This chapter contains sections titled: Fixation, staining and mounting Storage of slides Setting up and using a microscope Examining a blood film Test your knowledge
We have taken advantage of the permissive environment of the preimmune fetus to achieve engraftment of hematopoietic stem cells (HSC) from fetal and postnatal human sources in sheep. The resulting chimeric lambs exhibited long-term multilineage (erythroid, myeloid, lymphoid) expression of donor cells that remained responsive to human-specific cytokines. The small size of the fetus allowed the assessment of the engraftment potential of relatively small numbers of highly characterized human HSC populations, while the large size of the chimeric sheep permitted the long-term evaluation of the HSC activity and response in vivo. The human/sheep xenograft may offer a useful large animal model for the assay and long-term study of the human HSC subsets.
We have studied several features of pluripotent hematopoietic stem cells (PMSCs) and day-12 spleen colony-forming units (CFU-S) obtained from adult murine bone marrow. Single-cell suspensions of C57BL/6J mouse bone marrow were fractionated by counterflow centrifugal elutriation at flow rates (FR) of 15, 25, 30, and 35 ml/min, and with the rotor off (R/O), The fractions FR25 and FR35 contained approximately equal numbers of PHSC that could repopulate W/W-v mice. These PHSCs were further enriched by subtracting lineage-positive cells using monoclonal antibodies (MAb) and magnetic immunobeads. The resulting lineage-negative cells (Lin(-)) were then stained with a MAb for the c-kit receptor and sorted by flow cytometry. Both subsets were fractionated into cells expressing high (bright) (c-kit(BR)), low (dull) c-kit(DULL) and (negative, c-kit(NEG)) c-kit receptor. As few as 100 to 200 c-kit(BR) cells could repopulate the entire thymus and bone marrow in W/W-v mice. No PHSCs were present in the c-kit(DULL) and c-kit(NEG) fractions. We assayed fresh bone marrow and elutriation fractions FR25 and FR35 for gene expression by reverse transcriptase polymerase chain reaction. Using a semiquantitative protocol, we detected mRNA for beta-globin and flk-2, a protein tyrosine kinase receptor, in all samples except the FR25 Lin(-) c-kit(BR) subset. We consider the cells in FR25 Lin(-) c-kit(BR) to be the most primitive set of hematopoietic stem cells.
Human hematopoietic stem cells are contained within a population of marrow cells that expresses the CD34 antigen but not other antigens associated with commitment to specific lineages. Evidence that stem cells capable of maintaining long-term hematopoiesis are within this CD34+ lineage-negative (Lin-) population is reviewed, including in vivo studies in humans and nonhuman primates. In vitro studies of the CD34+ Lin- population have indicated that the blast-sized cells, which are presumably in cycle, proliferate and give rise to colony-forming cells in the presence of combinations of growth factors, including c-kit ligand and interleukin-3 (IL-3). Recent studies have examined the factors required for the growth of the quiescent subset of the CD34+ Lin- cells, identified as small to medium lymphocyte-sized cells that resist treatment with 4-hydroperoxycyclophosphamide, a known characteristic of the marrow-repopulating cell. These studies have shown that an interaction with marrow stromal cells is required, in addition to c-kit ligand and IL-3, to induce these cells to proliferate and form multiple colony-forming cells. These studies have further indicated that this effect of stroma is mediated by a soluble factor(s). This activity may represent a novel factor(s) and/or a novel combination of growth factors.
The ability to reliably transfer genes into hematopoietic stem cells with long-term repopulating potential and to selectively express such genes would allow genetic therapy for diseases such as sickle cell anemia and immunologic deficiencies due to T-cell defects, including acquired immune deficiency syndrome (AIDS). Understanding the biology of the hematopoietic stem cell is a key element in realizing the full therapeutic potential of gene insertion strategies. Current techniques have efficiency rates of gene insertion of approximately 10% to 20% into murine stem cells and 1% to 5% into primate stem cells. Many challenges, some biologic and some logistic, remain before gene transfer protocols that are successful in the mouse model can be extended to humans.
Recombinant retroviral vectors, engineered to express the beta-chain gene of swine major histocompatibility complex class II DR, were developed for the genetic modification of swine hematopoietic stem cells (HSC). The expression of these vectors in swine bone marrow has been studied both in culture and after bone marrow transplantation. In addition, myeloid progenitor colony assays were performed on swine umbilical cord blood as part of a study to identify alternative sources of HSC for somatic gene transfer, revealing the presence of both granulocyte macrophage colony forming-units (CFU-GM) and CFU-Mix at frequencies comparable to those found in juvenile swine bone marrow.
Human CD34(+) cells were isolated from bone marrow from normal volunteers and expanded under serum-free culture conditions, CD34(+) cells were cultured with interleukin-3 (IL-3), IL-1, and stem cell factor and expanded in granulocyte-macrophage colony-forming units, erythroid blast-forming units, and CD34(+) cell number during the first 7-14 days of incubation, By contrast, cultures maintained in fetal calf serum under identical conditions showed much reduced expansion, as measured by all of the above parameters, The level of expansion of the CD34(+) cells was dependent on the combination of growth factors used during culture, The data establish the feasibility of serum-free expansion of progenitors and suggest the clinical use of this procedure for the generation of expanded progenitor cell products for transfusion after chemotherapy to minimize treatment-related cytopenias.
Over the past 6 years, umbilical cord blood has emerged as an efficacious alternative source of hematopoietic stem cells in related bone marrow transplantation. These encouraging results led us to extend this technology to the mismatched related and unrelated settings in three high-risk leukemic children lacking a matched-related donor for transplantation. Two of the three children also lacked identifiable donors through the National Marrow Donor Program, while the third was in relapse and did not have time to wait for completion of a search. The first child was transplanted with haploidentical umbilical cord blood-derived mononuclear cells from his sister, while the remaining two children were transplanted with partially mismatched, unseparated, unrelated umbilical cord blood banked through the Placental Blood Project at the New York Blood Center. All three children demonstrated trilineage engraftment with donor cells within 6 weeks of transplantation. The patient transplanted with haploidentical marrow developed grade 2 graft vs. host disease (GVHD), which was controlled with steroid and anti-thymocyte globulin (ATG) therapy. One of the two patients grafted with unrelated umbilical cord blood developed mild grade 1 GVHD of the skin, which rapidly cleared with steroid therapy. One patient remains alive, in good health and disease-free 12 months from transplantation.
Cord blood is a recently recognized source of hematopoietic stem cells. It can be employed successfully to reconstitute hematopoiesis following allogeneic transplantation. One current drawback of cord blood as a treatment has been a risk of transfusion reactions attributable to ABO blood group mismatch. Removal of red cells from the cord blood has led to reduction of the stem cells by 30-50%. In this paper we report red cell depletion by a method that employs 3% gelatin to effectively sediment the erythrocytes and selectively deplete red cells but permits 94% recovery of nucleated cells and enrichment of colony-forming cells by granulocyte-macrophage colony-forming units, erythrocyte burst-forming units, and granulocyte-macrophage-megakaryocyte colony-forming units in the cord blood preparation. This technique has been employed in our study to remove red cells from the cord blood of a male infant delivered by cesarean section, which has permitted treatment of a female sibling suffering from leukemia. The recipient was 8 years old and weighted 36.7/kg. Complete HLA identity between the two siblings was established. A cord blood cell transplant of cryopreserved and later thawed cells (4 x 10(7) nucleated cells per kilogram) was administered to the patient after intensive myeloablative chemotherapy. The patient exhibited a prompt hematologic recovery (absolute neutrophil count > 500 by day 31, 100% male cells in bone marrow and peripheral blood by day 25) and has experienced a 13-month disease-free survival to date.(ABSTRACT TRUNCATED AT 250 WORDS)
We previously demonstrated stable integration of a transduced thymidine kinase (TK)-neo gene into immature and replatable stem and progenitor cells, as assessed by the presence of the gene in second-generation colonies. To evaluate whether this integration was still present in third- and fourth-generation colonies, nonadherent low-density T-lymphocyte-depleted (NALT-) cells from human umbilical cord blood were prestimulated with recombinant human (rhu) erythropoietin (Epo), steel factor (SLF), interleukin-3 (IL-3), granulocyte-macrophage (GM) colony-stimulating factor (CSF), and granulocyte (G)-CSF. Prestimulated NALT- cells were incubated with retroviral-containing supernatant obtained from TK-neo vector-producing cells, washed, and assayed for colony formation in the presence of Epo, SLF, IL-3, GM-CSF, and G-CSF -/+ G418. The results confirmed that the TK-neo gene could be efficiently introduced into hematopoietic progenitor cells without stromal cells as a source of virus. As previously reported, proviral integration was detected in primary G418R-colonies, and in second-generation replated colonies derived from G418R granulocyte erythroid macrophage megakaryocyte colony-forming units and high-proliferative potential colony-forming cells (HPP-CFCs). Moreover, we now document that proviral integration was apparent in cells from colonies derived from third- and fourth-generation replated HPP-CFC, suggesting a high degree of stable integration of the transduced gene.
Hematopoietic stem and progenitor cells present in umbilical cord blood at the birth of a child are efficiently transduced ex vivo by genes using retroviral vectors in combination with exposure of these cells to combinations of growth factors. Because retroviral-mediated gene transduction of adult bone marrow and blood hematopoietic stem and progenitor cells is greatly enhanced by growth factors, we evaluated the possibility that cord blood progenitors, which have extensive proliferative and replating capacity, could be efficiently transduced with a TK neo gene in a retroviral vector in the absence of growth factors, and also determined the influence of exogenously added growth factors on this transduction. Highly purified CD34+ (62% pure) cord blood cells isolated by magnetic bead separation were cultured in suspension for 72 hours with viral supernatant in the absence and presence of interleukin-3 (IL-3), IL-6, and steel factor. Evaluation of progenitor cell-derived colonies and polymerase chain reaction (PCR)/Southern analysis of the TK neo gene in resultant colony cells demonstrated that some gene transduction was apparent in the absence of growth factors (12.8-14.3% by PCR), but that this was greatly enhanced (40.0-44.4%) by addition of growth factors. Reverse transcription PCR analysis of the expression of IL-3, IL-6, and granulocyte-macrophage colony-stimulating factor genes in this population of cells suggested that the transduction, although at a lower efficiency, in the absence of added growth factors might in part be due to "constitutive" and viral supernatant-induced expression of these cytokine genes in the CD34(+)-enriched cell population.(ABSTRACT TRUNCATED AT 250 WORDS)
Human cord blood cells have been shown to highly engraft the marrows of sublethally irradiated SCID mice. Herein we report our experience with this system and the use of immunohistochemistry to identify human cell engraftment. Immunohistochemistry results correlated well with those of flow cytometry, human progenitor-cell cultures, and molecular analysis of human specific markers. Immunohistochemistry should play a useful role in the in vivo analysis of human stem/progenitor cell engraftment in xenogeneic transplantation models.
When 200 x 10(6) male BALB/c cells are given by tail vein injection to female nonmyeloablated hosts in one injection, a relatively low engraftment percentage is seen, but when the same total number of cells is given over five injections (separated by 24 hours), the observed engraftment is much higher. A further increase in engraftment appears to occur when the same number of cells is given in 10 injections separated by at least 24 hours, These data suggest that somewhere between 5 and 10% of marrow niches are available at intervals of 24 or more hours and that the keys to high levels of engraftment are the cell cycle status of the engrafting stem cell and the schedule of engraftment.
To carry out cord blood transplants from allogeneic unrelated donors, cord blood stem cell banks must be established. This report proposed the policies and procedures that can be used to establish cord blood banks. The areas covered include donor consent and suitability criteria; infectious and genetic disease testing; collection, processing, and preservation of the cord blood; retention of specimens for special testing; confidentiality; documentation and record keeping; establishment of a quality control program; and related regulatory issues. There are no technologic impediments to establishing cord blood banks. Agreement on some standard policies and procedures would facilitate exchange of cord blood stem cells among transplant centers and should increase the number of transplants that can be done.
It is becoming increasingly clear that the parvovirus-based vectors may prove to be a useful alternative to the more commonly used retroviral vectors in human gene therapy. Specifically, the adeno-associated virus 2 (AAV), a human parvovirus, has gained particular attention in view of its nonpathogenic nature as well as its remarkable site-specificity of integration into the human chromosome. Using the recombinant AAV vector system, it is feasible to obtain high-efficiency transduction of slow- or non-cycling primary hematopoietic stem and progenitor cells, without the need for prestimulation with cytokines, which could potentially lead to differentiation of these cells before transplantation.
For over 50 years the received wisdom has been that the shadow cells of Gumprecht otherwise known as basket cells (BC) are in artefact, produced during preparation of films when a drop of blood is spread on a slide. The assumption has been that they are therefore of no significance. They are commonly seen in blood films from patients with lymphoproliferative syndromes and particularly in chronic lymphocytic leukemia (CLL). In 96 patients with CLL a statistically significant correlation existed between the basket cells observed in films and the lymphocytes with dense chromatin (DC) determined by flow-cytometry. There was no statistically significant correlation between the number of BC and DC, and the anatomic-clinical stage of the disease.
