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Stem Cells, Vol. 15, No. 4, 314-323, July 1997
© 1997 AlphaMed Press

Growth of Human Hematopoietic Cells in Immunodeficient Mice Conditioned with Cyclophosphamide and Busulfan

Ross S. Basch, Francis L. Quito, Jane Beh, John A. Hirst

Department of Pathology and the Kaplan Cancer Center, New York University Medical Center, New York, New York, USA

Key Words. Hematopoiesis • SCID Mice • Bone marrow • Transplantation

Dr. Ross S. Basch, Department of Pathology, NYU School of Medicine, 550 First Avenue, New York, NY 10016, USA.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Human hematopoietic cells survive and proliferate for at least 10 weeks in severe combined immunodeficient mice prepared with the cytotoxic drugs busulfan and cyclophosphamide. The human cells growing in the mice can be detected by in situ hybridization using a probe detecting human repetitive DNA or by staining the cells with antihuman antibodies (anti-CD45 and anti-HLA I). Busulfan/cyclophosphamide-treated mice were injected with a wide range of cell doses, ranging from 5 to 50 million unfractionated bone marrow cells and 2 to 40 million low density bone marrow cells. Animals were killed at 1, 3, 5, 7 and 10 weeks after transplantation. Human cells were found in many animals and could be detected as early as one week after transplantation. The peak of repopulation was at two to five weeks, but in some animals human cells could be detected for as long as 10 weeks. Many of the human cells expressed high levels of glycophorin, but mature human erythrocytes were not found.

The human cells were not uniformly distributed throughout the marrow. They grew in small clusters in the subepiphyseal region. The extent of human hematopoietic repopulation in the mouse was extremely variable. At no time and at no dose was repopulation achieved in all of the animals. Treatment with human growth factors is not necessary for the survival of the human hematopoietic cells but, in their absence, normal hematopoiesis does not occur.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
For many years attempts to grow normal human hematopoietic cells in mice were largely unsuccessful [1]. The development of strains of congenitally immunodeficient mice altered this picture and beginning almost a decade ago, successes were achieved [2-8] using both SCID (severe combined immunodeficient) mice and triply deficient bg/nu/xid mice. Namikawa et al. [4, 5] reported long-term hematopoiesis in SCID-human mice which were constructed by implanting human fetal thymus and human fetal liver under the kidney capsule. In this system, the grafts create a marrow-like microenvironment consisting exclusively of human cells. Animals with a functional human hematopoietic system have been used for a variety of studies in recent years [4, 5, 9-11]. Attempts to grow human cells within the murine hematopoietic microenvironment have been less fruitful. Dick and associates achieved some success by treating irradiated SCID mice with human hematopoietic growth factors [6], but the survival of normal human hematopoietic cells in the murine environment has not been great. More substantial growth of human cells has been achieved when fetal or cord blood cells rather than adult bone marrow (BM) were used as the source of hematopoietic cells [9, 12, 13]. Barry et al. [7] suggested that the problem in maintaining adult tissues in SCID mice can be improved by treating the animals with anti-asialo GM-1 antibodies which reduce natural killer cell activity. As more experience has been accumulated with these chimeric systems, it has become clear that human hematopoietic cells, including T and B lymphocytes, can grow and differentiate in the murine environment [2, 14-17].

We have shown that human hematopoietic cells survive and proliferate for at least 10 weeks in SCID mice prepared with the cytotoxic drugs busulfan (BU) and cyclophosphamide (CY). These agents are known to suppress natural killer cell activity, and BU produces a specific loss of early stem cells [18-20]. If the concept that stem cells require biologic space is correct, then the reduction of mouse stem cells might prove helpful in promoting stem cell engraftment. The human cells in the mice can be detected by in situ hybridization using a probe detecting human repetitive DNA or by staining the cells with antihuman antibodies (anti-CD45 and anti-HLA I). Treatment with human growth factors is not necessary for the survival of the human hematopoietic cells but in their absence little if any normal hematopoiesis occurs.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Bone Marrow Cells
Human bone marrow cells (BMC) were obtained from discarded orthopedic specimens obtained after hip replacement. A total of 14 (10 men; 4 women) specimens were obtained. Donor ages ranged between 44 and 62 years. Specimens were obtained under an institutional review board approved protocol. Medullary bone was scraped into a sterile saline-filled pestle, ground, passed through a 50 µm-pore sieve and washed twice in Iscove's modified Dulbecco's medium (IMDM) containing 2% fetal bovine serum (FBS). In several experiments the BMC were cultured for 72 h in IMDM containing 20% FBS. When required, low-density BMC were prepared by flotation on a layer of isotonic Ficoll-Hypaque (sp g 1.077).

Construction of Chimeric Mice
CB.17 SCID mice and the congenic CB.17 mice [21] were purchased from Taconic Farms (Germantown, NY) and housed in an isolation facility in the NYU Medical Center animal facility. The animals were prepared for transplantation by injecting them with two series of three injections of BU (Myleran) one week apart (30 mg/kg, i.p. in 0.2 ml of 20% dimethylsulfoxide in saline). The BU was dissolved in dimethylsulfoxide and then diluted to the appropriate concentration in warm (42°C) saline. The injections were given 48-72 h apart. One to five days after the last injection of BU, the mice were given a single injection of CY (100 mg/kg in saline i.p.) and human BMC were injected i.v. 24-36 h later. Of the 14 experiments, 10 had CY injected 24-48 h after the last injection of BU. Because of delays in obtaining the human material, in two experiments the CY was injected after 72 h and in two others the interval was five days. No differences were found that could be attributed to the variation in the injection schedules. The regimen is moderately toxic: 7 out of 40 BU/CY SCID mice that were not injected with any human cells died during three months of observation. Mortality among immunodeficient animals after BU/CY is similar to that of normal BALB/C mice treated with the same regimen.

Immunofluorescence
Human cells in chimeric mice were detected by indirect immunofluorescence using biotinylated anti-CD45 (clone HI30, mouse IgG1; Caltag; S. San Francisco, CA) or biotinylated antimonomorphic HLA-ABC (clone B9.12.1, mouse IgG2a; Immunotech; Westbrook, ME) as the primary reagent and fluorochrome-conjugated avidin. Controls were stained with an isotype-matched normal immunoglobulin and the fluorescent conjugated secondary reagent.

In artificial mixtures of human and mouse BMC, 0.5%-0.7% human cells could be reliably detected. Anti-HLA staining was generally brighter than anti-CD45 staining and was used to identify human cells for most of the experiments. A three-color analysis was performed using FITC- (fluorescein isothiocyanate) and PE- (phycoerythrin) conjugated antibodies to detect differentiation markers in conjunction with streptavidin coupled to a tandem conjugate of PE and cyanine 5 (Tricolor, Caltag) to provide the third color. This tandem conjugate absorbs at 488 nm and emits at 670 nm. All three dyes are excited by a single laser. The antibodies used to identify differentiating hematopoietic cells were: CD34-PE (clone QBEND-10; Immunotech), CD19-PE (clone SJ25-C1; Caltag), CD3-FITC (clone S4.1; Caltag), CD33-PE (clone D3HL60.251; Immunotech), glycophorin A-FITC (clone D2.10; Immunotech) and CD45-FITC (clone ALB12, mouse IgG1; Immunotech).

In Situ Hybridization
Human cells in either cytocentrifuge preparations of murine hematopoietic cells or in tissue sections were detected by in situ hybridization using biotinylated COT-1 human DNA as a probe (GIBCO BRL; Rockville, MD). Suspensions of cells or sections were deposited on glass slides pretreated with 3-aminopropyltriethoxysilane. The slides were baked at 65°C for one h, deparaffinized when necessary and incubated with proteinase K (25 µg/ml in phosphate-buffered saline [PBS]) for 10 min at 37°C. The slides were then fixed with 4% paraformaldehyde and stored in 70% ethanol at 4°C. After removal from the ethanol, they were rinsed in 2x SSC (standard saline citrate) and prehybridized for six to eight h. Each slide was then incubated with 10 µl of hybridization mix (2x SSC, 1 mg/ml yeast tRNA, 50% formamide plus labeled probe) under a cover slip for 12 h at 42°C. Unhybridized probe was removed by three washes in 2x SSC, 50% formamide at 50°C. After washing, the bound probe was detected, after binding to a streptavidin-alkaline phosphatase conjugate (GIBCO BRL), by incubating the slides in a freshly prepared solution of NBT/BCIP containing 200 µg/ml of levamisole to block endogenous alkaline phosphatase.

Assay of Hematopoietic Progenitors (Colony Forming Unit Culture [CFU-C] Assay)
Colony forming cells were assayed in IMDM, and supplemented with 30% FBS, 1% bovine serum albumin and 10–4 M 2-mercaptoethanol in the presence of appropriate growth factors. The medium was made semisolid by the addition of methylcellulose. Cells (100,000-200,000) were cultured in three replicate cultures containing 1.0 ml of complete medium in 35 mm tissue culture dishes (Corning Glassware; Corning, NY). Recombinant growth factors (recombinant human GM-CSF, 20 ng/ml, and recombinant human interleukin 3, 20 ng/ml) were used to support the growth of human myeloid progenitors. The final medium contained 0.9% methylcellulose, 30% FBS, 1% bovine serum albumin, 10–4 M 2-mercaptoethanol and 2 mM L-glutamine in Iscove's Dulbecco's modified Eagle's medium (MethoCult H4230 [Stem Cell Technologies; Vancouver, Canada] or equivalent). This medium, unlike media prepared with phytohemagglutinin-L cell-conditioned medium or containing stem cell factor, G-CSF and interleukin 6, does not support the growth of murine hematopoietic cells. The cultures were scored after seven to nine days of incubation and again at two weeks. Only groups of more than 10 cells were counted as colonies. Three replicates were made for each sample and a mean (± SD) calculated.

Immunohistochemistry
Hematopoietic colonies growing in methylcellulose were deposited onto 3.0 µM Nucleopore membranes by gentle suction and then transferred to silanated glass slides and fixed. They were washed twice with PBS (10 min each), distilled water (2 x 10 min) and air dried. The slides were incubated with 10% normal goat serum in PBS for 10 min to block nonspecific binding and then incubated with the mouse anti HLA-ABC (biotin) for 60 min. After washing with PBS the cultures were incubated with streptavidin-alkaline phosphatase conjugate for 30 min. The alkaline phosphatase was visualized using naphthol AS-MX phosphate and Fast Red TR salt (Sigma Chemicals; St. Louis, MO). Levamisol (1 mM) was used to inhibit endogenous alkaline phosphatase. Control samples were incubated with mouse IgG instead of specific monoclonal antibodies.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
BU/CY-treated mice were injected with a wide range of cell doses, ranging from 5 to 50 million unfractionated BMC and 2 to 40 million low density BMC. Animals were killed 1, 3, 5, 7 and 10 weeks after transplantation. The results are summarized in Figures 1 -3GoGoGo. Human cells were serologically detected in many animals. The extent of human hematopoietic cell growth in the mice was extremely variable. At no time and at no dose was repopulation achieved in all of the animals. In 4 of 14 experiments no human cells were detectable at any time (results from these animals have been omitted from the figures). The proportion of mice in which human cells were found was dose dependent but the percentage of human cells recovered from the recipients did not reflect the number injected ( Fig. 2). Low density BMC, prepared by flotation over Ficoll-Hypaque (sp g 1.083), were used in most of the experiments. In several experiments these were compared with unfractionated BMC. Flotation over Ficoll-Hypaque produces a two- to threefold enrichment in CD34+ cells and, whenever possible, the dose of cells transferred was adjusted so that the animals received equivalent numbers of CD34+ cells. The low density cells were slightly less efficient than unfractionated marrow but the difference was not statistically significant ( Fig. 3). High density BMC (cells recovered from the pellet of the Ficoll-Hypaque separation) did not produce detectable progeny under any conditions (data not shown). The kinetics of repopulation after the transfer of low density BMC are shown in Figure 1. The results shown include all of the animals (total = 63) that received 20 million low density BMC in nine experiments in which at least one mouse was found to have significant numbers of human cells in the marrow after receiving this dose of cells. Each different symbol represents a separate donor sample. Human cells were detected in the marrow of 31/63 mice at this cell dose. If the 18 mice in the four experiments in which no human cells were detected are included, the rate of successful transfer falls to 31/81. The proportion of human cells in the mice that tested positive was generally in the 1%-4% range. In three animals the proportion of human cells exceeded 10%, and all of these mice received cells from the same donor. The recovery of human cells was maximal three to five weeks after transfer.



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  		Figure 1. Growth of human BMC in BU/CY SCID mice. Each symbol represents the percentage of HLA-positive cells found in an individual mouse that had been injected with 2 x 107 Ficoll-Hypaque separated human BMC. All of the mice injected with cells from the same donor are indicated with the same symbol. The dotted line above the filled area indicates the upper limit of values obtained in mice that received no human cells.

 


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  		Figure 2. Effect of varying number of human cells injected on the proportion of human cells recovered. Each symbol represents the percentage of HLA-positive cells found in the BM of an individual mouse. The results from three separate experiments are shown and each experiment is indicated by a different symbol. Low density BMC were used in all three experiments, and the recipient mice were killed five weeks after transfer. The fractions at the top of the figure indicate the number of mice in which human cells were identified divided by the total number examined.

 


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  		Figure 3. Growth of human hematopoietic cells in murine BM after repopulation with partially purified progenitors. The figure shows the proportion of HLA-positive cells recovered from the femoral BM five weeks after transfer. Each mouse received either 4 x 107 unfractionated BM: 2 x 107 ficoll-hypaque separated marrow or 1 x 106 sorted CD34+ BMC. Two different donors were used for this experiment, and these have been distinguished by the use of open and closed symbols. The + symbol indicates the geometric mean of the sample.

 
In two experiments CD34+ cells, purified by fluorescence activated cell sorting, were injected in numbers equivalent to those used with low density marrow. Minimal numbers of human cells were found in one of three animals injected with one sample and one of four mice injected with the other sample ( Fig. 3). Mixing CD34+ cells with high density BMC did not improve the recovery of human progeny (data not shown).

When human cells were found, they could be detected within a week after transplantation. Figure 4 illustrates the staining found in a mouse that was transplanted with 10 million low density BMC. Of the cells recovered from the BM, 3% were CD45+ and more than half (53%) of these were also stained with CD34. The CD45 staining was dim. Human cells were also detectable in the spleen at levels similar to those found in the marrow (data not shown). In one mouse whose spleen was severely atrophic (weight = 24 mg), 48% of the marrow and 55% of the spleen cells were of human origin and of these, ~60% of the marrow cells and 11% of the spleen cells expressed CD34.



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  		Figure 4. Detection of human hematopoietic cells in mouse BM. The figure is a correlated two-parameter histogram of cells stained with CD45 FITC and CD34 PE. The cluster in the lower left is produced by mouse cells which stain with neither CD45 nor CD34. CD45 is not expressed on nucleated RBC.

 
After three weeks an average of 7 x 106 cells/femur and 40 x 106/spleen were recovered from each mouse. The total number of cells recovered was not related to the number of human cells transferred. Since a femur represents 5%-7% of the total marrow, these mice have ~108 total marrow cells. In the best case, 15 x 106 HLA+ cells were recovered from a mouse that had received only 2.5 x 106 low density human BMC. This represents a sixfold increase compared to the number of human cells injected. Since most of the injected cells had only limited capacity for self-renewal and human cells presumably were also located in tissues other than those sampled, the extent of the multiplication of the human progenitors is probably much larger than this.

After five weeks the number declined in both the marrow and spleen. Few CD34+ cells were recovered from the marrow and this small number declined precipitously after the third week ( Fig. 5). In several mice, a CD34+ CD45very dim population was detected in the spleen three weeks after transfer of the human cells. A mouse in which this population was prominent is shown in Figure 5. This staining pattern was not found in most animals and was not detected in later samples.



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  		Figure 5. Human hematopoietic cells in BU/CY SCID mice three and five weeks after transplantation. Correlated two-parameter histogram of the expression of CD45 and CD34. The cells were stained simultaneously with anti-CD45 (FITC) and anti-CD34 (PE) and a biotinylated monoclonal antibody against a monomorphic determinant on HLA Class I. After washing they were stained with R613-streptavidin. The list mode data were gated so that only HLA I-positive cells (nucleated human cells) were analyzed. In this histogram the cluster in the lower right consists mainly of human (HLA I-positive) cells that did not stain with either CD34 or CD45.

 
The human cells were not uniformly distributed throughout the marrow. They grew in small clusters in the subepiphyseal region ( Fig. 6). Only occasional human cells were observed in diaphyseal regions. If the marrow was prepared by cutting off the ends of the long bones and flushing out the marrow "plug," all of the human cells would be lost unless care was exercised in obtaining the material located at the ends of the bones.



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  		Figure 6. Human hematopoietic cells in mouse BM. In situ hybridization using a cot-1 DNA probe to detect human cells growing in the BM of a BU/CY-treated mouse. The femurs were fixed in 10% buffered formaldehyde and then decalcified before sectioning. A biotinylated probe was used and the hybridized DNA was detected and visualized with streptavidin-alkaline phosphatase and NBT/BCIP. Panel A on the left is a section through the proximal end of the femur and Panel B is a section through the diaphysis of the same bone. Original magnification 100x.

 
Many of the human cells expressed high levels of glycophorin on their surface ( Fig. 7). The proportion in some animals reached 60% in marrow and 50% in spleen at three weeks but declined rapidly. This glycoprotein appears early and persists throughout erythroid development. Despite the presence of many glycophorin-positive cells in the spleen and marrow, only trace quantities of human hemoglobin were detected in the peripheral blood.




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  		Figure 7. Expression of glycophorin and CD33 by the human cells growing in BU/CY SCID mice five weeks after transplantation. The cells were stained simultaneously with directly conjugated antibodies to glycophorin and CD33 and a biotinylated monoclonal antibody against a monomorphic determinant on HLA Class I. After washing the cells were stained with R613-streptavidin, a covalent tandem conjugate of PE and Texas Red (GIBCO BRL) which is excited at 488 nm and emits at 613 nm. The three-color samples were then analyzed on a FACScan cytometer. The list mode data were gated so that only HLA I-positive cells (nucleated human cells) were analyzed for their expression of the other markers. Glycophorin is expressed by cells of the erythroid lineage and CD33 by developing myeloid cells. Controls showing the absence of staining of mouse spleen cells and the pattern obtained with normal human BM are shown at the top of the figure.

 
Human cells could be detected for at least 10 weeks. Myeloid differentiation, as indicated by the presence of the CD33 antigen, was not detected. Despite the absence of CD33(+) cells, human cells isolated from BU/CY-treated animals formed myeloid colonies in semisolid medium ( Table 1). In order to demonstrate that the myeloid colonies that developed after 9-14 days in these cultures were of human origin, selected colonies were transferred to microscope slides and stained immunohistochemically with the anti-HLA reagent. Under the conditions used, only human hematopoietic cells produced colonies.


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  		Table 1. Hematopoietic colony formation by human cells isolated from busulfan/cyclophosphamide (BU/CY) mice
 
In two animals, CD19, an antigen found on virtually all B cells, was detected on virtually all of the human cells (HLA+) recovered from the spleen and on 60% of peripheral blood lymphocytes (PBL) ( Fig. 8). CD2 was present on both BMC and spleen cells at five weeks. CD3(+) cells were not found in the blood at any time.




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  		Figure 8. Expression of CD2 and CD19 by human cells growing in BU/CY SCID five weeks after transplantation. The cells were stained simultaneously with anti-CD2 (FITC) and anti-CD19 (PE) and a biotinylated monoclonal antibody against a monomorphic determinant on HLA Class I. After washing they were stained with R613-streptavidin. The list mode data were gated so that only HLA I-positive cells (nucleated human cells) were analyzed. Controls showing the staining of normal human mononuclear cells and the absence of staining of mouse spleen cells are shown at the top of the figure. The two populations stained by anti-CD2 (representing T cells and natural killer cells) in the peripheral blood are clearly visible in the control figure.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Adult human hematopoietic progenitors can survive in mice in the absence of exogenous cytokines. The human cells survive for many weeks but do not undergo normal development. No mature myeloid lineage progeny were detectable serologically after three to five weeks, but CFU-C continued to be formed for at least 10 weeks. B cells (CD19+) were found in most animals and in a small number of mice—B cells proliferated extensively.

Human cells were found for as long as 10 weeks. It is possible that the first human cells detected (after one to three weeks) were the progeny of committed progenitors rather than pluripotential stem cells but, since CFU-C are the progeny of a short-lived "transit" population and are not self-replicating, the data suggest that the stem cells were among the human cells that survived and proliferated in these mice. Although it is difficult to establish the pedigree of the cells persisting for 7-10 weeks, in mouse repopulation studies, donor cells found at this time would be considered as evidence of long-term repopulation by donor stem cells. In retrospect, the fact that adult human hematopoietic stem cells can survive in an unmodified xeno-environment might have been predicted. In vivo long-term survival in a murine environment in the absence of exogenous cytokines is implicit in the results of Lapidot et al., who showed that delaying the administration of the cytokines for several weeks did not alter the number of human CFU that could be recovered from irradiated SCID mice that were the recipients of adult human BM [6]. Hematopoietic cells from fetal and cord blood grafts can survive for many weeks in immunodeficient animals in the absence of a preformed human microenvironment [12, 13]. Human hematopoietic stem cells also survive for relatively long periods ex vivo in cultures supported by murine stromal cell lines [22]. The factor(s) responsible for the survival of the human stem cells has not been identified [23]. In these cultures, as in intact mice, differentiation and the development of hematopoietic colonies (CFU) require the addition of human cytokines.

Survival of human cells in mice, or at a minimum, their detection, requires the use of immunodeficient mice as well as some kind of myeloablative conditioning regimen. Most of the work cited above involved the use of X-irradiation. We have used SCID mice and a combination of drugs (BU and CY) that has been used extensively in conditioning patients for BM transplantation [24, 25]. The BU/CY regimen is moderately toxic: 7 out of 40 SCID mice died before their tissues could be examined for the presence of human cells. Mortality among immunodeficient animals was similar to that of normal BALB/C mice treated with the same regimen. This mortality is comparable to that found in normal mice after irradiation with 400 rads. This dose appears to be the upper limit that can be given to SCID mice since these animals are extremely radiosensitive [3, 26, 27].

The growth of human cells in these mice is not a rare or unusual event—some indication of successful transfer was found in 10/14 experiments and even relatively small numbers of human cells (2.5 million total BMC) sometimes gave detectable progeny. On the other hand, the most striking feature of this repopulation is its inconsistency. Human cells were never detected in all of the recipient animals in any experiment, regardless of the time interval after transplantation or the quantity of cells transferred. The basis for this inconsistency is not apparent, but two observations may be relevant. First, highly enriched CD34 cells do not engraft as well as unfractionated BM. This suggests that either a product of other BMC (cytokine, chemokine, etc.) or another type of human cell may facilitate engraftment. The capacity of hematopoietic stem cells to engraft seems to be independent of their ability to function as stem cells [28], and release from their normal "quiescent" state seems required for engraftment as well as for colony formation [29, 30]. There is also evidence that cytokine treatment of hematopoietic progenitors alters the pattern of integrin expression by stem cells and thus affects mobilization and homing [31-33]. It seems reasonable to suggest that T cells or monocytes among the grafted cells could be activated and produce factors that enhance the ability of stem cells to engraft. The existence of a T cell, with the specific function of promoting or maintaining engraftment, has been postulated by Ildstad who calls these "facilitator" cells [34]. If a requirement for accessory cells is the explanation for the variability in this model, the apparent advantage of fetal or cord blood progenitors as donor in human to mouse xenografts might be the result of increased numbers of accessory cells rather than any inherent advantage of the progenitors.

The second factor that may contribute to the difficulty in detecting human cells in these mice is the remarkably restricted localization of the transplanted cells. Human cells were almost always detected in small clusters in the distal subepiphyseal metaphysis of the femur. Very few cells were found elsewhere. In early experiments in which the bone marrow cells were prepared by cutting off the ends of the long bones (discarding the ends) and then flushing out the core of marrow, no human cells were detected. Crushing the bones always gave higher yields of human cells. The reason for this localization has not been determined. The arterial supply of the bone consists of both vessels that enter at the ends and penetrating vessels along the length of the bone. The later vessels enter at almost a right angle, and plasma skimming is likely to occur so that relatively few of the transferred cells would be expected to enter at this sight. However in the metaphysis, branches of the nutrient artery become end-capillary loops. Because these capillary loops do not anastomose, cells or clumps of cells can be trapped. Weiss and his colleagues have described an unusual fibroblastic BM "barrier" cell, whose anatomic localization corresponds to the distribution that we have found for the human cells [35]. These barrier cells are thought to play an important role in early hematopoiesis but no evidence has been adduced to indicate that they have a role in regulating the circulation of hematopoietic stem cells. At this time it is not possible to distinguish between specific "homing" mechanisms and nonspecific trapping of the circulating cells.

The presence of a large number of CD19+ cells in some mice was not unexpected. Intraperitoneal injection of human PBL has been shown to consistently lead to B cell proliferation [36] and in Epstein-Barr virus (EBV), transformed B cells grow readily in SCID mice [37, 38]. The donors used in these experiments were not screened for EBV status. Since all of the donors were greater than 44 years old and most were in their 50s, it is probable that they were EBV-positive. The i.v. transfer of human fetal BMC has been shown to lead to the development of human B cells in the spleen and BM as well as the appearance of mature cells (CD20+, IgM+) in the peripheral blood [13, 17]. The absence of CD3+ cells in these mice was somewhat surprising since engraftment by human T cells has been found after transfer of both adult and cord blood PBL. The BM populations used in these transfers are somewhat different than those used in the experiments that have been previously reported. The cells were obtained from surgical specimens. The CD3 content of these preparations is always below 10% and is frequently below 5%. Samples prepared by aspiration from volunteers generally have much greater contamination with peripheral blood and in our experience have CD3 levels of up to 50%. The relatively small number of CD3+ cells transferred probably accounts for our failure to detect human T cells in the BU/CY SCID recipients.

The cells in both the spleen and BM that expressed glycophorin are presumably erythroid lineage cells. We have been unable to detect significant amounts of human hemoglobin in the peripheral blood of these mice (data not shown) and so we conclude that these cells are undergoing abortive development. The alternative possibility, that they complete their maturation but are cleared from the circulation extremely rapidly, has not been ruled out. The identity of the HLA I(+), glycophorin(–), CD33(–) cells found in the spleens and to a lesser extent, in the marrow, remains unknown. Some (approximately 15%) are CD19+ and 25% are CD2+ but the majority (55%) do not express any of the tested markers.

The erratic nature and low level of the repopulation described here contrasts with what appears to be more consistent results obtained with either cord blood or fetal BM [12, 13]. The basis for this difference is not clear. In in vitro studies, hematopoietic progenitors obtained from immature sources have been shown to have higher proliferative capacity and greater replating potential than the analogous material obtained from adults [39, 40]. Lansdorp et al. have suggested that fetal stem cells are better able to proliferate in culture [41]. The evidence that this is an intrinsic property of the stem cells, i.e., that aging, in some way, compromises the ability of primitive stem cells to self-replicate, is not fully developed. Several other explanations remain possible. One possibility is that cells obtained from immature humans respond better to a murine growth factor(s) than comparable adult cells. No evidence for this has been developed but it is important to remember that the adult cells survive in the mice without exogenous growth factors; it is their growth (increase in number and lineage specific differentiation) that requires the presence of human cytokines [6]. Paracrine effects of growth factors produced by cells in the graft other than the stem cells or cotransfer of an accessory cell required for engraftment may also play a role in either promoting the engraftment of the human cells or supporting their growth. The growth of hematopoietic progenitors is enhanced by factor(s) present in cord blood, and circulating fetal blood cells may produce growth factors that enhance stem cell growth or engraftment. Finally, it is also possible that fetal cells are in some way better able to avoid whatever residual immune response these hosts can mount.

These results demonstrate the ability of the BU/CY-treated immunodeficient mice to support human hematopoiesis and suggest that the model may be useful for studying hematopoiesis. These data indicate that the cytokines required for the survival of human PHSC are present in the immunodeficient mice, but also indicate that the further development of these cells requires the presence of species specific cytokines.


   Acknowledgments
 
This work was supported by grants R01DK43376 and R01DK49895 of the National Institutes of Health. The Flow Cytometry Laboratory is a core facility of the Kaplan Cancer Center. The gracious cooperation of Drs. Joseph Fetto and Noel Testa of the Department of Orthopedics at the NYU Medical Center made this work possible.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Steele AA, Sensenbrenner LL, Young MG. Growth and differentiation of normal and leukemic human bone marrow cells cultured in diffusion chambers. Exp Hematol 1977;5:199-210.[Medline]

  2. Mosier DE, Gulizia RJ, Baird SM et al. Transfer of a functional human immune system to mice with severe combined immunodeficiency. Nature 1988;335:256-259.[Medline]

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accepted for publication March 21, 1997.



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