprotein (DPP) is one of such proteins which is exclusively synthesized by the odontoblast cells and therefore a likely candidate to play a significant role in normal and abnormal dentine biomineralization. These studies are directed at characterizing the protein kinases involved in dentinogenesis and in particular the enzyme(s) responsible for DPP phosphorylation. In this report we present data which indicate that there are several different types of kinases in the odontoblast-enriched dental papilla mesenchyme (DPM), some of which can phosphorylate DPP, such as casein kinase I and II. However, a different DPP-kinase activity was identified. This enzyme(s) appears to be different from other reported kinases, and it is the only kinase that can phosphorylate both phosphorylated DPP and enzymatically dephosphorylated DPP.

Temperature sensitive simian virus 40 large T antigen immortalization of murine odontoblast cell cultures: establishment of clonal odontoblast cell line.

During tooth formation instructive epithelial-mesenchymal interactions result in the cytodifferentiation of ectomesenchymal cells into odontoblasts which produce the dentin extracellular matrix (DECM). The purpose of our study was to establish a stable murine odontoblast cell line by immortalization of odontoblasts using retrovirus transfection. In order to accomplish this goal, we utilized a previously characterized odontoblast monolayer cell culture system supportive of odontoblast cytodifferentiation from dental papilla mesenchyme (DPM), expression and secretion of a DECM and dentin biomineralization. First mandibular molars from E-18 Swiss Webster mice were dissected, the DPM isolated, and pulp cells dissociated. Pulp cells (5 x 10(5)/well) were plated as monolayers and grown in alpha-MEM supplemented with 10% FCS, 100 units/ml penicillin and streptomycin, 50 micrograms/ml ascorbic acid. Cultures were maintained for 6 days at 37 degrees C in a humidified atmosphere of 95% air and 5% CO2, with media changes every two days. Immortalization was performed using a recombinant defective retrovirus containing the temperature sensitive SV-40 large T antigen cDNA and the neomycin (G418) resistance gene recovered from CRE packaging cells. Cultures were infected for 24 h with CRE conditioned medium containing 8 micrograms/ml of polybrene, the media was replaced with selective media containing 300 micrograms/ml of G418, and the cultures incubated at 33 degrees C for one month with media changes every 3-5 days. Neomycin resistant cells were cloned by serial dilution to single cells in 96-well culture plates and grown in selection medium at 33 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Alternative splicing of the mouse amelogenin primary RNA transcript contributes to amelogenin heterogeneity.

A heterogeneous population of amelogenin proteins is derived from a single copy of the mouse amelogenin gene. To investigate the one gene--multiple protein enigma, we designed a study to distinguish between alternative splicing and proteolytic cleavage models. A pulse of [35S]methionine labeling demonstrated that multiple amelogenins are synthesized concurrently, a result consistent with an alternative splicing mechanism. Using reverse transcription and polymerase chain reaction we cloned a segment from the 5' end of a mouse amelogenin mRNA and connected it to a previously isolated abbreviated cDNA clone. Four additional cDNAs derived from alternatively spliced amelogenin mRNAs have been cloned and characterized. The five transcripts encode amelogenins 180, 156, 141, 74, and 59 amino acids in length.

The relationship of the internal security system to group level organization in Miller's living systems theory.

It has been suggested that a 20th subsystem, the internal security subsystem, should be added to Miller's 19 matter-energy and information processes that are critical to living systems (Bosserman, 1982). This subsystem protects all levels of living systems from error and disruption resulting from internal and external sources. Yet many characteristics of the proposed security subsystem already are found in existing ones, that is, the reproducer, channel and net, and distributor. Therefore, a discrete internal security subsystem is not justified. There are, however, dormant aspects of the security system that are not described within the existing taxonomy that perhaps best fit as a mirror system across the organizational levels.