No abstract

The purpose of this study was to evaluate human umbilical cord blood as an alternative to bone marrow in the provision of transplantable stem/progenitor cells for hematopoietic reconstitution. Although no direct quantitative assay for human hematopoietic repopulating cells is at present available, the granulocyte-macrophage progenitor cell (CFU-GM) assay has been used with success as a valid indicator of engrafting capability. We examined greater than 100 collections of human umbilical cord blood for their content of nucleated cells and granulocyte-macrophage, erythroid (BFU-E), and multipotential (CFU-GEMM) progenitor cells, in many cases both before and after cryopreservation. First it was determined that granulocyte-macrophage, erythroid, and multipotential progenitor cells remained functionally viable in cord blood untreated except for addition of anticoagulant for at least 3 days at 4 degrees C or 25 degrees C (room temperature), though not at 37 degrees C, implying that these cells could be satisfactorily studied and used or cryopreserved for therapy after transport of cord blood by overnight air freight carriage from a remote obstetrical service. Granulocyte-macrophage progenitor cells from cord blood so received responded normally to stimulation by purified recombinant preparations of granulocyte-macrophage, granulocyte, and macrophage colony-stimulating factors and interleukin 3. The salient finding, based on analysis of 101 cord blood collections, is that the numbers of progenitor cells present in the low-density (less than 1.077 gm/ml) fraction after Ficoll/Hypaque separation typically fell within the range that has been reported for successful engraftment by bone marrow cells. Another observation of practical importance is that procedures to remove erythrocytes or granulocytes prior to freezing, and washing of thawed cells before plating, entailed large losses of progenitor cells, the yield of unwashed progenitor cells from unfractionated cord blood being many times greater. The provisional inference is that human umbilical cord blood from a single individual is typically a sufficient source of cells for autologous (syngeneic) and for major histocompatibility complex-matched allogeneic hematopoietic reconstitution.

A polypeptide of 8500 molecular weight is described that induces the differentiation of T (thymusderived) cell and B (bone-marrow-derived) cell immunocytes in vitro, apparently via fl-adrenergic receptors and adenylate cyclase activation. This polypeptide shows a high degree of evolutionary conservation, exhibiting close structural, functional, and immunological similarity when isolated from such diverse origins as cells of mammals and higher plants. This polypeptide was detected in animal cells, yeast, bacteria, and higher plants, and so may well be a universal constituent of living cells.The isolation from bovine thymus of the polypeptide hormones thymopoietin I and II has been described previously (1 2). [We are now using the term thymopoietin (3) because valid objections (4) were raised to our previous term "thymin," which is easily confused with the pyrimidine base thymine. ] Pure thymopoietin I and II specifically induce prothymocytes to differentiate into thymocytes (5-7), but these polypeptides were recognized originally not by this evidently physiological faculty of T (thymus-derived) cell differentiation but by a presumably incidental property discovered in the course of studies related to the human disease myasthenia gravis, in which a characteristic neuromuscular disturbance is consistently associated with thymic pathology. When reliable assays of T-cell differentiation in vitro became available, based on the manifestation of T-cell antigens on precursor cells (5,8) it was realized that, although certain pure thymic products (thymopoietins) could induce this step in T-cell differentiation specifically, extracts of tissues other than thymus were also active (10).We describe here the isolation and purification from bovine thymus of a polypeptide subsequently detected in many other tissues, which like thymopoietin induces the differentiation of pro-thymocytes, but which unlike thymopoietin induces differentiation of B (bone-marrow-derived) cells, and is thus distinguished in its mode of action from the physiological inducer thymopoietin. This The isolation of UBIP from bovine thymus is summarized in Fig. 1. Bovine thymus was obtained fresh on wet ice, dissected clean and stored at -20°. Batches were homogenized 25% wet weight/volume in 0.1 M ammonium bicarbonate with a Waring blender. The extract was heated to 700 for 30 min and then centrifuged at 500 X g for 30 min. The supernatant was filtered through gauze and cotton and 0.1% thimerosal was added as a bacteriostatic agent, since the subsequent steps were carried out at room temperature. The extract was processed through a Diaflo XM1OOA membrane (Amicon) in a TCF10 apparatus. The dialysate was then concentrated over a Diaflo UM2 membrane at 55 lb./in.2, using a 402 stirred cell and a reservoir; 3 liters were concentrated to 15 ml. This retentate was fractionated on a 2.5 X 100-cm column of medium Sephadex G-50 (Pharmacia) in 0.1 M ammonium bicarbonate. The fractions shown in Fig. 1 were lyophilized and rerun on the same column. The lyophili...

Ly alloantigens coded by two unlinked genetic loci (Ly-1 and Ly-2/Ly-3) are expressed on lymphoid cells undergoing thymus-dependent differentiation. Peripheral Thy-1+ cells from C57BL/6 mice can be divided into three subclasses on the basis of differential expression of Ly-1, Ly-2, and Ly-3; about 50% express all three Ly antigens (Ly -123+), about 33% only Ly-1 (Ly-1+), and about 6-8% Ly-2 and Ly-3 (Ly-23+). Cells of the Ly-123+ subclasses are the first peripheral Thy-1+ cells to appear in ontogeny, and are reduced in the periphery shortly after adult thymectomy. In contrast, Ly-1+ and Ly-23+ subclasses appear later in the peripheral tissues than do Ly-123+ cells, and are resistant to the early effects of adult thymectomymperiheral lymphoid populations depleted of Ly-1+ cells and Ly-123+ cells (and thereby enriched for Ly-23+ cells) were incapable of developing significant helper activity to SRBC but generated substantial levels of cytotoxic activity to allogeneic target cells. The same lymphoid populations, depleted of Ly-23+ cells and Ly-123+ cells (and thereby enriched for Ly-1+ cells), produced substantial helper responses but were unable to generate appreciable levels of killer activity. These experiments imply that commitment of T cells to participate exclusively in either helper or cytotoxic function is a differentiative process that takes place before they encounter antigen, and is accompanied by exclusion of different Ly groups, Lu-23 or Ly-1 respectively, from TL+Ly-123+ T-cell precursors. It is yet to be decided whether the TL-phase by Ly-123+ subclass is a transitional form or a separately differentiated subclass with a discrete immunologic function.

Peripheral T cells can be subclassified on the basis of differential expression of cell surface antigens belonging to three Ly systems, Ly-1, Ly-2, and Ly-3 (1) . One subclass, bearing all three of these Ly determinants (Ly-123+), appears early in neonatal life and is selectively depleted shortly after adult thymectomy . Two other subclasses, Ly-1+ and Ly-23+, develop later in life and are unaffected shortly after adult thymectomy .Although both Ly-1+ and Ly-23+ subclasses recognize alloantigens of the major histocompatibility complex (MHC),' according to the criterion of thymidine incorporation in the mixed lymphocyte culture (MLC) test, only the Ly-23+ subclass develops killer activity (1) .We are concerned in this report with whether T-cell subclasses other than Ly-23 are indirectly involved in the genesis of killer cells, and we shall show: (a) that the development of killer activity by Ly-23+ T cells is amplified by Ly-1+ T cells [which we know provide helper activity during primary antibody responses (1) ], and (b) that the amplifying Ly-1+ T cells probably recognize alloantigens different from those recognized by the Ly-23+ prekiller cells. Materials and MethodsMice . The Ly congenic mouse strains B6/Ly-1