Human Hematopoietic Stem Cells

A. Clinical Transplantation Models

The ability of human HSC to repopulate all hematopoietic lineages has been demonstrated by the success of bone marrow transplantation for the treatment of hematological diseases. Since the initial descriptions of successful bone marrow transplants (6,7), there have been many improvements in the way the recipient's bone marrow is ablated and the management of histocompatibility differences between the donor and the recipient. These developments have made bone marrow transplantation a common treatment for many inherited and acquired hematological diseases (63). Similar to studies in mice, the properties of human HSC can be analyzed in selected patients transplanted with genetically distinguishable donor hematopoietic cells (64). Through the use of isozyme polymorphisms, DNA polymorphisms, and sex chromosome differences, it has been shown that the donor bone marrow can repopulate the lymphoid, myeloid, and erythroid lineages of human recipients. In a classic example, analysis of transplant recipients infused with bone marrow from female donors heterozygous for the X-linked isozymes glucose-6-phosphate dehydrogenase (G-6-PD), phosphopglycerate kinase (PGK), or hypoxanthine phosphoribosyltransferase (HGPRT) has shown that human hematopoiesis can be reconstituted from a limited number of HSC (64).

Several sources of human HSC have been used successfully for transplantation. While bone marrow and mobilized peripheral blood are the most common sources of HSC, fetal liver and cord blood HSC are also used [for reviews see (65-67)]. Human HSC are mobilized into the peripheral blood by treating the donor with cytokines such as G-CSF alone or in combination with other growth factors. Apheresis of the donor after 5-7 days of treatment gives a very high yield of cells, which generally exceeds the average number of stem and progenitor cells that can be harvested from bone marrow (67). Cord blood collected after delivery has also been shown to be a rich source of transplantable HSC. Recent work has shown that the HSC content of approximately 100 mL of cord blood is sufficient to repopulate 80-kg recipients (66).

B. In Vitro Assays for Human HSC

The development of in vitro assays for the most primitive human hematopoietic cells has greatly facilitated the study of human hematopoiesis. The long-term bone marrow culture (LTBMC) (68,69) and the ''extended'' LTBMC (70) areiniti-ated by growing an adherent layer of stromal cells consisting of fibroblasts, endothelial cells, and macrophages. The stromal layers are then seeded with bone marrow cells to start the bone marrow culture. At biweekly intervals, a portion of the culture medium is replaced, and the nonadherent cells in the aspirated medium can be analyzed for myeloid progenitor colony formation. Human long-term bone marrow cultures initiated by single cells generate myeloid-colony-forming cells for periods of 40-60 days (71). After several weeks of culture, the standard LTBMC medium can be replaced with medium that supports the growth of lymphoid progenitor colonies. Following the medium change, the same culture will begin to produce lymphoid progenitor cells (72). In the extended LTBM, the remaining hematopoietic cells are reseeded onto a fresh layer of stromal cells where hematopoiesis continues for another 50-60 days. The continuous generation of hemato-poietic progenitor cells over a long period of time from the extended LTBMC suggests strongly that these in vitro assays are good surrogates for the transplant experiments used to study mouse HSC (70,71).

Clonal hematopoiesis in vitro has been demonstrated using long-term culture initiating cell (LTCIC) assay (69). In this assay, hematopoietic cells are enriched for HSC, and single cells are cultured on preexisting stromal layers and analyzed for proliferation and colony formation for 40-60 days. In mouse models, the behavior of purified murine HSC has been compared in both in vivo transplant models and in LTCIC. The number of LTCICs and the number of repopulating HSC were directly proportional (73). These studies suggest that human LTCIC assays are also recognizing the most primitive hematopoietic cells. In the extended LTCIC assay, hematopoietic cells are replated onto a ''fresh'' layer of stromal cells, which are cultured for an additional 40-60 days. The cells that have the capacity to generate colony-forming cells in the extended LTCIC assay have the most primitive phenotype, and the rate at which LTCICs are marked by transduction with retrovirus vectors closely resembles the rate at which human HSC are marked with retrovirus vectors (70).

C. Immune Deficient Animal Models

Attempts to develop an in vivo transplantation assay for human hematopoietic stem cells have focused on immune-deficient sheep [reviewed in (74,75)] or mice [reviewed in (76,77)] as recipients for human hematopoietic cells. During development, the sheep hematopoietic system undergoes a rapid expansion between days 50 and 60 of gestation, while the sheep immune system does not become functional until days 67 to 77 of gestation. The window between days 55 and 63 of gestation provides an opportunity to introduce human hematopoietic cells into fetal sheep. The transplantation of human cells during the ''expansion'' period facilitates en-graftment, and the presence of human cells in the fetal sheep before the immune system becomes active induces tolerance to human antigens [Fig. 2; (74,75)].

In a large series of sheep generated over the last 10 years, approximately 70% of the animals transplanted with human fetal liver cells had human hematopoietic cells in their peripheral blood and bone marrow. The human cells accounted for approximately 5% of the total number of peripheral blood and bone marrow cells, and all lineages were represented. Human cells were identified at all time points for periods of up to 4 years (78-82). To evaluate HSC self-renewal, bone marrow cells from primary animals are transplanted into preimmune fetuses. In approximately 1/3 of the recipients, human cells were detected demonstrating self-renewal of the original engrafted cells (82,83).

Human bone marrow or cord blood HSC can also engraft into fetal sheep. Approximately 50% of recipient sheep transplanted with human marrow or cord blood cells show long-term persistence of human cells. The levels of human cells in sheep transplanted with either bone marrow or cord blood HSC were as good (bone marrow) or better (cord blood) than that seen in the recipients of human fetal liver cells (84-86). Approximately 80% of these recipients showed signs of Graft vs. Host Disease (GVHD), as might be expected from transplants including mature human T-lymphocytes (84-86). The transplantation of T-cell-depleted bone marrow or cord blood prevented GVHD, but was associated with lower levels of engraftment and a lower percentage of animals engrafted (87). These observations are nearly identical to what are observed in human bone marrow transplant recipients and validate the fetal sheep model as an assay for the most primitive human hematopoietic cells.

The low percentage of human cells in the chimeric sheep can be increased by the injection of recombinant human cytokines. Injection of human Interleukin-3 (IL-3) and Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF), or injection of Stem Cell Factor (SCF) increased the number of human cells in the blood or marrow, 5- and 2-fold respectively (82,83).

Although the fetal sheep model satisfies all of the criteria for a xenograft model for human hematopoiesis, the sheep model is not practical for most research laboratories. As a result, many groups have sought to develop small animal models for human hematopoiesis that combine the advantages

Figure 2 The fetal sheep model for the engraftment of human hematopoietic stem cells. The hematopoietic system of the developing sheep begins a rapid expansion around day 50 of gestation, but the immune system does not begin to develop until around day 70 of gestation. Injection of human hematopoietic cells around day 60 of gestation leads to engraftment and expansion of human cells that can be recovered from newborn and older sheep.

Figure 2 The fetal sheep model for the engraftment of human hematopoietic stem cells. The hematopoietic system of the developing sheep begins a rapid expansion around day 50 of gestation, but the immune system does not begin to develop until around day 70 of gestation. Injection of human hematopoietic cells around day 60 of gestation leads to engraftment and expansion of human cells that can be recovered from newborn and older sheep.

demonstrated in the fetal sheep model with the cost effectiveness of a small animal model. These efforts have focused on either beige, nude, xid (BNX) mice or mice homozygous for the severe combined immunodeficiency (scid) mutation (reviewed in (88-90).

BNX mice are homozygous for 3 mutations causing immune deficiency in the mouse. The combination of the natural killer cell deficiency caused by the beige mutation, the lack of a thymus caused by the nude mutation, and the loss of some B-cell functions caused by the xid mutation renders BNX mice almost completely immune-deficient. In the original report, human bone marrow cells were injected into sublethally irradiated BNX mice (91). The recipient mice contained low levels of human cells in the bone marrow and spleen (<1%), which could be detected by the presence of human repetitive DNA in these organs. In addition, human colony-forming cells from the same tissues could be identified by their selective growth in medium supplemented with human IL-3 and GM-CSF. Since hIL-3 and hGM-CSF do not support the growth of mouse colony-forming cells, it was possible to rescue a low number of human progenitor cells for up to 8 weeks posttransplantation (91). The BNX model has been refined by the injection of human stromal cells engineered to produce human IL-3 along with human bone marrow cells (92). Human hemato-poietic progenitors were recovered from the spleens and bone marrow of recipient mice for up to 9 months after transplantation. The coinjection of engineered human stromal cells im proved the level of human cells in the bone marrow to approximately 6% (92).

The ability of a single cell to give rise to both lymphoid and myeloid progeny is a unique property associated with HSC. This property has been demonstrated in the BNX mouse model by transplanting human CD34 + umbilical cord blood cells transduced with a retrovirus containing a neomycin resistance (neor) gene (93). Following transplantation, a small number of human hematopoietic progenitor cells containing the neor provirus in the recipient animals were detected. If a myeloid progenitor cell and a T-lymphocyte containing the neor provirus are derived from the same progenitor cell, the provirus should be integrated into exactly the same genomic site in each type of cell. DNA was extracted from human myeloid colonies grown in semisolid medium and from individual human T cells. The insertion site of the provirus was demonstrated using inverse PCR. Inverse PCR amplifies circular fragments generated by digestion of DNA with an enzyme that cuts once inside the provirus and at other random sites throughout the genome. Using primers specific to a single region of the provirus for PCR, the circular fragments can be amplified, generating a specific fragment for each proviral insertion that can be identified by its DNA sequence (94). Many insertion sites were detected in the myeloid colonies, 4 of which were shared by T-lymphocyte clones isolated from the same mouse. DNA sequence analysis demonstrated that the proviruses were integrated into the same spot in the ge nome. These studies provided the first and most definitive evidence that among the human cells that engraft into the BNX mouse are cells that have the properties associated with HSC (93).

Mice homozygous for the scid mutation lack functional B and T lymphocytes due to defects in V(D)J recombination and double-stranded, break repair due to mutations in the gene encoding the catalytic subunits of DNA-PK and DNA-PKCS (95). Improved levels of engraftment and proliferation of human bone marrow cells transplanted into CB.17 scid/scid (SCID) mice. Human cells were detected in the bone marrow of transplanted CB.17 SCID mice at levels of 3% or higher for 8-10 weeks (96). Higher levels of human cells were detected in CB.17 SCID mice treated with human stem cell factor (SCF) and PIXY 321, a fusion molecule between human IL-3 and GM-CSF (96). Human myeloid and lymphoid cells were detected in the peripheral blood of transgenic SCID mice transplanted with human bone marrow cells for up to 24 weeks in a line of transgenic mice that expressed the human GM-CSF, IL-3, and SCF genes (97).

The meticulous work of Shultz and colleagues showed that the NOD strain of mice, which is susceptible to Non-Obese Diabetes, was NK-cell-deficient (98). When the scid mutation was crossed onto this strain, the resulting NOD-SCID mouse was more immune-deficient than any other strain carrying the scid mutation (98). A number of groups have demonstrated that NOD-SCID mice make superior hosts for engraftment of human hematopoietic cells (76). NOD-SCID carrying the knockout allele of p-2 microglobulin are even more immune-deficient and are further improved recipients of transplanted human cells (99).

NOD-SCID mice have been further engineered to provide the optimal microenvironment for human hematopoiesis. SCID mouse models in which pieces of human fetal thymus and fetal liver are implanted under the kidney capsule demonstrated the presence of human T-lymphocytes and myeloid cells are detected in the peripheral blood of transplanted SCID-hu animals for 6-12 months (100). Examination of the engrafted organs revealed the presence of multipotential mye-loid and erythroid progenitor cells, and a full complement of differentiating human T-lymphocytes (100,101). Further refinement of the SCID-hu model implanted human fetal bone, thymus, and spleen fragments (abbreviated BTS) into SCID mice. The SCID-hu BTS mouse that can support human hematopoietic cells of all lineages for 36 weeks or more (102). The fetal bone fragments can be directly injected with purified hematopoietic cells that can then be analyzed for their capacity to repopulate and proliferate (102).

The demonstration of clonal hematopoiesis on the SCID models has not been as clear as in the BNX mouse model. For example, Josephson et al. examined multiple clones of CD19 + B-lymphocytes, and expanded populations of single myeloid cells obtained from NOD-SCID mice transplanted with foamy virus-transduced cells 6 weeks earlier. Less than 20% of the proviral insertions present in the B-lymphocyte clones were also represented in myeloid cells (103). It is clear that NOD-SCID mice allow the engraftment and proliferation of both HSC and more committed progenitor cells. Recent data analyzing NOD-SCID mice at 12 weeks posttransplantation suggest that the hematopoietic cells present at the later stages of transplantation are derived from the most primitive cells (104).

D. Phenotype of Human HSC

The enrichment of human HSC has used strategies that are similar to those used to enrich mouse HSC. Human hemato-poietic cells expressing lineage markers have no ability to form colonies in vitro and are inactive in the LTCIC assay (105). All human hematopoietic colony-forming cells express the glycoprotein CD34. Human CD34+ cells isolated from bone marrow, mobilized peripheral blood, umbilical cord blood, or fetal liver are used for clinical transplantation, indicating that the CD34+ population contains HSC. Human HSC can be separated from colony-forming cells based on the presence of the CD38 antigen (106). Colony-forming cells express CD38 (Lin - CD34 + CD38 +), while the more rare Lin- CD34+ CD38- cells are the only cell type capable of generating extended LTCIC in vitro (70).

As described above, human CD34 + cells engraft and proliferate in the BNX, SCID, and fetal sheep models (92,107-110). Recently, several lines of evidence have demonstrated the existence of human CD34- HSC. Differential Hoechst staining of human bone marrow cells reveals a substantial number of CD34 - cells in the side population (111). (Lin - CD34 - CD38 -) bone marrow cells have been shown to engraft and proliferate in the fetal sheep (112) and NOD-SCID mouse models (113). These results indicate that at least some human HSC are CD34 -, while others are CD34 +. The difficulty in separating the repopulating CD34- CD38- cell from the large number of other CD34 - cells may have prevented the injection of sufficient numbers of CD34- cells in previous studies.

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