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Mononuclear cells from human peripheral blood were purified by semicontinuous flow centrifugation (SCFC) using the Haemonetics model 30 blood cell separator; 64% +/- 7% of the mononuclear cells in 600 ml of peripheral blood were collected in a 30-ml volume. Analysis of sequential 5-ml aliquots of the mononuclear cell concentrate revealed that both immunocompetent cells and granulocytic progenitor cells (CFU-C) were proportional to the cell count throughout the buffy coat. In vitro pheresis of large volumes of human bone marrow resulted in recovery of 63% of the cells, 12% of the hemoglobin, and 84% of the CFU-C in 20% of the original volume. Further centrifugation eliminated 80% of the platelets without loss of cells or CFU-C. SCFC of peripheral blood or bone marrow selectively concentrated mononuclear cells and reduced the contamination by granulocytes and erythrocytes. Large numbers of mononuclear cells can thus be collected for studies in vitro or for cryopreservation and the autologous reconstitution of immunosuppressed or myelosuppressed patients undergoing intensive antitumor therapy.
Recent studies suggest that megakaryocytopoiesis is governed by a dual-level regulatory process, with megakaryocyte colony-stimulating factor (Meg-CSF) primarily influencing proliferation of the committed precursors and thrombopoietin required for megakaryocyte ploidy amplification and for maturation. The authors have examined different sources of Meg-CSF in a microagar culture system with a view to their capacity to enhance megakaryocyte colony formation directly or via an indirect T-lymphocyte- or monocyte-mediated effect. The comparative influences of phytohemagglutinin-stimulated leukocyte-conditioned medium (PHA-LCM), erythropoietin (Epo), sera of patients with severe aplastic anemia, and direct PHA addition to the culture were evaluated for their capacity to enhance megakaryocytic colony formation as well as for the maturation rate of megakaryocytes (Mk) grown in our microagar culture system. Each treatment by itself enhanced colony formation from unseparated low-density cells. Removal of T-lymphocytes and monocytes from the bone marrow sample caused a cessation of the enhancing effect of direct PHA addition to cultures stimulated with Epo, but did not influence the enhancing activities of severe aplastic anemia serum (SAA), PHA-LCM, and Epo. The results show that SAA serum, Epo, and PHA-LCM induced Mk colony formation directly and therefore may act via a common mechanism. Differences, however, were observed concerning their colony-stimulating potency and their influence on the Mk maturation rate.
Cytogenetic examination of bone marrow cells was performed in 43 patients with myelodysplastic syndrome (MDS). MDS was diagnosed from bone marrow biopsies and smears according to the FAB classification. Of all 43 patients 24 (56%) had clonal karyotype changes including frequently monosomies of chromosomes #5 and #7 as well as interstitial deletions of the long arm of #5, 5 q-. Chromosome aberrations were observed in patients belonging to all FAB-subgroups, e.g. 11/15 patients with RA, 6/10 with RAEB, 5/9 with RAEB/T, and 1/9 with CMMoL. Complex chromosome aberrations involving more than 2 chromosomes occurred predominantly in patients with RAEB/T (4/9) but also in patients with RA (2/15) and RAEB (2/10), and correlated significantly (p less than 0.002) with a shorter survival. No correlation was found between chromosome aberrations and the development of ANLL in 9/43 patients (21%).
Embryonal carcinoma(EC) cells, the undifferentiated stem cells of teratocarcinomas, have many properties in common with pluripotent embryonic cells, and thus provide an excellent system for studying the early events involved in embryonic development and stem cell differentiation. We have isolated three novel mutants with temperature-sensitive(ts) cell growth that were able to differentiate at a non-permissive temperature for cell growth. These mutations affect the progression of the cell cycle, leading to the transient accumulation of cells in a specific phase, the S phase, of the cell cycle, which is likely to be the primary cause of stem cell differentiation of EC cells at non-permissive temperature. Isolation of these mutants strongly supports the notion that there is a close association between the inhibition of DNA synthesis and EC cell differentiation.
This report describes an allogeneic peripheral blood stem cell transplant in a patient who had received marrow ablative therapy. The patient was an 18-year-old white male with acute lymphocytic leukemia in third remission for whom an allogeneic bone marrow transplant was recommended. His HLA-identical sibling preferred to donate peripheral blood stem cells rather than marrow. The donor cells were collected with 10 apheresis procedures and depleted of T lymphocytes to prevent excessive graft-versus-host disease. Nine collections were cryopreserved. The patient received high-dose cytosine arabinoside and 12 Gy of total body irradiation, followed by infusion of all cryopreserved donor cells. A portion of the tenth apheresis product collected on the day of transplant containing 1.8 x 10(9) T lymphocytes was infused without further processing to approximate the number of T lymphocytes given in an allogeneic bone marrow transplant; the remainder was T lymphocyte depleted and infused. More than 1 x 10(9)/l granulocytes were present on day +11. A bone marrow biopsy on day +27 showed trilineage engraftment. Cytogenetic studies demonstrated that the recipient's marrow and peripheral blood were populated exclusively with donor cells. Allogeneic peripheral stem cell transplantation produced an early hematopoietic engraftment. Since the patient died on day +32, sustained engraftment could not be evaluated.
Murine embryonal carcinoma and embryonic stem cell lines were investigated with regard to the occurrence of the small heat shock protein hsp25 during cell growth and differentiation. In the embryonal carcinoma cell line F9 considerable constitutive levels of hsp25 were observed which could be slightly increased by treatment with retinoic acid. No hsp25 was found, however, in the embryonal carcinoma cell line PCC4. When analyzing the pluripotent embryonal carcinoma cell line P19 and the pluripotent embryonic stem cell line BLC6, both characterized by high differentiation capacity, no hsp25 was observed under cell culture conditions maintaining the undifferentiated state. Induction of differentiation caused by prolonged cell culture, retinoic acid treatment, or embryoid body formation, however, resulted in an increase of the level of hsp25. The finding that hsp25 is accumulated in a differentiation-dependent manner suggests that this protein is associated with processes involved in differentiation. Therefore, hsp25 can be regarded as a marker of differentiation in the investigated embryonal carcinoma cell line P19 and the embryonic stem cell line BLC6.
The influence of bilateral adrenalectomy on hemopoietic stem cell (CFU) migration in mice has been studied. Formation of endogenous spleen colonies in lethally irradiated, leg-shielded mice was sharply increased by prior adrenalectomy, and this increase was not dependent on the volume of shielded bone marrow. Adrenalectomy was shown to increase endogenous spleen colony formation in sublethally irradiated mice as well. However, it had no affect on formation of spleen colonies in lethally irradiated mice injected with syngeneic bone marrow. The CFU content of murine bone marrow decreased acutely after removal of the adrenals, and this decrease was accompanied by a concomitant increase in the peripheral blood and splenic CFU. Thus, adrenalectomy appeared to have no affect on the splenic plating efficiency or proliferative rate of hemopoietic stem cells, but it did result in increased migration of stem cells from the bone marrow to the blood, and thence to the spleen. It is concluded that the adrenal steroids may be of physiologic importance in the regulation of ehmopoietic stem cell migration.
Systematic comparison of the properties of oligodendrocyte-type-2 astrocyte (O-2A) progenitor cells derived from optic nerves of perinatal and adult rats has revealed that these two populations differ in many fundamental properties. In particular, O-2A(perinatal) progenitor cells are rapidly dividing cells capable of generating large numbers of oligodendrocytes over a relatively short time span. Oligodendrocyte differentiation generally occurs synchronously in all members of a clone, thus leading to elimination of that clone from the pool of dividing cells. However, some O-2A(perinatal) progenitors are also capable of giving rise to O-2A(adult) progenitors. These latter cells express many of the characteristics of stem cells of adult animals, including the capacity to undergo asymmetric division and differentiation. We suggest that precursors which function during early development give rise to terminally differentiated end-stage cells and to a second generation of precursors with properties more appropriate for later developmental stages. It is this second generation of precursors which express the properties of stem cells in adult animals, and we therefore further suggest that our work offers novel insights into the possible developmental origin of stem cells.
The present investigation is concerned with the morphological changes observed in human testicular tissue following prolonged estrogen administration. Testicular material obtained from 11 transsexual patients who had been submitted to long-term estrogen treatment prior to sex-reversal surgery was studied by means of light- and electron microscopy. The testes of all patients examined present a more or less uniform appearance: There are narrow seminiferous cords surrounded by an extensively thickened lamina propria. They contain Sertoli cells and spermatogonia exclusively. There is no evidence of typical Leydig cells. The persisting spermatogonia show the characteristic features of pale type-A spermatogonia, whereas dark type-A spermatogonia are almost completely eliminated from the epithelium. In view of the fact that spermatogonia that survived radiotherapy and treatment with various noxious agents have recently been regarded as the stem cells of the human testis, it is suggested that also the majority of those spermatogonial types that are less sensitive to disturbances of the endocrine balance may consist of stem cells. The present results, therefore, corroborate the concept that the stem cells of the human testis may be derived from pale type-A spermatogonia or the variants of this cell type. Sertoli cells display two types of ovoid nuclei. In contrast to untreated material the nuclei lie adjacent to the basal lamina, and organelles and telolysosomes are confined to the apical cytoplasm. The apico-basal differentiation of mature cells, therefore, is not observed. Moreover, typical organelles and inclusions of mature cells are absent, as are the junctional specializations. Thus, Sertoli cells have transformed into immature cells, resembling precursors prior to puberty. Fibroblast-like cells in the interstitial tissue, which display strongly lobulated nuclei, a well-developed smooth endoplasmic reticulum, lipid droplets, and numerous inclusions are assumed to represent dedifferentiated Leydig cells. Since after estrogen treatment serum testosterone and gonadotropin levels are known to be reduced, it appears that the morphological changes correlate well with the endocrine status.
It is widely believed that hemopoietic stem cells have to be mobilized from extravascular sites into the circulation to guarantee a sufficient and safe blood stem cell autograft. The question of using mobilized or non-mobilized stem cells for transplantation purposes addresses the quality of hemopoietic engraftment rather than its feasibility. Two aspects are of clinical relevance: 1. The increment of peripheral cell concentration per time, and 2. shortening the duration of total aplasia following myeloablation and stem cell transplantation. When comparing the various stem cell mobilization techniques the CFU-GM yield per apheresis was highest during rh GM-CSF application (250 micrograms/m2/day continuous i.v. infusion), whereas the MNC yield was not greatly affected. More severe side effects were seen during rh GM-CSF infusion: One patient experienced an axillary phlebothrombosis. In a series of 15 advanced stage Hodgkin's lymphoma patients the reconstitutive ability of the various stem cell autografts, whether chemotherapy-, cytokine-, or non-mobilized, did not vary. Particularly in acute leukemias, mobilization of hemopoietic precursor cells does not necessarily exclude a concomitant mobilization of clonogenic tumor cells, and, therefore, the probability of disease-free survival after ABSCT might be lower when using mobilized stem cells for transplant.
Circulating stem cells exist in sufficient numbers in mouse, dog, and man to allow collection and transplantation after ablative treatment. Preclinical studies in the mouse have shown a low concentration, with a transplantation potential ratio of bone marrow to blood of 1:100. The ratio of circulating stem cells to bone marrow stem cells is more favorable in the dog (1:10-20). Recent pilot studies carried out in different centers with 10 patients have shown that this approach is feasible in man, too. It appears that 5 X 10(8) mononuclear cells/kg of body weight collected by seven or eight leukapheresis procedures of about 4 hrs each is sufficient for fast hemopoietic recovery after marrow ablative treatment. Potential advantages of the use of blood stem cells over bone marrow stem cells are the decreased likelihood of contamination with malignant cells, the avoidance of general anesthesia, and the infusion of immunocompetent cells, which might hasten immunorecovery in the autologous setting.
We studied the capacity of bone marrow hemopoietic stem cells to form colonies (colony forming units, CFU) in the spleens of irradiated recipient mice following incubation of the cells with T cell-derived mediators in short-term culture in vitro. The mediators were a) stem cell-activating factor (SAF) present in crude form in the supernatant from phytohemagglutinin (PHA); stimulated lymphocytes, and b) immunoenhancing factor (IEF) obtained in partially purified form from the supernatant of antigen-stimulated lymphocytes and characterized by its ability to enhance antibody formation in vitro. Both SAF and IEF increased the number of CFU in the cultured bone marrow cell suspension. However, only SAF, and not IEF, significantly stimulated DNA synthesis in the cultured cells, Furhter more, SAF appears to activate CFU in vitro, rather than merely to promote the survival of active CFU stem cells. Experiments with SAF and bone marrow from different strains of mice indicated that the biologic activity was not restricted by K and Ia regions of H complex.
The in vitro study of stem-cell-stromal-cell interactions has previously been made possible by the existence of a technique for long-term murine marrow culture, but the occurrence of both stem cells and stromal cells in fresh bone marrow (BM) explants and the heterogeneity of stromal cells have delayed functional categorization. Therefore, single-cell suspensions of murine BM were passed over nylon wool columns to allow fractionation of cells having distinctive function for in vitro hemopoiesis. A subpopulation of nylon-column-nonadherent (NNA) cells (20% +/- 1% total cells) is devoid of stromal progenitors that form the in vitro microenvironment, but the NNA subpopulation has control numbers of hemopoietic colony-forming cells, GM-CFU-C, and high-proliferative-potential colony forming cells (HPP-CFC). This subpopulation also produces control numbers of in vivo spleen colony-forming cells, CFU-S, and has control numbers of primitive, noncycling colony-forming cells that resist in vitro treatment with 5-fluorouracil (5-FU). By contrast, nylon-adherent populations, when eluted by mechanical agitation (MA) (41% +/- 1%) or by subsequent EDTA treatment (CA) (6% +/- 1%) could reform the hemopoietic microenvironment in vitro. Stromal progenitor cells were negative for surface markers Thy-1 and Mac-1. When NNA stem cells were added to culture with nylon-adherent stromal fractions, lodgement of stem cells occurred, resulting in stem cell proliferation for up to three months.
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A mixed transplantation of bone marrow cells, and lymph nodes or thymic cells of mice CBA strain into lethally irradiated hybrid recipients (CBAXC57B1)F1 is accompanied with changes in the differentiation pattern from a mainly erythroid to a mainly granuloid way. Thymectomy of either donor of bone marrow cells or recipients, or both, destroys the stem cell differentiation in the direction of granulopoieseis. Intact syngeneic lymphocytes normalize differentiation of the stem cells, but in the presence of tissue antigens these provide for the stem cell differentiation mainly in the direction of granulopoiesis. The differentiation of stem haemopoietic cells is accomplished under the thymic and lymphocyte control. T-differentiating lymphocytes (Td) are the lymphocytes controlling the stem cell differentiation.
Stem cell activities of bone marrow, spleen, thymus, and fetal liver cells for T cell lineage were studied comparatively by transferring the cells from these organs through i.v. or intrathymus (i.t.) route into right leg- and tail-shielded (L-T-shielded) and 900 R-irradiated recipient mice, which were able to survive without supplying hemopoietic stem cells. Cells from B10.Thy-1.1 (H-2b, Thy-1.1) mice were serially diluted and were transferred into L-T-shielded and irradiated C57BL/6 (H-2b, Thy-1.2) mice, and 21 days later the thymus cells of recipient mice were assayed for Thy-1.1+ cells by flow cytofluorometry. The percentage of recipient mice possessing donor-type T cells was plotted against the number of cells transferred, and the stem cell activity in each cell source was expressed as the 50% positive value, the number of donor cells required for generating donor-type T cells in the thymuses of 50% of recipient mice. In i.v. transfer experiments, the activity of bone marrow cells was similar to that of fetal liver cells, and about 100 times and nearly 1000 times higher than those of spleen cells and thymus cells, respectively. In i.t. transfer experiments, the number of cells required for generating donor-type T cells was much lower than that in i.v. transfer experiments, although the ratio in 50% positive values between i.v. and i.t. transfers differed among cell sources. In i.t. transfers, the 50% positive value of bone marrow cells was five times, 400 times, and 500 times higher than that of fetal liver cells, spleen cells, and thymus cells, respectively. Our previous finding that stem cells are enriched in the spleens of mice which were whole body-irradiated and marrow-reconstituted 7 days earlier was confirmed also by the present limiting dilution assay carried out in i.v. as well as i.t. transfers.