Manipulating hematopoietic stem cells for clinical use

Mobilization of HSCs

Mobilization of HSCs in response to chemotherapy or cytokines was first documented in the 1970s and 1980s. This process may be induced by a variety of molecules, including cytokines such as G-CSF, GM-CSF, IL-7, IL-3, IL-12, stem cell factor and flt-3 ligand; and chemokines such as IL-8, Mip-1a, Gro-P and SDF-1. The one that is most often used clinically is G-CSF, which may be combined with chemotherapeutic agents for added benefit. This mobilizing capability has resulted in a dramatic change in the manner by which HSCs are harvested for transplantation. Up to 25% of candidates for autologous transplantation are unable to mobilize sufficient cells to enable the procedure to be safely performed. The study of mobilization and its counterpart, engraftment, has implications of great significance for patient care. The ability of G-CSF to mobilize bone marrow HSCs has several apparent mechanisms. The first is reported to be the activation of neu-trophils, causing the release of neutrophil elastases capable of cleaving CXCR4 on HSCs, thus reducing HSC-bone marrow interaction. Other receptors that undergo cleavage are VCAM-1 and c-kit. A second mechanism of G-CSF-induced mobilization is via CD26, an extracellular dipeptidase present on primitive HSCs that is able to cleave SDF-1 to an inactive form. Other proposed options for improving mobilization include co-administration of G-CSF and kit ligand, antibodies directed against VLA-4, and infusion of IL-8.

Isolating stem cells for manipulation

Characteristics of HSCs used for isolation

Physical Early attempts to isolate HSCs were based on cell size and density. In order to clarify whether the heterogeneity of CFU-S was due to differences in the input cells used, velocity sedimentation was performed to separate cells by size, demonstrating that smaller cells were more likely to produce secondary CFU-S than larger cells. HSCs are similar in size to mature lymphocytes and, when flow cytometry is performed, overlap the lymphocyte region on plots of forward and side scatter.

Using cell-cycle-active drugs Because HSCs are largely in a quiescent portion of the cell cycle (G0 or Gt), investigators have used cell-cycle-active drugs to deplete bone marrow populations of cycling cells and thereby enrich for primitive HSCs. Treatment of mice with nitrogen mustard resulted in a 30-fold enrichment in CFU-S. HSCs may be isolated by in vitro treatment with 5-fluorouracil, and this remains the most commonly used agent. In addition, HSC populations may be further enriched by first stimulating cells to enter the cell cycle with the early-acting cytokines c-kit ligand and IL-3 before forcing them to metabolic death. This strategy is useful for human cells but not murine cells, probably because of different cycling characteristics. It should be noted that these techniques might result in a decrease in the quality of HSCs obtained.

Markers of primitive HSCs A variety of strategies have been used to identify potential HSC markers. CFU-S in rat bone marrow, fetal liver and neonatal spleen express Thy-1 antigen at high levels, and this was the first important HSC marker discovered. Pluripotent stem cells could be enriched from the bone marrow 150-fold on the basis of combination of size and Thy-1 expression. Negative selection of cells using soybean agglutination resulted in enrichment in the colony-forming unit culture assay (CFU-C). In addition, a FACS-based negative selection strategy involving labeling hematopoietic cells with a cocktail of antibodies directed against mature hematopoietic cell antigens has been developed. These lineage-directed antibodies included B220, against mature B lymphocytes, CD8, against T cells, Mac-1, against macrophages, and Gr-1, against granulocytes. When this negative selection protocol (Linneg) was combined with a positive selection protocol to enrich for cells that expressed low levels of Thy-1 (Thy-1low), 200-fold enrichment of day-10 CFU-S could be achieved.

Using a magnetic bead selection strategy to enrich for Thy-1-expressing cells followed by a FACS-based strategy to deplete cells expressing lineage markers, murine cells were isolated that were found to express a newly defined stem cell antigen, Sca-1. These LinnegThy-1lowSca-1pos cells represented 1 in 1000 bone marrow cells and had heightened stem cell activity compared with whole bone marrow in the CFU-S assay. This highly selected cell population produced day-13 CFU-S at 1 colony per 10 cells and day-8 CFU-S at 1 per 100 cells. Also, these cells were 1000- to 2000-fold enriched in their ability to rescue irradiated animals and could give rise to all blood cell lineages. Self-renewal capability was demonstrated by the ability of these cells to rescue lethally irradiated animals upon secondary transplantation. Interestingly, the Sca-1neg population had similar CFU-S activity but could not produce T cells or confer radioprotection.

More recently, the receptor for stem cell factor, c-kit, was demonstrated to be present on HSCs. Populations expressing this phenotype (c-kitP°sThy-1.1lowLinnegSca-1P°s), also known as KTLS, are 2000-fold enriched in HSC activity compared with unfractionated bone marrow. Thus, KTLS has come to be regarded by many as a profile that represents, but is not specific for, HSCs in the mouse. Equivalent markers are not as well defined in the human, though it is apparent that cells expressing kit ligand without lineage markers (including the CD38 antigen) are enriched in stem cells. The antigen CD34 has long been regarded as a marker for a stem cell population, but it is now clear that the vast majority of CD34+ cells are progenitors, and stem cells may or may not express CD34. CD133 is a marker more recently shown to be expressed on primitive human hematopoietic cells. A summary of proposed HSC markers for the mouse and human is presented in Table 3.2.

Supravital stains Since HSCs are inherently quiescent, spend most of their time in inactive portions of the cell cycle and are resistant to toxins, exclusion of dyes has been used as a method of isolation. The DNA dye Hoechst 33342 was first used to separate quiescent cells from the bone marrow. Cells with low-intensity staining were enriched for HPP-CFC and day-12 CFU-S. The red and blue emissions from this dye have been recently used to define a small subset of bone marrow cells known as the side population (SP). SP cells have extremely low fluorescence emission in these channels, resulting from efflux of Hoechst 33342 by multidrug resistance pumps that are highly expressed on HSCs. SP cells constitute approximately 0.1% of the bone marrow and are highly enriched in reconstitution potential.

The mitochondrial dye rhodamine-123 (Rh-123) has also been used to subdivide primitive stem cells. Mitochondria in quiescent cells bind low levels of Rh-123 and FACS can be used to separate Rh-123low cells. These cells were enriched for day-13 CFU-S and multilineage reconstituting potential.

The combination of supravital stains with fluorescent antibodies against cell surface markers provides the ability to enrich for highly primitive HSCs such that fewer than ten cells are required to reconstitute hematopoiesis.

Table 3.2 Proposed markers of primitive HSCs.

Hematopoietic stem cell surface markers

Mouse

Human

CD34low/-

CD34+

Sca-1+

CD59+

Thy1+/low

Thy1 +

CD38+

CD38low/-

C-kit+

C-kit-/low

lin-

lin-

Methods of isolation of HSCs

FACS While the flow cytometer may be used for analysis of cells, the apparatus may also physically sort cells of desired fluorescence or fluorescence pattern, size and granularity characteristics. Using a magnetic field, these cells may be diverted to a collection tube during analysis and later analyzed using techniques of molecular and cellular biology. Sorting is both expensive and labor-intensive as it requires costly machines, a high degree of expertise, and time to sort samples consisting of single-cell suspensions. Many FACS machines are now available with high-speed sorting. This was once a technique available to only a few laboratories, but many centers are developing 'core' laboratories to provide cell analysis and sorting services for investigators. FACS may be used to isolate HSCs using both positive and negative selection strategies with fluorescence-labeled antibodies directed against primitive hematopoietic cell antigens, as described above.

Magnetic bead columns Large-volume isolation of HSC subsets has been facilitated by the use of magnetic bead columns. Using this system, cells are incubated with antibodies directed against primitive hematopoietic cells. These antibodies are typically coupled to a hapten. A second-step incubation is then performed using a magnetic microbead conjugated to a hapten that is able to bind the first-step hapten. The effect is to label HSCs with a magnetic bead. Cells are then passed through a column mounted adjacent to a magnet. Labeled cells are retained within the column and unbound cells can be washed through. Then, the column is removed from the magnet and the desired cells may be eluted.

Alternatively, negative selection may be performed by capturing only the cells that pass through the column. For example, a sample may be depleted of mature cells by labeling with antibodies directed against mature blood cell antigens (Linpos). Cells can then be passed over a column in which the mature cells adhere and immature cells pass through and may be isolated.

Systems of these types permit rapid isolation of large numbers of primitive cells of relatively high purity.

Ex vivo expansion

Given the possible clinical applications of HSCs for such uses as bone marrow transplantation, there is increasing interest in strategies that both result in an increase in the quantity of HSCs and the ability to manipulate HSCs ex vivo. Thus, ex vivo expansion of HSCs represents a highly prioritized goal of clinically oriented HSC research.

The first benefit of expanding HSCs is to provide sufficient cells for transplantation when insufficient numbers exist. For example, cord blood represents a rich source of primitive

CD34+ cells that are less immunocompetent and are therefore transplantable across partial HLA disparity barriers. However, the absolute quantity of HSCs within a single cord blood is low and transplantation is followed by periods of aplasia. Ex vivo expansion would thereby facilitate cord blood transplantation. Similarly, selective expansion of HSC subsets would permit the extension of tumor-free cells from patients with limited quantities of normal bone marrow due to bone marrow-infiltrating diseases, such as leukemia, for the purpose of autologous transplantation.

The second benefit of ex vivo manipulation is that HSCs have a relative growth advantage over other cell types, such as tumor cells. Therefore, ex vivo growth provides a purging effect. Furthermore, specific tumor cell purging may be achieved via the application of certain cytokines (IL-2, IFN-y), antitumor agents such as 5-fluorouracil or cyclophosphamide, tumor-specific antibodies combined with complement-mediated lysis, and oncogene-specific tyrosine kinase inhibitors, in addition to other targeted therapies, such as antisense oligonu-cleotides, prior to use of the graft.

The third benefit is the support of gene transfer into HSCs for the purpose of gene therapy. A variety of gene-transfer mechanisms, including retroviral infection, are conveyed during mitosis. Thus, the ex vivo stimulation of cells using cytokines results in heightened transfer of exogenous genes to HSCs.

Strategies to expand HSCs ex vivo have used cytokine cocktails such as IL-11, flt3-ligand and steel factor, stimulation with the purified WNT-3a glycoprotein, neutralizing antibodies of TGF-P alone or in combination with inhibition of CDKI p27, inhibition of the CDKI p21, and stimulation with Notch ligands. While these efforts have resulted in encouraging laboratory results, none to date has translated into accepted clinical practice. Testing regarding these methods continues with intensity and relies heavily on specific functional analyses.

Functional analysis of HSCs

Functional assays for HSCs do not actually measure the activity of HSCs but instead assess more differentiated progeny, such as progenitor and precursor cells. Whereas in vitro assays measure mature populations, in vivo assays detect the activity of primitive cells capable of homing and engrafting in the proper microenvironment to produce functional hematopoi-etic progeny.

In vitro assays The CFU-C measures hematopoietic progenitor function and is performed by plating cells in semisolid media containing methylcellulose and one or more cytokines. After 5-14 days, colonies comprising mature cell populations committed to either myeloid or lymphoid lineages may be observed. While most colonies obtained using this assay are composed of cells of a single lineage, less frequently multipotent progenitors can yield colonies containing multiple lineages. Another type of primitive cell, known as the 'high proliferative potential colony-forming cell' (HPP-CFC), which possesses a high degree of proliferative and multiline-age potential, may be detected in this culture system. Formation of HPP-CFC colonies, characterized by size greater than 0.5 mm and multilineage composition, requires the use of multiple cytokines in order to proliferate.

The LTC-IC assay correlates more closely to HSCs. Here, hematopoietic cells are plated on top of stromal cell lines or irradiated primary bone marrow stroma. Primitive HSCs are able to initiate growth and to generate progeny in vitro for up to 12 weeks. Progenitor cells and mature myeloid cells are removed weekly to prevent overgrowth. Ultimately, HSCs, characterized by high proliferative and self-renewal capabilities, are able to sustain long-term culture and may be enumerated at the conclusion of the assay.

The cobblestone area-forming cell assay represents a type of LTC-IC that similarly measures the ability of cells to initiate growth and generate progeny in vitro for up to 12 weeks. However, the readout is slightly different. Hematopoietic cells are plated at limiting dilution on top of a monolayer consisting of irradiated bone marrow stroma or a stromal cell line. The growth of colonies consisting of at least five small, non-refractile cells reminiscent of cobblestones, found underneath the stromal layer, are counted. Such cultures are maintained using weekly half-media changes until up to 5 weeks after seeding. In this assay, more primitive cells appear later, and day-35 cobblestone area-forming cells (CAFCs) represent a close correlate of a cell with in vivo long-term multilineage repopulating potential. LTC-ICs may be enumerated after day 35 by completely removing the CAFC medium, overlaying methylcellulose and counting the number of colonies produced after 8-10 days.

In vivo assays The CFU-S assay, first developed by Till and McCulloch in 1961, is described earlier in this chapter (see Hematopoietic stem cell concepts and their origin). Bone marrow or spleen cells are transplanted to irradiated recipients and animals are killed after 8 or 12 days for analysis of spleen colonies, termed 'CFU-S8' and 'CFU-S ', respectively. Cells that give rise to CFU-S8 are predominantly unipotential and produce erythroid colonies. CFU-S12 colonies consist of several types of myeloid cells, including erythrocytes, megakary-ocytes, macrophages and granulocytes. Cells giving rise to CFU-S12 represent a more primitive population of multipotent cells than those that result in CFU-Sg.

The long-term repopulation assay (LTRA) is a more accurate measure of HSC activity. Whole collections of hemat-opoietic cells or fractionate subpopulations are transplanted to lethally irradiated syngeneic mice, typically by tail vein injection. Recipients are screened for ongoing hematopoiesis 8-10 weeks after transplantation. By this time, hematopoiesis is firmly established and donor-derived blood is produced by transplanted HSCs. This assay requires that cells fulfill the two central features of HSCs: multilineage reconstitution, consistent with multipotentiality, and indefinite hematopoiesis, indicative of self-renewal.

Tracking of transplanted cells was originally conducted using radiation-induced chromosomal abnormalities or by retrovirally marking donor cells. However, a major advance in the ability to track transplanted cells has been the development of congenic mice with minor allelic differences in the leukocyte common antigen (Ly5), which is expressed on all nucleated blood cells. The C57/BL6 ('black-6') strain contains the Ly5.2 antigen while the BL6/SJL strain contains a separate allele, Ly5.1. However, these syngeneic strains may be transplanted interchangeably. Both antibodies are available with distinct fluorescent labels. FACS analysis using these antibodies permits measurement of donor-derived reconstitution of the nucleated blood lineages. However, erythrocytes and platelets do not express the Ly5 antigen and cannot be tracked using this technique. Instead, investigators use con-genic strains with allelic variants of hemoglobin and glucose phosphate isomerase to track erythroid and platelet engraft-ment, respectively.

A modification of this assay permits quantitation of HSCs within the graft. Here, HSCs are quantified by transplanting limiting-dilution numbers of bone marrow into lethally irradiated recipients. Each recipient also receives 1 X 105 cells of the host's marrow to ensure survival during the period of pancytopenia immediately after irradiation. At 10-12 weeks, host peripheral blood is assessed to determine whether donor-derived reconstitution has occurred. Donor cells must constitute at least 1% of the peripheral blood to contend that at least one HSC was present in the donor population. Also, both lymphoid and myeloid lineages must demonstrate at least 1% donor derivations. The percentage of reconstituted animals in each group may be plotted against the number of input cells to determine a limiting-dilution estimate of the frequency of HSCs within the donor population. This assay is termed a 'competitive repopulation assay', as transplanted HSCs compete with the host's HSCs that survive irradiation-induced death, in addition to host cells transplanted with the graft. The HSCs detected are termed 'competitive repopulation units' (CRU). The competitive repopulation assay using congenic mouse strains is depicted in Figure 3.5.

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