Isolation of the Bone Marrow or Cord Blood Mononuclear Fraction

The mononuclear fraction of bone marrow includes lymphocytes, monocytes, several types of blast cells, and at least three populations of stem/progenitor cells: HSCs, MSCs and endothelial progenitor cells (EPCs). EPCs were first described as occurring in circulating blood , and their origin in bone marrow was later proposed . However, some studies indicate that bone marrow-derived EPCs do not significantly contribute to neovascularization/angiogenesis, which could be attributed to poor knowledge of the hierarchy of the EPC compartment . The application of EPCs in kidney disease has been reviewed .

It has been suggested that the bone marrow also contains a plethora of versatile tissue-committed, non-hematopoietic stem cells . It is accepted today that the main therapeutic activity of bone marrow cells for non-hematological diseases is due to MSCs . The frequency of MSCs is much more limited in the UCB , so that the mononuclear fraction of cord blood is more appropriate for the treatment of hematological diseases than other pathologies.

The isolation of mononuclear cells from human blood was first reported by Dr Arne Bøyum in 1968 , and has been used successfully ever since. The method is based on the lower buoyant density of mononuclear cells compared to erythrocytes and polymorphonuclear leukocytes (granulocytes). Most mononuclear cells have densities below 1.077 g/ml, so that they can be isolated by centrifugation on an isoosmotic medium with this density. The two types of most widely used reagents are Lymphoprep™ (AXIS-SHIELD PoC AS) and Ficoll-Paque™ (GE Healthcare), which have recently been shown to yield BMMNC fractions equivalent in terms of the composition and quantity of cell types .

For clinical trials, bone marrow is usually collected by aspiration from the anterosuperior iliac crest, performed approximately 3 h before the reinfusion and with the patient under sedation. The volume aspirated is around 80 ml, in a medium containing anticoagulant preservative. When the cells are to be used for research purposes, the procedure may be different. Although some institutions have access to bone marrow samples donated for research, in most cases alternatives to whole bone marrow must be implemented, including the use of bone marrow remnants in bags and nylon meshes used for collecting bone marrow for transplantation . When bone marrow is collected for research purposes, it is usually obtained by a single puncture of the iliac crest using a 20 ml syringe. The quality of the sample depends on some factors. The site where the bone marrow is aspirated from can influence the amount of cells obtained as more or less medullar blood or fat can be present.

Human UCB is collected after clamping of the cord during vaginal deliveries. The blood is collected into sterile blood bags containing anticoagulant. The volume of blood collected varies usually between 70 and 120 ml. In either type of sample, the anticoagulant used is an important factor, which is sometimes overlooked. Heparin is a very popular (and effective) anticoagulant. However, heparin has soluble factor-binding activities that can influence the quality of the cells obtained. Hence, the amount of heparin should be just enough to prevent coagulation. Alternatives to heparin include sodium citrate and ethylene-diaminetetraacetic acid (EDTA), which are Ca ²⁺ chelating agents and tend not to interfere with the quality of the cells when used at the right concentrations.

Bone marrow or cord blood is then diluted 1:1 with 2 mM EDTA in phosphate-buffered saline (PBS-EDTA), and light-density mononuclear cells are separated by centrifugation on Ficoll-Hypaque 1.077, at 400 × g for 30 min. If the method, which is conventionally used for the isolation of mononuclear cells from peripheral blood, results in contamination with red cells, an enrichment step with 3% gelatin in 0.9% saline may be used before centrifugation on Ficoll-Hypaque. For that, the sample is mixed 1:1 with gelatin solution and incubated at room temperature for 2 h. The mononuclear-enriched supernatant is collected and centrifuged (10 min, 400 × g ) and the pellet is centrifuged on Ficoll-Hypaque.

Cells are then counted using a hemocytometer or Neubauer chamber. Although intact cells can be discerned from damaged cells by counting under a phase-contrast microscope, viable cells can be more accurately identified using a stain such as trypan blue. To assess cell viability, an aliquot of the parent cell suspension is mixed with an equal volume of 0.4% trypan blue in PBS and placed in the hemocytometer. Cells permeable to trypan blue are stained blue and correspond to non-viable cells. These are scored along with the viable cells and cell viability is calculated by dividing the number of viable cells by the total number of cells; red blood cells (RBCs) are recognized by eye and not included.

For clinical studies, the mononuclear fraction is collected, washed in a heparinized saline solution containing 5% autologous serum and filtered for removal of cellular adjunctive. The cells are counted, suspended in saline/5% autologous serum in the concentration determined and administered to the patient by local or systemic injection. A small fraction should be separated for sterility and viability tests, immunophenotyping by flow cytometry to determine the cell subpopulations present, and for functional analyses.

Isolation and Cultivation of Hematopoietic Stem Cells

Human HSCs are characterized as CD34- or CD133-positive, CD38- and lineage-negative cells. For clinical applications, even in cases when the treatment is referred to as hematopoietic stem-cell transplantation, the cells which are used are actually populations enriched, but not purified, for HSCs . A usual method of enrichment for HSCs which avoids the need for bone marrow collection is mobilization with differentiation/growth factors such as the granulocyte colony-stimulating factor (G-CSF), followed by blood collection by leukapheresis . More recently, combinations of growth factors and other molecules, such as plerixafor, have been developed for more efficient concentration of HSCs in the peripheral blood .

With much less frequency, patients receive purified HSCs , which may be isolated from bone marrow, peripheral blood after mobilization or UCB . In these cases, the cells are selected by reaction with anti-CD34 or anti-CD133 antibodies followed by two alternative methods of isolation of positive cells. The first one is FACS, as described below for the isolation of pericytes. The second method, which preserves more of the biological characteristics of the cells, is magnetic cell sorting (MACS). In this case, the cells are collected, washed and incubated with an antibody conjugated to magnetic microbeads at 4°C. The cells are then washed, resuspended in the appropriate buffer and loaded on a magnetic separation column which is placed in a magnetic field. Cells negative for the marker are then eluted from the column, which preferentially retains the positive cells. After removal from the magnetic field, the positive cells are eluted. To increase purity of the sorted cells, the eluted cells may be subjected to an additional purification cycle.

All blood cells are derived, through a series of maturational cell divisions regulated by the hematopoietic microenvironment, from a small common pool of HSCs. HSCs are of interest not only because of their developmental capacity but also because of their therapeutic role in hematological disorders and gene therapy. They share the main characteristics presented by adult stem cells: (i) they constitute a very small compartment, estimated between less than 0.05% and up to 0.5% of cells in the bone marrow; (ii) they are normally quiescent or slowly cycling, as shown for instance by their resistance to treatment with 5-fluorouracil or 4-hydroperoxycyclophosphamide. Estimates of periodicity of mitosis vary widely, and the direct examination of the cell cycle of long-term cells indicates that at any moment only 4% of them are in the S/G2/M phases. Besides bone marrow, HSCs can be found in UCB and in peripheral blood, particularly after mobilization treatments.

When stem cells divide, they may return to the G0 phase of the cell cycle, generating more stem cells, or generate large numbers of committed progenitors with a progressively restricted differentiation potential. Homeotic genes have a fundamental role in the regulation of these and other cellular processes . Intermediate steps, characterized by the progressive loss of the self-renewal capacity and the commitment to a specific cell lineage, result in the transition from stem cells to mature hematopoietic cells.

Despite much effort, the in vitro cultivation of HSCs is still poorly developed . Many different combinations of growth/differentiation factors , or functional parameters , are under investigation to define the conditions allowing expansion of this cell population with maintenance of stemness. However, as different assays detect and analyze hematopoietic cell types specifically stimulated by the experimental conditions used, and the correspondence among the assays is not always easily established, different names have been given to the cell types observed, as detailed below.

Till and McCulloch established, in 1961, the first quantitative assay for cells with a radioprotective effect. Although these cells, which were denominated spleen colony-forming units (CFU-S), do not represent the more primitive stem cell pool, the assay is useful for the investigation of early events in hematopoiesis. In vivo assays involving the engraftment of human hematopoietic cells into immunodeficient mice have shown the existence of pluripotent cells, either by limiting dilution analysis or by clonal integration of a retroviral marker gene . The expression “marrow repopulating ability”, derived from in vivo studies, refers to primitive HSCs capable of repopulating the bone marrow of lethally irradiated mice .

Two different types of cells with marrow repopulating ability have been distinguished in the mouse. Initial engraftment (short-term repopulation) is a characteristic of CFU-S. Long-term engraftment is attributed to a different cell type and is possible only if the animals also receive short-term repopulating cells. A cell type which is more primitive than CFU-S (pre-CFU-S) is considered to be responsible for long-term marrow repopulating ability [reviewed in Ref. ].

The development of in vitro cultivation systems for the study of hematopoiesis allowed the identification and quantification of several different types of hematopoietic precursor cells. The colony formation assay allows the identification and quantification of early progenitor cells, capable of forming colonies when cultured in semisolid medium. In this assay, cells are grown in vitro in methylcellulose or other highly viscous media, containing also different combinations of growth and differentiation factors. These semisolid media reduce cell movement and allow individual cells to develop into cell clones that are identified as single clusters (< 50 cells) or colonies (> 50 cells) of differentiated cells after a culture period of 7–14 days. These colonies are the progeny of single cells called colony-forming cells (CFCs) or colony-forming units (CFUs), and the composition of the colonies determines which CFU is being assessed. These clonogenic assays allow the identification, for instance, of CFU-blasts which give rise to colonies composed of cells with blast-like morphology, of CFU-GEMM which originate multilineage colonies (granulocytes, erythrocytes, monocytes and megakaryocytes), CFU-meg which give rise to megakaryocyte colonies, and so on . Other types of stem/precursor cells identified by this culture system include the high proliferative potential colony-forming cells (HPP-CFC), defined by the ability to form very large colonies (> 5 mm) containing approximately 50,000 cells, and the low proliferative potential colony-forming cells (LPP-CFC), that give rise to colonies smaller than 1 mm. These primitive HSC pools are considered to comprise cell types such as BFU-E (erythrocyte blast-forming unit) and CFU-GM (precursor of granulocytes and monocytes) .

Another culture system currently used for the study of hematopoietic progenitors is the delta assay . In this system, cells are grown first in liquid culture for 1 week and then replated onto semisolid medium. Colonies observed after this period indicate the number of hematopoietic progenitors more primitive than those normally obtained after 14 days of growth in semisolid medium.

The development of hematopoietic cells in these culture systems depends on the continuous presence of colony-stimulating factors (CSFs). The investigation of results obtained with addition of different types of stimulating molecules has been the key to the discovery and characterization of many of the hematopoietic growth factors . Colony-forming assays, therefore, allow the study of the role of growth factors or cytokines in the determination of the lineage along which CFCs differentiate.

Expansion of HSCs is not possible with the use of standard semisolid systems; consequently, alternative culture assays have been developed for the sustained production or self-renewal of clonogenic cells. In the long-term culture (LTC) system originally described by Dexter for murine cells and later adapted for human cells , cells are cultivated at a lower temperature in a culture medium containing high concentrations of horse serum and hydrocortisone. This supportive microenvironment allows the self-renewal of stem cells over a period of several months. The long-term culture of bone marrow cells employs primary adherent layers of stromal cells which include MSCs as important components. These stromal cells provide a complex functional extracellular matrix which allows direct cell–cell contacts between different cell types and secrete cytokines and low molecular weight substances which are required for the controlled differentiation and proliferation of hematopoietic progenitor cells. This system can maintain primitive stem cells, induce early differentiation of a fraction of the primitive progenitors and prevent their terminal differentiation.

A second type of widely used culture system for hematopoietic cells is the Whitlock–Witte long-term bone marrow culture , which was initially developed for murine bone marrow. In this lymphoid culture system, a “poor” culture medium containing 5% fetal calf serum and no cortisone permits the growth of freshly isolated bone marrow cells that develop into a confluent adherent stromal cell layer within 2–3 weeks. Whitlock–Witte cultures can reconstitute the B-lymphocyte compartment in immunocompromised mice, but do not maintain primitive multilineage hematopoietic precursors such as CFU-S . Pluripotent HSCs and early precursors can also be identified by the LTC assays as cobblestone area-forming cells (CAFCs) and long-term culture-initiating cells (LTC-ICs). Limiting dilution analysis is used for the quantification of these cells .

The transduction of CFU cells and LTC-ICs using retroviral vectors indicates that the in vitro progenitor assays currently available measure functionally different, and presumably less quiescent, populations than the long-term repopulating stem cells . However, none of these culture systems is able to maintain hematopoiesis indefinitely, showing that the in vivo niche of HSCs has not yet been adequately reproduced in vitro. It is not probable, therefore, that clinical application of isolated, cultivated HSCs will be available in the near future.