- Email: [email protected]
Functional differences between transplantable human hematopoietic stem cells from fetal liver, cord blood, and adult marrow
Experimental Hematology 27 (1999) 1418–1427
Functional differences between transplantable human hematopoietic stem cells from fetal liver, cord blood, and adult marrow Tessa L. Holyoake,* Franck E. Nicolini,* and Connie J. Eaves Terry Fox Laboratory, British Columbia Cancer Agency and Department of Medical Genetics, University of British Columbia, Vancouver, BC, Canada. (Received 2 April 1999; revised 28 April 1999; accepted 4 May 1999)
The purpose of this study was to develop a simple assay for quantitating transplantable human lymphomyeloid stem cells (competitive repopulating units [CRU]) to enable comparison among the numbers and types of progeny generated in NOD/ SCID mice by such cells from different ontologic sources. Sublethally irradiated NOD/SCID mice were transplanted with varying numbers of CD341 cell-enriched suspensions of human fetal liver, cord blood, or adult marrow cells. The types and numbers of human cells present in the marrow of the mice were measured 6 to 8 weeks later using flow cytometry, in vitro progenitor assays, and secondary transplant endpoints. Frequencies of human CRU obtained by limiting dilution analysis of mice repopulated 6 to 8 weeks posttransplant were the same when the lymphoid and myeloid progeny of CRU were both detected by specific immunophenotypic endpoints as when in vitro myeloid progenitor assays were used to detect CRU myelopoietic activity. The average output per injected CRU of very primitive cells (CD341CD382 cells, LTC-IC, and secondary CRU) was found to be highest for fetal liver CRU and progressively decreased (up to .100-fold) for ontologically older CRU. In contrast, the average output of mature cells was highest for cord blood CRU and lowest for fetal liver CRU, despite equivalent production of intermediate progenitors. Differences in the relative numbers of mature lymphoid, myeloid, and erythroid progeny produced by CRU from different ontologic sources were also seen. Finally, evidence of a transplantable human lymphoid-restricted cell present throughout ontogeny was obtained. A simpler and easier assay for enumerating transplantable human stem cells with lymphomyeloid reconstituting activity has been described, and its specificity and sensitivity validated. The use of this assay has revealed ontogenyassociated differences in a variety of functional attributes of human stem cells proliferating and differentiating in an in vivo, but xenogeneic, setting. © 1999 International Society for Experimental Hematology. Published by Elsevier Science Inc. Keywords: Ontogeny—Human competitive repopulating unit—NOD/SCID mice—LTC-IC—Self-renewal
Offprint requests to: Connie J. Eaves, Terry Fox Laboratory, 601 West 10th Avenue, Vancouver, British Columbia, Canada V5Z 1L3. E-mail: [email protected] *Both authors contributed equally to this work.
Introduction Primitive normal human hematopoietic cells can engraft the bone marrow of various types of immunodeficient xenogeneic hosts, including fetal sheep [1,2] and certain strains of mutant mice [3–6]. In the latter, the highest overall levels of human hematopoiesis reported to date have been obtained in sublethally irradiated nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice [5–9]. The human cells produced in this model include large numbers of human B-lineage cells as well as myeloid cells and their precursors. This has allowed the technique of limiting dilution analysis, which previously was used for quantitating transplantable lymphomyelopoietic murine stem cells [10,11], to be applied to studies of an analogous population of human cells, hence their similar designation as competitive repopulating units (CRU) [9]. Detection of human DNA in the marrow of NOD/SCID mice transplanted with human cells [7,12], or simply of human cells expressing CD45, the common leukocyte antigen [13], have both been used as evidence of transplantable human hematopoietic stem cell activity. However, the inherent lack of specificity of either of these endpoints poses obvious risks to investigations of heterogeneous or manipulated cell suspensions that may include cells that do not possess both lymphoid and myeloid repopulating potential. To circumvent this problem, we proposed the adoption of more specific measures of the type of engraftment obtained. This ensures detection of significant numbers of human lymphoid and myeloid progeny in every mouse scored as positive [9]. In previous studies, the presence of CD342CD191 human cells was used to indicate production of human lymphoid-restricted cells (as it has been reported that some human CD341CD191 cells can display myelopoietic potential [14]). The presence of myeloidrestricted progeny in the same mice was demonstrated by first isolating any human CD341 cells present and then assessing their granulopoietic and/or erythroid colony-forming ability in vitro [9]. The detection of human lymphoid and myelopoietic activity thus was similarly rigorous, although the requirement for a cell sorting and culture step limited the number of mice that could be evaluated and de-
0301-472X/99 $–see front matter. Copyright © 1999 International Society for Experimental Hematology. Published by Elsevier Science Inc. PII S0301-472X(99)0 0 0 7 8 - 8
T.L. Holyoake et al./Experimental Hematology 27 (1999) 1418–1427
layed the acquisition of data, thereby reducing the utility of this assay to address many questions of interest in human hematopoietic stem cell biology. We now describe a simple modification that improves both the rapidity and ease of measuring transplantable human lymphomyeloid stem cell numbers in a given cell suspension without altering the defining features of the cell detected or compromising the sensitivity of method by which it is quantitated. These modifications involve the direct immunophenotypic detection by flow cytometry of the myeloid as well as the lymphoid progeny that human CRU produce in NOD/SCID mice. In addition, we show that the average size that both these compartments achieve by 6 to 8 weeks posttransplant is a linear function of the number of human CRU injected for grafts of up to 10 CRU per recipient. Using this modified assay, a comparison of the different in vivo differentiation and self-renewal properties of human CRU present in fetal liver, cord blood, and adult marrow was undertaken.
Materials and methods Human cells Human bone marrow cells were either aspirate samples from normal donors of allogeneic transplants or samples of cryopreserved normal cadaveric marrow obtained from the Northwest Tissue Center (Seattle, WA). Cord blood samples were obtained from mothers undergoing cesarean delivery of normal, full-term infants. Livers were obtained from 14- to 21-week-old aborted human fetuses. In all cases, approved institutional procedures for obtaining informed consent were followed. Single cell suspensions were prepared from the fetal liver samples by pushing tissue fragments through a coarse sieve. Low density (,1.077g/cm3) cells were isolated from all samples by Ficoll-Hypaque density centrifugation (Pharmacia Biotech AB, Uppsala, Sweden) and washed twice in Hanks balanced salt solution (StemCell Technologies Inc., Vancouver, BC, Canada) supplemented with 5% fetal calf serum (FCS; StemCell) (HF). Cells to be transplanted were first depleted of cells expressing a number of lineage (lin) markers using an immunomagnetic column system as described by the manufacturers (StemSep™, StemCell). Animals NOD/LtSz-scid/scid (NOD/SCID) mice were bred and maintained in the animal facility of the British Columbia Cancer Research Centre (Vancouver, BC, Canada) in microisolators under defined sterile conditions and according to institutional guidelines. Animals 6 to 8 weeks of age were sublethally irradiated with 350 cGy from a 137Cs source less than 24 hours prior to receiving an intravenous injection of human cells. To assess engraftment, marrow cells in the four large hind leg bones of each transplanted mouse were harvested using a syringe and a 21-gauge needle. Cell suspensions for analysis and sorting were prepared in cold HF supplemented with 5% pooled normal human serum (HF/5% HS). In some experiments, mice were given six intraperitoneal injections of human growth factors spread over a 2-week interval just prior to sacrifice, as indicated.
1419
Flow cytometry Cells to be analyzed were first incubated for 10 minutes at 48C with HF/5% HS supplemented with 3 mg/mL 2.4G2 anti-mouse Fc receptor antibody to block Fc receptors and prevent nonspecific binding [15]. Separate aliquots of cells were incubated for 30 minutes at 48C with the following combinations of antibodies against human antigens: (1) anti-human CD34-FITC (8G12) [16] plus anti-CD19-PE and anti-CD20-PE (both from Becton Dickinson [BD], San Jose, CA); (2) anti-CD71-PE (OKT-9) plus anti-CD45PE, anti-CD15-FITC, and anti-CD66b-FITC (all from Pharmingen, Mississauga, Ontario, Canada); (3) anti-glycophorin-A-FITC (10F7); (4) anti-CD41 [17]; and (5) anti-CD34-FITC and antiCD38-PE (BD). Cells were washed twice with HF, the second wash containing 1 mg/mL propidium iodide (PI; Sigma Chemical Co., St Louis, MO). Acquisition and analysis of the cells were performed on a FACSort using LYSIS II software (BD). Positive cells were defined as those exhibiting a level of fluorescence that exceeded 99.98% of that obtained with irrelevant isotype-matched control antibodies labeled with the same fluorochromes. All antibodies used for positive staining were checked for their nonreactivity with primary NOD/SCID mouse bone marrow cells. In addition, as indicated, human CD341 cells were purified by FACS, or by removal by immunomagnetic depletion (see following) of murine cells as well as human lineage marker-positive (lin1) cells [18]. Human CRU assay For each test cell sample (Table 1), five to six groups of irradiated mice (four mice per group) were injected with threefold dilutions of lin2 cells plus 106 irradiated (1500 cGy) normal human bone marrow cells as carrier cells. Mice were killed 6 to 8 weeks later and their marrow cells removed and stained with anti-human CD34 and CD19 antibodies (“old” CRU assay), as previously described [8,9]. Other aliquots were stained with anti-human CD45 1 CD71 and CD15 1 CD66b antibodies or anti-human CD34 and CD19 1 CD20 antibodies (“new” CRU assay). For human CRU determinations using the old assay, mice were considered negative if there were ,5 CD342CD191 (B-lineage) human cells per 5 3 103 viable cells and/or no CD341 human colony-forming cells (CFC) per 106 total viable (PI2) cells [8,9]. For the new CRU assay (used throughout this study unless otherwise indicated), mice were considered negative if there were ,5 CD342CD19/201 human cells and/or ,5 CD45/711CD15/66b1 human cells per 2 3 104 viable cells analyzed. Old and new CRU frequencies were calculated using the L-calc software program (StemCell) from the proportions of negative mice in each similarly treated group, using Poisson statistics and the method of maximum likelihood [10]. In vitro assays CFC and LTC-IC assays were performed as described previously [6,19]. Briefly, for CFC assays, the FACS-sorted CD341 human cells were cultured in methylcellulose medium (H4330, StemCel) supplemented with 50 ng/mL human steel factor (SF, purified from supernatants of cos cells transfected with human SF cDNA) and 20 ng/mL each of human interleukin 3 (IL-3; Novartis, Basel, Switzerland), interleukin 6 (IL-6; Cangene, Missassauga, ON), granulocyte-macrophage colony-stimulating factor (GM-CSF; Novartis), G-CSF (StemCell), and 3 U/mL erythropoietin (Epo; StemCell). CD341 cells were assayed for LTC-IC by culturing them on preestablished irradiated murine fibroblasts genetically
1420
T.L. Holyoake et al./Experimental Hematology 27 (1999) 1418–1427
Table 1. Frequency of CRU relative to the total CD341 population decreases during ontogeny CRU frequency (no. of CRU per 105 injected CD341 cells)* Source of cells assayed (no. of lin2 cells/mouse) Fetal liver (300–105)
Cord blood (1.2 3 104–3 3 105) Adult bone marrow (104–7.2 3 105)
Endpoint of CRU activity
Mean
95% confidence interval
No. of experiments
New assay Old assay CD45/711 New assay Old assay CD45/711 New assay Old assay CD45/711
9.1 6.1 29.4 6.0 4.2 26.6 0.8 2.0 2.0
5.4–15.4 3.7–10.2 17.2–50.1 3.1–11 2.1–8.0 15–46 0.5–1.3 1.1–3.3 1.2–3.3
3 3 3 3 1 3 3 3 3
*As described in the Methods, each CRU determination was calculated from the combined results of three independent experiments, in each of which a total of 20 mice were transplanted with a 100-fold range of lin2 cells of the type shown (five groups of four mice each). In each experiment, all injected mice were analyzed and CRU frequencies calculated using each of the three sets of engraftment criteria indicated to distinguish positive and negative recipients (see Methods for details). To reduce the variability in the data due simply to different efficiencies of lin1 cell depletion in different experiments, results have been calculated on a per injected CD341 cell basis.
engineered to produce human IL-3, G-CSF, and SF and then determining the number of CFC present 6 weeks later. The number of LTC-IC present in the input innoculum was calculated by dividing the total number of CFC present in the 6-week-old cultures by 18 for cells derived from adult human bone marrow, by 28 for cells derived from human cord blood [19], and by 72 for cells derived from fetal liver [20].
Results Quantitation of human CRU by direct immunophenotyping of regenerated lymphoid and myeloid progeny In a first series of experiments, we compared the FACS profiles obtained when cells from NOD/SCID mice engrafted with human bone marrow cells were stained with various combinations of anti-human antibodies to identify those that gave the best discrimination of the human B lineage and myeloid cells present. The most sensitive combination was
found to be anti-CD34-FITC 1 anti-CD19/20-PE to identify CD342CD19/201 B-lineage cells and anti-CD45/71-PE 1 anti-CD15/66b-FITC to identify CD45/711CD15/66b1 mature myeloid cells (see representative example in Fig. 1). We then used this approach to identify mice that showed dual lymphoid and myeloid engraftment with human cells 6 to 8 weeks after the injection of decreasing numbers of lin2 human fetal liver, cord blood, or adult marrow cells. Human CRU frequencies were calculated, using five positive cells of each type (myeloid and lymphoid, from a total of 2 3 104 cells analyzed) as the criterion for distinguishing positive and negative recipients for the “new” assay (as described in the Methods). For the “old” assay, 5 3 103 cells were analyzed for the presence of human B-lineage cells and an additional 106 were assessed for the presence of human CD341 granulopoietic or erythroid CFC [8,9]. As shown in Table 1, the results obtained with both procedures were similar (p . 0.05) for each of the three sources of cells assayed. However, when CRU frequencies
Figure 1. Representative dot plots for the marrow cells obtained from a NOD/SCID mouse 6 weeks after being transplanted with 1.5 3 105 lin2 human cord blood cells (z10 CRU). Left: Staining with isotype control Ig; middle: extent of total (CD45/711) and myeloid-specific (CD45/711 CD15/66b1) human cell engraftment; right: extent of B-lineage-specific (CD342CD19/201) and primitive (CD341) human cell engraftment.
T.L. Holyoake et al./Experimental Hematology 27 (1999) 1418–1427
were calculated using just the presence of human CD45 and/or CD711 cells ($5 per 2 3 104 cells analyzed) to identify positive recipients, the values obtained for both cord blood and fetal liver CRU were three- to fourfold higher, although the value obtained for adult marrow CRU was similar. The higher frequency of cord blood and fetal liver CRU obtained with the less discriminating (CD45/711 cell) readout presumably reflects the presence of transplantable cord blood and fetal liver cells that can generate lymphoid progeny but not detectable numbers of myeloid progeny in this model. Analysis of mice injected with doses of cells calculated retrospectively to have contained, on average, #1 repopulating cell of any kind (based on the results for the entire dataset) showed that a proportion of the recipients transplanted with any of the three human cell sources contained detectable numbers of human B-lineage cells only (21%, 39%, and 30% of recipients of #1 CRU from human fetal liver, cord blood, or adult marrow, respectively). None contained myeloid cells only. This situation is different from what we previously reported when only 5 3 103 cells were analyzed (#6% mice with only human myeloid or lymphoid cells detected) [9]. Thus, the increased sensitivity of detecting human B-lineage cells obtained by evaluating four times more cells did not increase the sensitivity of the CRU assay over that previously reported [9], but allowed the detection of an apparently lymphoid-restricted population of transplantable human cells present at a slightly higher frequency but characterized by a lower proliferative activity. Comparison of the number of CRU per 105 CD341 cells in fetal liver, cord blood, and adult marrow populations shows that this value decreases during ontogeny by a factor of approximately 10 (Table 1).
Human lymphomyeloid engraftment is linearly related to transplant dose In addition to monitoring the proportions of mice transplanted with varying numbers of cells that had detectable numbers of human B-lineage, myeloid, or any human hematopoietic (CD45/711) cells in their marrow, the average size of these populations produced in the same groups of mice was calculated. Figure 2 shows a plot of the results obtained for each of the three types of transplants evaluated using the total CD45/711 cell endpoint and assuming that the number of cells present in two femurs plus two tibias represents 25% of the total marrow cellularity of the mouse [21]. To facilitate a comparison of the three plots, each cell dose injected was converted to a CRU number based on the frequency values determined in Table 1. It can be seen that there was a linear increase in the total number of human hematopoietic cells generated (as assessed 6 to 8 weeks posttransplant) with increasing numbers of human CRU transplanted for transplants containing up to 10 CRU per mouse, regardless of their origin. However,
1421
there was a marked difference in the average total number of cells produced per human CRU for different sources of these cells. The largest populations were produced by cord blood CRU, the second largest were generated by adult marrow CRU, and the smallest populations were those derived from fetal liver CRU. A similar analysis of the human B-lineage (CD342CD19/201) and myeloid (CD45/711CD15/ 66b1 ) subpopulations showed these also to be linearly related to CRU input (data not shown).
Figure 2. Cell dose response data for the absolute number of human (CD45/711) cells present in the marrow of NOD/SCID mice transplanted 6 to 8 weeks previously with lin2 human cells of fetal liver (FL), cord blood (CB), and adult bone marrow (BM) origin. Each point represents pooled data from 3 to 12 mice from a total of three experiments for each kind of transplant, assuming the marrow present in two tibias and two femurs represents 25% of the total marrow of a mouse [21]. R values are derived correlation ratios.
1422
T.L. Holyoake et al./Experimental Hematology 27 (1999) 1418–1427
Human CRU from different ontologic sources produce different numbers of various primitive and mature progeny in the marrow of NOD/SCID mice In the experiments described earlier, additional aliquots of cells harvested from the marrow of each of the NOD/SCID mice showing engraftment with human lymphoid and myeloid cells were labeled with anti-human CD38 and CD34, glycophorin A, or CD41 antibodies and the cells positive for these markers also quantitated. In addition, LTC-IC assays were performed on the human CD341 cells isolated. The results of each of these additional measurements were used to calculate the corresponding number of cells of each type present in each mouse and average 6- to 8-week outputs per CRU then derived. The results of these calculations are summarized in Table 2. Because maturing B-lineage (CD342CD19/201) cells constituted the largest population generated by all sources of human CRU, the average output of these cells per CRU mirrors the corresponding average output of total cells per CRU. The average output of mature human granulopoietic (CD45/711CD15/66b1) cells in the mice also was greater for cord blood and adult marrow CRU than for fetal liver CRU. In contrast, the average output of human cells belonging to the megakaryocyte lineage (CD411 cells) appeared less affected by the origin of the stem cells from which they were derived. The average output of mature human erythroid (glycophorin A1) cells was
significantly (p , 0.05) higher for fetal liver CRU than cord blood CRU and undetectable in NOD/SCID mice engrafted with adult marrow cells (despite the confirmed presence of similar numbers of erythroid-restricted CFC in the same mice, as found previously [8]). In mice engrafted with human fetal liver cells, the output of mature erythroid progeny was, on average, twice the output of mature myeloid cells. In contrast, the reverse was true in the cord blood-engrafted mice, in which the output of human myeloid cells was five times the output of mature human erythroid cells. Interestingly, these differences at the level of the mature compartments were not predicted by the size of the more primitive cell populations. Thus, the total numbers of both CD341 cells and CFC produced from fetal, newborn, and adult human CRU were similar but, as shown in Fig. 3, the output of more primitive human cells (CD341CD382 cells and LTC-IC) decreased markedly with the ontologic age of the human CRU transplanted. Human fetal liver CRU produced, on average, z6- and z30-fold more CD341CD382 cells than cord blood and adult marrow CRU, respectively, and z7 and z300-fold more LTC-IC. It should be noted that, in these calculations, the numbers of LTC-IC were determined assuming that their individual CFC outputs were the same as those of the LTC-IC in the tissue transplanted (although these values decrease during normal human ontogeny [19,20]). If a similar trend occurs over time posttransplant, this would increase even further the differences, shown in Fig. 3.
Table 2. Calculated output per injected CRU of phenotypically and functionally defined human cell subsets assessed 6 to 8 weeks after transplantation of human fetal liver, cord blood, and adult marrow lin2 cells into sublethally irradiated NOD/SCID mice Human cell subsets CD45/711 CD342CD19/201 CD45/711 CD15/66b1 Glycophorin A1 CD411 CD341 CFC CD341CD382 LTC-IC
Fetal liver
Cord blood
Bone marrow
35 6 18 22 6 12 362 762 0.7 6 0.2 18 6 8 0.6 6 0.3 0.6 6 0.3 2100 6 880
144 6 37 137 6 54 661 1.2 6 0.6 1.8 6 0.5 23 6 5 0.4 6 0.2 0.1 6 0.1 280 6 210
64 6 26 49 6 20 663 ,0.02 6 0.01 1.2 6 0.4 35 6 9 0.8 6 0.1 0.02 6 0.01 765
Values are given as mean (3105) 6 SEM, except for the LTC-IC, which are as shown. These values were calculated (as described in the text) from the results obtained in mice injected with up to z10 CRU per mouse (based on the frequencies shown in Table 1) as follows. For each subset of human cells analyzed, the total number of these cells present in each positive recipient of #10 CRU (from a given source) was first calculated by multiplying their frequency in the marrow (e.g., number of human CD45/ 711 cells per 20,000 total marrow cells analyzed) by the total number of marrow cells per mouse (assuming the contents of two femurs and two tibias represent 25% of the total marrow volume [21]). The total number of human cells of this subset for all positive recipients of #10 CRU was obtained by adding the results for the individual mice together. Finally, this value was divided by the total number of CRU injected into these recipients (as determined in Table 1) to yield the output of each subset per CRU.
Figure 3. Yield of human CD341CD382 cells (top) and LTC-IC (bottom) per NOD/SCID mouse per human CRU injected (calculated as described in the legend to Table 3) for CRU of fetal liver (open bars), cord blood, (black bars), or adult marrow origin (gray bars). Values shown are the mean 6 SEM from data pooled from three experiments for each type of CRU.
T.L. Holyoake et al./Experimental Hematology 27 (1999) 1418–1427
1423
cells. However, in this case, the output of human CRU was several-fold higher than the input value (again of approximately 10 CRU per primary mouse). Assessment of the effects of the growth factor injections on other cell types confirmed previous findings for adult marrow-engrafted primary mice [6]. Fetal liver-engrafted mice showed the same (lack of) response by less primitive cell types (data not shown). Because of the high levels of human multilineage engraftment generally attained in all groups of secondary recipients of cells from primary mice transplanted with human fetal liver cells (6 growth factors), aliquots of the marrow cells from each group of these secondary recipients were transferred to tertiary irradiated NOD/SCID mice. Another 6 weeks later, the marrow of the tertiary animals was assessed for the presence of human B-lineage (CD342 CD19/201 ) and myeloid (CD45/711CD15/66b1 ) cells. As shown in Table 4, 3 of 15 of these tertiary recipients were positive for both cell types and three others contained human B-lineage cells but no detectable myeloid cells. This indicates continued maintenance of a detectable human CRU population in the secondary mice (none of which received injections of human growth factors). An example of a primary, secondary, and tertiary recipient of serially transplanted human fetal liver cells resulting each time in the production of both human lymphoid and myeloid cells is shown in Fig. 4.
Enhanced factor-independent CRU regeneration in NOD/SCID recipients of human fetal liver (vs cord blood or adult marrow cells) In a final series of experiments, we compared the ability of equivalent transplants (z10 CRU per recipient) of human lin2 fetal liver or adult marrow cells to regenerate progeny CRU 4 weeks after injection of the cells into sublethally irradiated NOD/SCID mice. We had previously found that a 2-week course of three alternate daily intraperitoneal injections of human SF (10 mg/mouse/injection), IL-3, and GMCSF (both at 6 mg/mouse/injection), and Epo (10 U/mouse/ injection) enhanced the number of human CRU obtained from mice transplanted 4 to 6 weeks previously with human cord blood cells [22], even though there was no effect on the output of later cell types [6,23], with the exception of marrow-derived erythroblasts [20]. Therefore, we also tested the effect of these growth factors on the regeneration in NOD/SCID mice of CRU derived from human fetal liver and adult marrow. The number of these present in the marrow of the primary mice (at 4 weeks posttransplant) was determined by performing secondary CRU assays on the cells harvested from the marrow of each group of primary mice. The details of these experiments and the results of the secondary limiting dilution assays are summarized in Table 3. Human CRU were not present at detectable levels in primary mice transplanted with adult human marrow cells irrespective of whether or not they also were injected with growth factors. This represents a .25-fold decrease in CRU numbers below the estimated 10 initially injected per primary mouse. There also was no evidence of an effect of the growth factors injected on the number of human CRU regenerated in the mice transplanted with human fetal liver
Discussion Much interest has focused recently on the potential of xenotransplant models to address questions about normal and
Table 3. Number of human CRU detected in the marrow of primary NOD/SCID mice transplanted 4 weeks previously with z10 CRU of human fetal liver, cord blood, and adult bone marrow origin Primary recipients
Initial human cells transplanted (no. of lin2 cells/mouse)* Adult marrow Fetal Liver
Secondary recipients
GF injected† 2 1 2 1
Percentage of primary mouse BM cells per secondary mouse
Positive secondary mice/total secondary mice
25 25 25 5 1 25 5 1
0/4 0/4 2/2 4/5 4/4 4/4 4/5 2/5
Change in CRU no. posttransplant in primary mice‡ (95% CI) .25-fold decrease .25-fold decrease 5-fold increase (3-10-fold increase) 3-fold increase (1- to 10-fold increase)
*There were 1.25 3 106 and 1.1 3 105 CD341 cells, respectively, present in the adult marrow and fetal liver lin2 cells injected into each mouse (i.e. z10 CRU, based on the frequencies derived in Table 1). † Growth factors (GF) were injected, as described in the text. ‡ This was determined by dividing the average number of human CRU calculated to be present in the entire marrow of the primary recipients (measured CRU frequency 3 total marrow cells assuming two femurs plus two tibias represent 25% of the total marrow volume [21]) by 10 (the estimated number of human CRU injected into each primary recipient). This calculation does not take into consideration the fact that only a small proportion of the regenerated cells with CRU activity would be detected in the secondary mice and hence all “changes” shown would be underestimated by this factor.
1424
T.L. Holyoake et al./Experimental Hematology 27 (1999) 1418–1427
Table 4. Detection of human lymphoid (CD342CD19/201) and myeloid (CD45/711CD15/66b1) cells in tertiary NOD/SCID recipients of serially transplanted human fetal liver-derived CRU Percentage human subsets in individual tertiary mice GF injected into primary mice 2 1
Percentage of primary mouse BM cells per secondary mouse
CD342CD19/201
CD45/711CD15/66b1
25 1 1 25 25 5
0.11 0.03 0.34 0.05 0.82 0.03
,0.03 0.12 ,0.03 0.03 0.74 ,0.03
*Each tertiary mouse received the equivalent of 25% of the marrow of a secondary mouse from the group of secondary recipients shown in Table 3. Only mice in which some human cells were detected (either lymphoid or myeloid, .0.025% positive) are shown. The total number of tertiary mice analyzed after transplantation of cells from secondary recipients of variable numbers of cells from primary non–GF-treated primary mice was as follows: 25% 5 one, 5% 5 one, 1% 5 three; and from corresponding recipients of cells originally regenerated in 1GF primary mice was as follows: 25% 5 three, 5% 5 three, 1% 5 three.
malignant human hematopoietic stem cells, including their responses to various cytokine and genetic manipulations in vitro. Central to such investigations are the endpoints used to detect and quantify human stem cells, as these determine the specificity and precision of responses observed. The ability of human hematopoietic cells to home to the marrow
of xenogeneic recipients and respond to species–cross-reactive factors was first indicated by the engraftment of irradiated adult bg/nu/xid (bnx) mice with injected human cells [3]. Many murine stromal cell lines now have been shown to support the long-term maintenance of human hematopoiesis in vitro [24–28], and a number of specific cytokines
Figure 4. Representative dot plots showing total (CD451/71) and myeloid-specific (CD45/711CD15/66b1) human engraftment of the marrows of primary, secondary, and tertiary NOD/SCID recipients of human fetal liver cells (top three panels) and corresponding results from the same mice for primitive (CD341) and B-lineage-specific (CD342CD19/201) human cells (bottom three panels).
T.L. Holyoake et al./Experimental Hematology 27 (1999) 1418–1427
with cross-species activity on hematopoietic stem cells have been identified [29–31]. More recent in vivo studies have confirmed that the major barrier to the xenotransplantation of human hematopoietic stem cells is immunologic, as shown by improvements using naive (fetal) [1,2] or other genetically compromised recipients [4–8]. The reduced or absent B, T, NK, macrophage. and complement activities of the NOD/SCID mouse makes it the most receptive host thus far described. When these mice are additionally treated with a sublethal dose of irradiation (325–400 cGy) prior to the transplantation of normal human hematopoietic cells, both human lymphoid and myeloid progeny typically can be found when the total number of human cells regenerated per mouse 6 to 8 weeks posttransplant is $105 [9]. In the present studies we show that the human stem cells (CRU) capable of producing this multilineage engraftment can be enumerated by limiting dilution analysis of mice defined as repopulated using an appropriate set of phenotypic markers to identify specific subpopulations of human B-lineage and myeloid cells in the marrow cells harvested from the mice. However, by increasing the sensitivity of human cell detection by a factor of 4 over that previously reported [8,9] (i.e., from z0.1% to z0.02%, which allows the detection of transplantable human cells with reduced proliferative potential, i.e., z 2 3 104 vs 105 cells), we also obtained evidence of a cell type that appears to exhibit a lymphoid-restricted program in this assay. Such cells were demonstrable in adult human marrow, cord blood, and fetal liver, and at frequencies slightly higher than those measured for cells with both lymphoid and myeloid reconstituting activities. These findings underscore the importance of adopting specific endpoints to measure the differentiation potential of the cells being assayed, particularly after exposure to factors that may influence their proliferative and/or differentiation responses. A less specific, but much easier and less expensive, method to assess effects on transplantable human stem cells is to infer quantitative changes in the initial innoculum from the average total level of NOD/SCID marrow engraftment obtained 6 to 8 weeks posttransplant. The validity of this approach is supported by the demonstration of a linear relationship between the number of human CRU injected and the average level of engraftment seen, at least for transplants containing #10 human CRU. This was true for all three sources of human CRU studied. A simple modification to improve the specificity of the readout for lymphomyeloid repopulating cells might then be to simply prolong the time posttransplant prior to assessing the level of engraftment, as validated by previous in vivo studies in mice [32] and sheep [33], and by in vitro studies using the long-term culture system [34,35]. In the present experiments, the development of a relatively simple procedure for quantitating transplantable human cells with lymphomyeloid repopulating potential made it possible to identify intrinsic differences imposed by ontogeny on the self-renewal and differentiation activity of
1425
human hematopoietic cells and their progeny. Interestingly, CRU from human fetal liver, cord blood, and adult marrow all produced a similarly sized population of intermediate progenitors as indicated by measurements of human CD341 cells and in vitro clonogenic cells. However, marked differences in the output of terminally differentiating erythroid and developing B cells were noted in mice that received fetal vs adult transplants. This disparity in the production of various differentiated cell populations is likely to reflect the stable transmission through successive generations of cells of previously documented ontogeny-determined differences in the cytokine responsiveness or requirements of lineagerestricted progenitor cells and their terminally differentiating progeny [36–38]. Significant differences also were noted in the output of very primitive cells by transplanted CRU derived from different ontologic sources. This was evident from an examination of the size of the human CD341CD382 population regenerated as well as in the number of human cells present that could initiate long-term hematopoiesis in stromal feeders in vitro (LTC-IC) or engraft secondary irradiated NOD/ SCID recipients (CRU). The magnitude of these differences increased progressively with ontogeny and were .100-fold for all three endpoints when fetal and adult CRU were compared. As a result, a several-fold net expansion of human CRU in vivo could be demonstrated for the first time with transplants of fetal liver cells, and these could be serially transplanted two more times. The greater self-renewal activity of fetal human hematopoietic stem cells mimics previous data from studies of murine stem cells [39,40]. The finding that the proliferation and/or self-renewal of fetal human stem cells were not further enhanced by exogenous human growth factors (in contrast to cord blood CRU where 10fold increases in CRU yields were stimulated by such treatments [22]) reinforces the concept of an altered state that persists throughout the differentiation of fetal hematopoietic cells and exaggerates the type of response elicited by certain cytokines. It also should be noted that failure to detect regeneration of CRU from injected adult or pediatric human sources does not preclude the occurrence of this activity, as implied by long-term xenotransplant studies in sheep [33] or retroviral marking studies [41]. However, in the present experiments, the reduced sensitivity of the serial transplantation procedure may have precluded detection of the smaller numbers of CRU regenerated by adult marrow cells. This necessarily involves an underestimation of the magnitude of CRU regeneration posttransplant, because the seeding efficiency of human CRU in NOD/SCID mice (which is likely to be low [6,8]) has not been factored into the present studies. That NOD/SCID mice transplanted with similar numbers of human stem cells at the peak of the initial engraftment phase [6,8] contain similar numbers of cells at intermediate stages of differentiation, despite large differences in the size of the stem cell population from which they would be con-
1426
T.L. Holyoake et al./Experimental Hematology 27 (1999) 1418–1427
tinuously originating, suggests the operation in the mice of mechanisms that also must differentially regulate this process. In other studies, we have shown that the later decline of adult human marrow-derived clonogenic progenitors may represent a response of these cells (or their immediate precursors) to endogenously produced chemokines such as MCP-1 and MIP-1 [42]. It is possible that the relatively reduced output of clonogenic cells from fetal liver or cord blood stem cells may reflect a greater sensitivity of these developmentally earlier cells to factors with this type of activity. It is interesting to note that evidence of analogous regulatory mechanisms responsible for normalizing intermediate populations of murine progenitors from differentially regenerated stem cell compartments has been obtained [43,44]. In summary, we have described a simpler and more rapid methodology for quantifying transplantable human stem cells with lymphomyeloid repopulating activity and have used it to demonstrate both ontogeny and stage-specific differences in the regulation of human hematopoiesis in an in vivo, but xenogeneic, model of engraftment. These findings set the stage for future investigation of both extrinsic and intrinsic molecular mechanisms responsible for these differences and how they may be altered in disease states. Acknowledgments We thank the staff of the Stem Cell Assay Service for their assistance in the initial preparation of primary human cell samples, Jessyca Maltman and Maya Sinclaire for their assistance in the animal studies, Pamela Austin for additional technical assistance, Gayle Thornbury and Giovanna Cameron for operating the FACS, and Tara Palmater for preparation of the manuscript. We are also grateful to P. Lansdorp (Terry Fox Laboratory), and Cangene, Novartis, and StemCell Technologies, Inc., for valuable gifts of reagents. This work was supported by grants from Novartis (Basel, Switzerland), the National Cancer Institute of Canada (NCIC) with funds from the Terry Fox Run, and the NIH (POI HL 55435). T.L. Holyoake holds a United Kingdom Leukemia Research Fund Senior Lecturership, and C.J. Eaves is a Terry Fox Cancer Research Scientist of the NCIC.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16. 17.
18.
References 1. Zanjani ED, Flake AW, Rice H, Hedrick M, Tavassoli M (1994) Longterm repopulating ability of xenogeneic transplanted human fetal liver hematopoietic stem cells in sheep. J Clin Invest 93:1051 2. Zanjani ED, Pallavicini MG, Ascensao JL, Flake AW, Langlois RG, Reitsma M, MacKintosh FR, Stutes D, Harrison MR, Tavassoli M (1992) Engraftment and long-term expression of human fetal hemopoietic stem cells in sheep following transplantation in utero. J Clin Invest 89:1178 3. Kamel-Reid S, Dick JE (1988) Engraftment of immune-deficient mice with human hematopoietic stem cells. Science 242:1706 4. Lapidot T, Pflumio F, Doedens M, Murdoch B, Williams DE, Dick JE (1992) Cytokine stimulation of multilineage hematopoiesis from immature human cells engrafted in SCID mice. Science 255:1137 5. Pflumio F, Izac B, Katz A, Shultz LD, Vainchenker W, Coulombel L (1996) Phenotype and function of human hematopoietic cells engraft-
19.
20.
21. 22.
23.
ing immune-deficient CB17-severe combined immunodeficiency mice and nonobese diabetic-severe combined immunodeficiency mice after transplantation of human cord blood mononuclear cells. Blood 88:3731 Cashman JD, Lapidot T, Wang JCY, Doedens M, Shultz LD, Lansdorp P, Dick JE, Eaves CJ (1997) Kinetic evidence of the regeneration of multilineage hematopoiesis from primitive cells in normal human bone marrow transplanted into immunodeficient mice. Blood 89:4307 Larochelle A, Vormoor J, Hanenberg H, Wang JCY, Bhatia M, Lapidot T, Moritz T, Murdoch B, Xiao XL, Kato I, Williams DA, Dick JE (1996) Identification of primitive human hematopoietic cells capable of repopulating NOD/SCID mouse bone marrow: implications for gene therapy. Nature Med 2:1329 Cashman J, Bockhold K, Hogge DE, Eaves AC, Eaves CJ (1997) Sustained proliferation, multi-lineage differentiation and maintenance of primitive human haematopoietic cells in NOD/SCID mice transplanted with human cord blood. Br J Haematol 98:1026 Conneally E, Cashman J, Petzer A, Eaves C (1997) Expansion in vitro of transplantable human cord blood stem cells demonstrated using a quantitative assay of their lympho-myeloid repopulating activity in nonobese diabetic-scid/scid mice. Proc Natl Acad Sci U S A 94:9836 Szilvassy SJ, Humphries RK, Lansdorp PM, Eaves AC, Eaves CJ (1990) Quantitative assay for totipotent reconstituting hematopoietic stem cells by a competitive repopulation strategy. Proc Natl Acad Sci U S A 87:8736 Miller CL, Eaves CJ (1997) Expansion in vitro of adult murine hematopoietic stem cells with transplantable lympho-myeloid reconstituting ability. Proc Natl Acad Sci U S A 94:13648 Bhatia M, Wang JCY, Kapp U, Bonnet D, Dick JE (1997) Purification of primitive human hematopoietic cells capable of repopulating immune-deficient mice. Proc Natl Acad Sci U S A 94:5320 Hogan CJ, Shpall EJ, McNulty O, McNiece I, Dick JE, Shultz LD, Keller G (1997) Engraftment and development of human CD341-enriched cells from umbilical cord blood in NOD/LtSz-scid/scid mice. Blood 90:85 Tjonnfjord GE, Steen R, Veiby OP, Egeland T (1996) Lineage commitment of CD341 human hematopoietic progenitor cells. Exp Hematol 24:875 Unkeless JC (1979) Characterization of a monoclonal antibody directed against mouse macrophage and lymphocyte Fc receptors. J Exp Med 150:580 Schmitt C, Eaves CJ, Lansdorp PM (1991) Expression of CD34 on human B cell precursors. Clin Exp Immunol 85:168 Hogge D, Fanning S, Bockhold K, Petzer A, Lambie K, Lansdorp P, Eaves A, Eaves C (1997) Quantitation and characterization of human megakaryocyte colony-forming cells using a standardized serum-free agarose assay. Br J Haematol 96:790 Holyoake TL, Cameron G, Horrocks C, Thomas T, Eaves C, Eaves A (1998) Development of immunomagnetic negative depletion procedures for obtaining enriched populations of primitive cells from CML blood samples or their progeny regenerated in NOD/SCID mice. Exp Hematol 26: 757 (abstr) Hogge DE, Lansdorp PM, Reid D, Gerhard B, Eaves CJ (1996) Enhanced detection, maintenance and differentiation of primitive human hematopoietic cells in cultures containing murine fibroblasts engineered to produce human Steel factor, interleukin-3 and granulocyte colony-stimulating factor. Blood 88:3765 Nicolini FE, Holyoake TL, Cashman JD, Chu PPY, Lambie K, Eaves CJ. Unique differentiation programs of human fetal liver stem cells revealed both in vitro and in vivo in NOD/SCID mice. Blood (in press) Boggs DR (1984) The total marrow mass of the mouse: a simplified method of measurement. Am J Hematol 16:277 Cashman JD, Eaves CJ (1999) Human growth factor-enhanced regeneration of transplantable human hematopoietic stem cells in nonobese diabetic/severe combined immunodeficient mice. Blood 93:481 Vormoor J, Lapidot T, Pflumio F, Risdon G, Patterson B, Broxmeyer
T.L. Holyoake et al./Experimental Hematology 27 (1999) 1418–1427
24.
25.
26.
27.
28.
29.
30.
31.
32. 33.
HE, Dick JE (1994) Immature human cord blood progenitors engraft and proliferate to high levels in severe combined immunodeficient mice. Blood 83:2489 Xu M, Tsuji K, Ueda T, Mukouyama Y, Hara T, Yang F-C, Ebihara Y, Matsuoka S, Manabe A, Kikuchi A, Ito M, Miyajima A, Nakahata T (1998) Stimulation of mouse and human primitive hematopoiesis by murine embryonic aorta-gonad-mesonephros-derived stromal cell lines. Blood 92:2032 Baum CM, Weissman IL, Tsukamoto AS, Buckle AM, Péault B (1992) Isolation of a candidate human hematopoietic stem-cell population. Proc Natl Acad Sci U S A 89:2804 Sutherland HJ, Eaves CJ, Lansdorp PM, Thacker JD, Hogge DE (1991) Differential regulation of primitive human hematopoietic cells in long-term cultures maintained on genetically engineered murine stromal cells. Blood 78:666 Issaad C, Croisille L, Katz A, Vainchenker W, Coulombel L (1993) A murine stromal cell line allows the proliferation of very primitive human CD3411/CD38 progenitor cells in long-term cultures and semisolid assays. Blood 81:2916 Thiemann FT, Moore KA, Smogorzewska EM, Lemischka IR, Crooks GM (1998) The murine stromal cell line AFT024 acts specifically on human CD341CD38 progenitors to maintain primitive function and immunophenotype in vitro. Exp Hematol 26:612 Martin FH, Suggs SV, Langley KE, Lu HS, Ting J, Okino KH, Morris CF, McNiece IK, Jacobsen FW, Mendiaz EA, Birkett NC, Smith KA, et al (1990) Primary structure and functional expression of rat and human stem cell factor DNAs. Cell 63:203 Lyman SD, James L, Vanden Bos T, de Vries P, Brasel K, Gliniak B, Hollingsworth LT, Picha KS, McKenna HJ, Splett RR, Fletcher FA, Maraskovsky E, et al (1993) Molecular cloning of a ligand for the flt3/ flk-2 tyrosine kinase receptor: a proliferative factor for primitive hematopoietic cells. Cell 75:1157 Wendling F, Maraskovsky E, Debili N, Florindo C, Teepe M, Titeux M, Methia N, Breton-Gorius J, Cosman D, Vainchenker W (1994) c-Mpl ligand is a humoral regulator of megakaryocytopoiesis. Nature 369:571 Jordan CT, Lemischka IR (1990) Clonal and systemic analysis of longterm hematopoiesis in the mouse. Genes Dev 4:220 Civin CI, Almeida-Porada G, Lee M, Olweus J, Terstappen LWMM, Zanjani ED (1996) Sustained, retransplantable, multilineage engraftment of highly purified adult human bone marrow stem cells in vivo. Blood 88:4102
1427
34. Prosper F, Stroncek D, Verfaillie CM (1996) Phenotypic and functional characterization of long-term culture-initiating cells present in peripheral blood progenitor collections of normal donors treated with granulocyte colony-stimulating factor. Blood 88:2033 35. Hao QL, Thiemann FT, Petersen D, Smogorzewska EM, Crooks GM (1996) Extended long-term culture reveals a highly quiescent and primitive human hematopoietic progentior population. Blood 88:3306 36. Rich IN, Kubanek B (1976) Erythroid colony formation (CFU-e) in fetal liver and adult bone marrow and spleen from the mouse. Blut 33:171 37. Peschle C, Migliaccio AR, Migliaccio G, Ciccariello R, Lettieri F, Quattrin S, Russo G, Mastroberardino G (1981) Identification and characterization of three classes of erythroid progenitors in human fetal liver. Blood 58:565 38. Weekx SFA, van Bockstaele DR, Plum J, Moulijn A, Rodrigus I, Lardon F, De Smedt M, Nijs G, Lenjou M, Loquet P, Berneman ZN, Snoeck H-W (1998) CD3411CD38 and CD341CD381 human hematopoietic progenitors from fetal liver, cord blood, and adult bone marrow respond differently to hematopoietic cytokines depending on the ontogenic source. Exp Hematol 26:1034 39. Micklem HS, Ford CE, Evans EP, Ogden DA, Papworth DS (1972) Competitive in vivo proliferation of foetal and adult haematopoietic cells in lethally irradiated mice. J Cell Physiol 79:293 40. Rebel VI, Miller CL, Eaves CJ, Lansdorp PM (1996) The repopulation potential of fetal liver hematopoietic stem cells in mice exceeds that of their adult bone marrow counterparts. Blood 87:3500 41. Brenner MK, Rill DR, Holladay MS, Heslop HE, Moen RC, Buschle M, Krance RA, Santana VM, Anderson WF, Ihle JN (1993) Gene marking to determine whether autologous marrow infusion restores long-term haemopoiesis in cancer patients. Lancet 342:1134 42. Cashman JD, Clark-Lewis I, Eaves A, Eaves CJ (1998) Differentiation stage-specific regulation of primitive human hematopoietic progenitor cycling by exogenous and endogenous inhibitors in an in vivo model. Blood (in press) 43. Sauvageau G, Thorsteinsdottir U, Eaves CJ, Lawrence HJ, Largman C, Lansdorp PM, Humphries RK (1995) Overexpression of HOXB4 in hematopoietic cells causes the selective expansion of more primitive populations in vitro and in vivo. Genes Dev 9:1753 44. Pawliuk R, Eaves C, Humphries RK (1996) Evidence of both ontogeny and transplant dose-regulated expansion of hematopoietic stem cells in vivo. Blood 88:2852
Functional differences between transplantable human hematopoietic stem cells from fetal liver, cord blood, and adult marrow Tessa L. Holyoake,* Franck E. Nicolini,* and Connie J. Eaves Terry Fox Laboratory, British Columbia Cancer Agency and Department of Medical Genetics, University of British Columbia, Vancouver, BC, Canada. (Received 2 April 1999; revised 28 April 1999; accepted 4 May 1999)
The purpose of this study was to develop a simple assay for quantitating transplantable human lymphomyeloid stem cells (competitive repopulating units [CRU]) to enable comparison among the numbers and types of progeny generated in NOD/ SCID mice by such cells from different ontologic sources. Sublethally irradiated NOD/SCID mice were transplanted with varying numbers of CD341 cell-enriched suspensions of human fetal liver, cord blood, or adult marrow cells. The types and numbers of human cells present in the marrow of the mice were measured 6 to 8 weeks later using flow cytometry, in vitro progenitor assays, and secondary transplant endpoints. Frequencies of human CRU obtained by limiting dilution analysis of mice repopulated 6 to 8 weeks posttransplant were the same when the lymphoid and myeloid progeny of CRU were both detected by specific immunophenotypic endpoints as when in vitro myeloid progenitor assays were used to detect CRU myelopoietic activity. The average output per injected CRU of very primitive cells (CD341CD382 cells, LTC-IC, and secondary CRU) was found to be highest for fetal liver CRU and progressively decreased (up to .100-fold) for ontologically older CRU. In contrast, the average output of mature cells was highest for cord blood CRU and lowest for fetal liver CRU, despite equivalent production of intermediate progenitors. Differences in the relative numbers of mature lymphoid, myeloid, and erythroid progeny produced by CRU from different ontologic sources were also seen. Finally, evidence of a transplantable human lymphoid-restricted cell present throughout ontogeny was obtained. A simpler and easier assay for enumerating transplantable human stem cells with lymphomyeloid reconstituting activity has been described, and its specificity and sensitivity validated. The use of this assay has revealed ontogenyassociated differences in a variety of functional attributes of human stem cells proliferating and differentiating in an in vivo, but xenogeneic, setting. © 1999 International Society for Experimental Hematology. Published by Elsevier Science Inc. Keywords: Ontogeny—Human competitive repopulating unit—NOD/SCID mice—LTC-IC—Self-renewal
Offprint requests to: Connie J. Eaves, Terry Fox Laboratory, 601 West 10th Avenue, Vancouver, British Columbia, Canada V5Z 1L3. E-mail: [email protected] *Both authors contributed equally to this work.
Introduction Primitive normal human hematopoietic cells can engraft the bone marrow of various types of immunodeficient xenogeneic hosts, including fetal sheep [1,2] and certain strains of mutant mice [3–6]. In the latter, the highest overall levels of human hematopoiesis reported to date have been obtained in sublethally irradiated nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice [5–9]. The human cells produced in this model include large numbers of human B-lineage cells as well as myeloid cells and their precursors. This has allowed the technique of limiting dilution analysis, which previously was used for quantitating transplantable lymphomyelopoietic murine stem cells [10,11], to be applied to studies of an analogous population of human cells, hence their similar designation as competitive repopulating units (CRU) [9]. Detection of human DNA in the marrow of NOD/SCID mice transplanted with human cells [7,12], or simply of human cells expressing CD45, the common leukocyte antigen [13], have both been used as evidence of transplantable human hematopoietic stem cell activity. However, the inherent lack of specificity of either of these endpoints poses obvious risks to investigations of heterogeneous or manipulated cell suspensions that may include cells that do not possess both lymphoid and myeloid repopulating potential. To circumvent this problem, we proposed the adoption of more specific measures of the type of engraftment obtained. This ensures detection of significant numbers of human lymphoid and myeloid progeny in every mouse scored as positive [9]. In previous studies, the presence of CD342CD191 human cells was used to indicate production of human lymphoid-restricted cells (as it has been reported that some human CD341CD191 cells can display myelopoietic potential [14]). The presence of myeloidrestricted progeny in the same mice was demonstrated by first isolating any human CD341 cells present and then assessing their granulopoietic and/or erythroid colony-forming ability in vitro [9]. The detection of human lymphoid and myelopoietic activity thus was similarly rigorous, although the requirement for a cell sorting and culture step limited the number of mice that could be evaluated and de-
0301-472X/99 $–see front matter. Copyright © 1999 International Society for Experimental Hematology. Published by Elsevier Science Inc. PII S0301-472X(99)0 0 0 7 8 - 8
T.L. Holyoake et al./Experimental Hematology 27 (1999) 1418–1427
layed the acquisition of data, thereby reducing the utility of this assay to address many questions of interest in human hematopoietic stem cell biology. We now describe a simple modification that improves both the rapidity and ease of measuring transplantable human lymphomyeloid stem cell numbers in a given cell suspension without altering the defining features of the cell detected or compromising the sensitivity of method by which it is quantitated. These modifications involve the direct immunophenotypic detection by flow cytometry of the myeloid as well as the lymphoid progeny that human CRU produce in NOD/SCID mice. In addition, we show that the average size that both these compartments achieve by 6 to 8 weeks posttransplant is a linear function of the number of human CRU injected for grafts of up to 10 CRU per recipient. Using this modified assay, a comparison of the different in vivo differentiation and self-renewal properties of human CRU present in fetal liver, cord blood, and adult marrow was undertaken.
Materials and methods Human cells Human bone marrow cells were either aspirate samples from normal donors of allogeneic transplants or samples of cryopreserved normal cadaveric marrow obtained from the Northwest Tissue Center (Seattle, WA). Cord blood samples were obtained from mothers undergoing cesarean delivery of normal, full-term infants. Livers were obtained from 14- to 21-week-old aborted human fetuses. In all cases, approved institutional procedures for obtaining informed consent were followed. Single cell suspensions were prepared from the fetal liver samples by pushing tissue fragments through a coarse sieve. Low density (,1.077g/cm3) cells were isolated from all samples by Ficoll-Hypaque density centrifugation (Pharmacia Biotech AB, Uppsala, Sweden) and washed twice in Hanks balanced salt solution (StemCell Technologies Inc., Vancouver, BC, Canada) supplemented with 5% fetal calf serum (FCS; StemCell) (HF). Cells to be transplanted were first depleted of cells expressing a number of lineage (lin) markers using an immunomagnetic column system as described by the manufacturers (StemSep™, StemCell). Animals NOD/LtSz-scid/scid (NOD/SCID) mice were bred and maintained in the animal facility of the British Columbia Cancer Research Centre (Vancouver, BC, Canada) in microisolators under defined sterile conditions and according to institutional guidelines. Animals 6 to 8 weeks of age were sublethally irradiated with 350 cGy from a 137Cs source less than 24 hours prior to receiving an intravenous injection of human cells. To assess engraftment, marrow cells in the four large hind leg bones of each transplanted mouse were harvested using a syringe and a 21-gauge needle. Cell suspensions for analysis and sorting were prepared in cold HF supplemented with 5% pooled normal human serum (HF/5% HS). In some experiments, mice were given six intraperitoneal injections of human growth factors spread over a 2-week interval just prior to sacrifice, as indicated.
1419
Flow cytometry Cells to be analyzed were first incubated for 10 minutes at 48C with HF/5% HS supplemented with 3 mg/mL 2.4G2 anti-mouse Fc receptor antibody to block Fc receptors and prevent nonspecific binding [15]. Separate aliquots of cells were incubated for 30 minutes at 48C with the following combinations of antibodies against human antigens: (1) anti-human CD34-FITC (8G12) [16] plus anti-CD19-PE and anti-CD20-PE (both from Becton Dickinson [BD], San Jose, CA); (2) anti-CD71-PE (OKT-9) plus anti-CD45PE, anti-CD15-FITC, and anti-CD66b-FITC (all from Pharmingen, Mississauga, Ontario, Canada); (3) anti-glycophorin-A-FITC (10F7); (4) anti-CD41 [17]; and (5) anti-CD34-FITC and antiCD38-PE (BD). Cells were washed twice with HF, the second wash containing 1 mg/mL propidium iodide (PI; Sigma Chemical Co., St Louis, MO). Acquisition and analysis of the cells were performed on a FACSort using LYSIS II software (BD). Positive cells were defined as those exhibiting a level of fluorescence that exceeded 99.98% of that obtained with irrelevant isotype-matched control antibodies labeled with the same fluorochromes. All antibodies used for positive staining were checked for their nonreactivity with primary NOD/SCID mouse bone marrow cells. In addition, as indicated, human CD341 cells were purified by FACS, or by removal by immunomagnetic depletion (see following) of murine cells as well as human lineage marker-positive (lin1) cells [18]. Human CRU assay For each test cell sample (Table 1), five to six groups of irradiated mice (four mice per group) were injected with threefold dilutions of lin2 cells plus 106 irradiated (1500 cGy) normal human bone marrow cells as carrier cells. Mice were killed 6 to 8 weeks later and their marrow cells removed and stained with anti-human CD34 and CD19 antibodies (“old” CRU assay), as previously described [8,9]. Other aliquots were stained with anti-human CD45 1 CD71 and CD15 1 CD66b antibodies or anti-human CD34 and CD19 1 CD20 antibodies (“new” CRU assay). For human CRU determinations using the old assay, mice were considered negative if there were ,5 CD342CD191 (B-lineage) human cells per 5 3 103 viable cells and/or no CD341 human colony-forming cells (CFC) per 106 total viable (PI2) cells [8,9]. For the new CRU assay (used throughout this study unless otherwise indicated), mice were considered negative if there were ,5 CD342CD19/201 human cells and/or ,5 CD45/711CD15/66b1 human cells per 2 3 104 viable cells analyzed. Old and new CRU frequencies were calculated using the L-calc software program (StemCell) from the proportions of negative mice in each similarly treated group, using Poisson statistics and the method of maximum likelihood [10]. In vitro assays CFC and LTC-IC assays were performed as described previously [6,19]. Briefly, for CFC assays, the FACS-sorted CD341 human cells were cultured in methylcellulose medium (H4330, StemCel) supplemented with 50 ng/mL human steel factor (SF, purified from supernatants of cos cells transfected with human SF cDNA) and 20 ng/mL each of human interleukin 3 (IL-3; Novartis, Basel, Switzerland), interleukin 6 (IL-6; Cangene, Missassauga, ON), granulocyte-macrophage colony-stimulating factor (GM-CSF; Novartis), G-CSF (StemCell), and 3 U/mL erythropoietin (Epo; StemCell). CD341 cells were assayed for LTC-IC by culturing them on preestablished irradiated murine fibroblasts genetically
1420
T.L. Holyoake et al./Experimental Hematology 27 (1999) 1418–1427
Table 1. Frequency of CRU relative to the total CD341 population decreases during ontogeny CRU frequency (no. of CRU per 105 injected CD341 cells)* Source of cells assayed (no. of lin2 cells/mouse) Fetal liver (300–105)
Cord blood (1.2 3 104–3 3 105) Adult bone marrow (104–7.2 3 105)
Endpoint of CRU activity
Mean
95% confidence interval
No. of experiments
New assay Old assay CD45/711 New assay Old assay CD45/711 New assay Old assay CD45/711
9.1 6.1 29.4 6.0 4.2 26.6 0.8 2.0 2.0
5.4–15.4 3.7–10.2 17.2–50.1 3.1–11 2.1–8.0 15–46 0.5–1.3 1.1–3.3 1.2–3.3
3 3 3 3 1 3 3 3 3
*As described in the Methods, each CRU determination was calculated from the combined results of three independent experiments, in each of which a total of 20 mice were transplanted with a 100-fold range of lin2 cells of the type shown (five groups of four mice each). In each experiment, all injected mice were analyzed and CRU frequencies calculated using each of the three sets of engraftment criteria indicated to distinguish positive and negative recipients (see Methods for details). To reduce the variability in the data due simply to different efficiencies of lin1 cell depletion in different experiments, results have been calculated on a per injected CD341 cell basis.
engineered to produce human IL-3, G-CSF, and SF and then determining the number of CFC present 6 weeks later. The number of LTC-IC present in the input innoculum was calculated by dividing the total number of CFC present in the 6-week-old cultures by 18 for cells derived from adult human bone marrow, by 28 for cells derived from human cord blood [19], and by 72 for cells derived from fetal liver [20].
Results Quantitation of human CRU by direct immunophenotyping of regenerated lymphoid and myeloid progeny In a first series of experiments, we compared the FACS profiles obtained when cells from NOD/SCID mice engrafted with human bone marrow cells were stained with various combinations of anti-human antibodies to identify those that gave the best discrimination of the human B lineage and myeloid cells present. The most sensitive combination was
found to be anti-CD34-FITC 1 anti-CD19/20-PE to identify CD342CD19/201 B-lineage cells and anti-CD45/71-PE 1 anti-CD15/66b-FITC to identify CD45/711CD15/66b1 mature myeloid cells (see representative example in Fig. 1). We then used this approach to identify mice that showed dual lymphoid and myeloid engraftment with human cells 6 to 8 weeks after the injection of decreasing numbers of lin2 human fetal liver, cord blood, or adult marrow cells. Human CRU frequencies were calculated, using five positive cells of each type (myeloid and lymphoid, from a total of 2 3 104 cells analyzed) as the criterion for distinguishing positive and negative recipients for the “new” assay (as described in the Methods). For the “old” assay, 5 3 103 cells were analyzed for the presence of human B-lineage cells and an additional 106 were assessed for the presence of human CD341 granulopoietic or erythroid CFC [8,9]. As shown in Table 1, the results obtained with both procedures were similar (p . 0.05) for each of the three sources of cells assayed. However, when CRU frequencies
Figure 1. Representative dot plots for the marrow cells obtained from a NOD/SCID mouse 6 weeks after being transplanted with 1.5 3 105 lin2 human cord blood cells (z10 CRU). Left: Staining with isotype control Ig; middle: extent of total (CD45/711) and myeloid-specific (CD45/711 CD15/66b1) human cell engraftment; right: extent of B-lineage-specific (CD342CD19/201) and primitive (CD341) human cell engraftment.
T.L. Holyoake et al./Experimental Hematology 27 (1999) 1418–1427
were calculated using just the presence of human CD45 and/or CD711 cells ($5 per 2 3 104 cells analyzed) to identify positive recipients, the values obtained for both cord blood and fetal liver CRU were three- to fourfold higher, although the value obtained for adult marrow CRU was similar. The higher frequency of cord blood and fetal liver CRU obtained with the less discriminating (CD45/711 cell) readout presumably reflects the presence of transplantable cord blood and fetal liver cells that can generate lymphoid progeny but not detectable numbers of myeloid progeny in this model. Analysis of mice injected with doses of cells calculated retrospectively to have contained, on average, #1 repopulating cell of any kind (based on the results for the entire dataset) showed that a proportion of the recipients transplanted with any of the three human cell sources contained detectable numbers of human B-lineage cells only (21%, 39%, and 30% of recipients of #1 CRU from human fetal liver, cord blood, or adult marrow, respectively). None contained myeloid cells only. This situation is different from what we previously reported when only 5 3 103 cells were analyzed (#6% mice with only human myeloid or lymphoid cells detected) [9]. Thus, the increased sensitivity of detecting human B-lineage cells obtained by evaluating four times more cells did not increase the sensitivity of the CRU assay over that previously reported [9], but allowed the detection of an apparently lymphoid-restricted population of transplantable human cells present at a slightly higher frequency but characterized by a lower proliferative activity. Comparison of the number of CRU per 105 CD341 cells in fetal liver, cord blood, and adult marrow populations shows that this value decreases during ontogeny by a factor of approximately 10 (Table 1).
Human lymphomyeloid engraftment is linearly related to transplant dose In addition to monitoring the proportions of mice transplanted with varying numbers of cells that had detectable numbers of human B-lineage, myeloid, or any human hematopoietic (CD45/711) cells in their marrow, the average size of these populations produced in the same groups of mice was calculated. Figure 2 shows a plot of the results obtained for each of the three types of transplants evaluated using the total CD45/711 cell endpoint and assuming that the number of cells present in two femurs plus two tibias represents 25% of the total marrow cellularity of the mouse [21]. To facilitate a comparison of the three plots, each cell dose injected was converted to a CRU number based on the frequency values determined in Table 1. It can be seen that there was a linear increase in the total number of human hematopoietic cells generated (as assessed 6 to 8 weeks posttransplant) with increasing numbers of human CRU transplanted for transplants containing up to 10 CRU per mouse, regardless of their origin. However,
1421
there was a marked difference in the average total number of cells produced per human CRU for different sources of these cells. The largest populations were produced by cord blood CRU, the second largest were generated by adult marrow CRU, and the smallest populations were those derived from fetal liver CRU. A similar analysis of the human B-lineage (CD342CD19/201) and myeloid (CD45/711CD15/ 66b1 ) subpopulations showed these also to be linearly related to CRU input (data not shown).
Figure 2. Cell dose response data for the absolute number of human (CD45/711) cells present in the marrow of NOD/SCID mice transplanted 6 to 8 weeks previously with lin2 human cells of fetal liver (FL), cord blood (CB), and adult bone marrow (BM) origin. Each point represents pooled data from 3 to 12 mice from a total of three experiments for each kind of transplant, assuming the marrow present in two tibias and two femurs represents 25% of the total marrow of a mouse [21]. R values are derived correlation ratios.
1422
T.L. Holyoake et al./Experimental Hematology 27 (1999) 1418–1427
Human CRU from different ontologic sources produce different numbers of various primitive and mature progeny in the marrow of NOD/SCID mice In the experiments described earlier, additional aliquots of cells harvested from the marrow of each of the NOD/SCID mice showing engraftment with human lymphoid and myeloid cells were labeled with anti-human CD38 and CD34, glycophorin A, or CD41 antibodies and the cells positive for these markers also quantitated. In addition, LTC-IC assays were performed on the human CD341 cells isolated. The results of each of these additional measurements were used to calculate the corresponding number of cells of each type present in each mouse and average 6- to 8-week outputs per CRU then derived. The results of these calculations are summarized in Table 2. Because maturing B-lineage (CD342CD19/201) cells constituted the largest population generated by all sources of human CRU, the average output of these cells per CRU mirrors the corresponding average output of total cells per CRU. The average output of mature human granulopoietic (CD45/711CD15/66b1) cells in the mice also was greater for cord blood and adult marrow CRU than for fetal liver CRU. In contrast, the average output of human cells belonging to the megakaryocyte lineage (CD411 cells) appeared less affected by the origin of the stem cells from which they were derived. The average output of mature human erythroid (glycophorin A1) cells was
significantly (p , 0.05) higher for fetal liver CRU than cord blood CRU and undetectable in NOD/SCID mice engrafted with adult marrow cells (despite the confirmed presence of similar numbers of erythroid-restricted CFC in the same mice, as found previously [8]). In mice engrafted with human fetal liver cells, the output of mature erythroid progeny was, on average, twice the output of mature myeloid cells. In contrast, the reverse was true in the cord blood-engrafted mice, in which the output of human myeloid cells was five times the output of mature human erythroid cells. Interestingly, these differences at the level of the mature compartments were not predicted by the size of the more primitive cell populations. Thus, the total numbers of both CD341 cells and CFC produced from fetal, newborn, and adult human CRU were similar but, as shown in Fig. 3, the output of more primitive human cells (CD341CD382 cells and LTC-IC) decreased markedly with the ontologic age of the human CRU transplanted. Human fetal liver CRU produced, on average, z6- and z30-fold more CD341CD382 cells than cord blood and adult marrow CRU, respectively, and z7 and z300-fold more LTC-IC. It should be noted that, in these calculations, the numbers of LTC-IC were determined assuming that their individual CFC outputs were the same as those of the LTC-IC in the tissue transplanted (although these values decrease during normal human ontogeny [19,20]). If a similar trend occurs over time posttransplant, this would increase even further the differences, shown in Fig. 3.
Table 2. Calculated output per injected CRU of phenotypically and functionally defined human cell subsets assessed 6 to 8 weeks after transplantation of human fetal liver, cord blood, and adult marrow lin2 cells into sublethally irradiated NOD/SCID mice Human cell subsets CD45/711 CD342CD19/201 CD45/711 CD15/66b1 Glycophorin A1 CD411 CD341 CFC CD341CD382 LTC-IC
Fetal liver
Cord blood
Bone marrow
35 6 18 22 6 12 362 762 0.7 6 0.2 18 6 8 0.6 6 0.3 0.6 6 0.3 2100 6 880
144 6 37 137 6 54 661 1.2 6 0.6 1.8 6 0.5 23 6 5 0.4 6 0.2 0.1 6 0.1 280 6 210
64 6 26 49 6 20 663 ,0.02 6 0.01 1.2 6 0.4 35 6 9 0.8 6 0.1 0.02 6 0.01 765
Values are given as mean (3105) 6 SEM, except for the LTC-IC, which are as shown. These values were calculated (as described in the text) from the results obtained in mice injected with up to z10 CRU per mouse (based on the frequencies shown in Table 1) as follows. For each subset of human cells analyzed, the total number of these cells present in each positive recipient of #10 CRU (from a given source) was first calculated by multiplying their frequency in the marrow (e.g., number of human CD45/ 711 cells per 20,000 total marrow cells analyzed) by the total number of marrow cells per mouse (assuming the contents of two femurs and two tibias represent 25% of the total marrow volume [21]). The total number of human cells of this subset for all positive recipients of #10 CRU was obtained by adding the results for the individual mice together. Finally, this value was divided by the total number of CRU injected into these recipients (as determined in Table 1) to yield the output of each subset per CRU.
Figure 3. Yield of human CD341CD382 cells (top) and LTC-IC (bottom) per NOD/SCID mouse per human CRU injected (calculated as described in the legend to Table 3) for CRU of fetal liver (open bars), cord blood, (black bars), or adult marrow origin (gray bars). Values shown are the mean 6 SEM from data pooled from three experiments for each type of CRU.
T.L. Holyoake et al./Experimental Hematology 27 (1999) 1418–1427
1423
cells. However, in this case, the output of human CRU was several-fold higher than the input value (again of approximately 10 CRU per primary mouse). Assessment of the effects of the growth factor injections on other cell types confirmed previous findings for adult marrow-engrafted primary mice [6]. Fetal liver-engrafted mice showed the same (lack of) response by less primitive cell types (data not shown). Because of the high levels of human multilineage engraftment generally attained in all groups of secondary recipients of cells from primary mice transplanted with human fetal liver cells (6 growth factors), aliquots of the marrow cells from each group of these secondary recipients were transferred to tertiary irradiated NOD/SCID mice. Another 6 weeks later, the marrow of the tertiary animals was assessed for the presence of human B-lineage (CD342 CD19/201 ) and myeloid (CD45/711CD15/66b1 ) cells. As shown in Table 4, 3 of 15 of these tertiary recipients were positive for both cell types and three others contained human B-lineage cells but no detectable myeloid cells. This indicates continued maintenance of a detectable human CRU population in the secondary mice (none of which received injections of human growth factors). An example of a primary, secondary, and tertiary recipient of serially transplanted human fetal liver cells resulting each time in the production of both human lymphoid and myeloid cells is shown in Fig. 4.
Enhanced factor-independent CRU regeneration in NOD/SCID recipients of human fetal liver (vs cord blood or adult marrow cells) In a final series of experiments, we compared the ability of equivalent transplants (z10 CRU per recipient) of human lin2 fetal liver or adult marrow cells to regenerate progeny CRU 4 weeks after injection of the cells into sublethally irradiated NOD/SCID mice. We had previously found that a 2-week course of three alternate daily intraperitoneal injections of human SF (10 mg/mouse/injection), IL-3, and GMCSF (both at 6 mg/mouse/injection), and Epo (10 U/mouse/ injection) enhanced the number of human CRU obtained from mice transplanted 4 to 6 weeks previously with human cord blood cells [22], even though there was no effect on the output of later cell types [6,23], with the exception of marrow-derived erythroblasts [20]. Therefore, we also tested the effect of these growth factors on the regeneration in NOD/SCID mice of CRU derived from human fetal liver and adult marrow. The number of these present in the marrow of the primary mice (at 4 weeks posttransplant) was determined by performing secondary CRU assays on the cells harvested from the marrow of each group of primary mice. The details of these experiments and the results of the secondary limiting dilution assays are summarized in Table 3. Human CRU were not present at detectable levels in primary mice transplanted with adult human marrow cells irrespective of whether or not they also were injected with growth factors. This represents a .25-fold decrease in CRU numbers below the estimated 10 initially injected per primary mouse. There also was no evidence of an effect of the growth factors injected on the number of human CRU regenerated in the mice transplanted with human fetal liver
Discussion Much interest has focused recently on the potential of xenotransplant models to address questions about normal and
Table 3. Number of human CRU detected in the marrow of primary NOD/SCID mice transplanted 4 weeks previously with z10 CRU of human fetal liver, cord blood, and adult bone marrow origin Primary recipients
Initial human cells transplanted (no. of lin2 cells/mouse)* Adult marrow Fetal Liver
Secondary recipients
GF injected† 2 1 2 1
Percentage of primary mouse BM cells per secondary mouse
Positive secondary mice/total secondary mice
25 25 25 5 1 25 5 1
0/4 0/4 2/2 4/5 4/4 4/4 4/5 2/5
Change in CRU no. posttransplant in primary mice‡ (95% CI) .25-fold decrease .25-fold decrease 5-fold increase (3-10-fold increase) 3-fold increase (1- to 10-fold increase)
*There were 1.25 3 106 and 1.1 3 105 CD341 cells, respectively, present in the adult marrow and fetal liver lin2 cells injected into each mouse (i.e. z10 CRU, based on the frequencies derived in Table 1). † Growth factors (GF) were injected, as described in the text. ‡ This was determined by dividing the average number of human CRU calculated to be present in the entire marrow of the primary recipients (measured CRU frequency 3 total marrow cells assuming two femurs plus two tibias represent 25% of the total marrow volume [21]) by 10 (the estimated number of human CRU injected into each primary recipient). This calculation does not take into consideration the fact that only a small proportion of the regenerated cells with CRU activity would be detected in the secondary mice and hence all “changes” shown would be underestimated by this factor.
1424
T.L. Holyoake et al./Experimental Hematology 27 (1999) 1418–1427
Table 4. Detection of human lymphoid (CD342CD19/201) and myeloid (CD45/711CD15/66b1) cells in tertiary NOD/SCID recipients of serially transplanted human fetal liver-derived CRU Percentage human subsets in individual tertiary mice GF injected into primary mice 2 1
Percentage of primary mouse BM cells per secondary mouse
CD342CD19/201
CD45/711CD15/66b1
25 1 1 25 25 5
0.11 0.03 0.34 0.05 0.82 0.03
,0.03 0.12 ,0.03 0.03 0.74 ,0.03
*Each tertiary mouse received the equivalent of 25% of the marrow of a secondary mouse from the group of secondary recipients shown in Table 3. Only mice in which some human cells were detected (either lymphoid or myeloid, .0.025% positive) are shown. The total number of tertiary mice analyzed after transplantation of cells from secondary recipients of variable numbers of cells from primary non–GF-treated primary mice was as follows: 25% 5 one, 5% 5 one, 1% 5 three; and from corresponding recipients of cells originally regenerated in 1GF primary mice was as follows: 25% 5 three, 5% 5 three, 1% 5 three.
malignant human hematopoietic stem cells, including their responses to various cytokine and genetic manipulations in vitro. Central to such investigations are the endpoints used to detect and quantify human stem cells, as these determine the specificity and precision of responses observed. The ability of human hematopoietic cells to home to the marrow
of xenogeneic recipients and respond to species–cross-reactive factors was first indicated by the engraftment of irradiated adult bg/nu/xid (bnx) mice with injected human cells [3]. Many murine stromal cell lines now have been shown to support the long-term maintenance of human hematopoiesis in vitro [24–28], and a number of specific cytokines
Figure 4. Representative dot plots showing total (CD451/71) and myeloid-specific (CD45/711CD15/66b1) human engraftment of the marrows of primary, secondary, and tertiary NOD/SCID recipients of human fetal liver cells (top three panels) and corresponding results from the same mice for primitive (CD341) and B-lineage-specific (CD342CD19/201) human cells (bottom three panels).
T.L. Holyoake et al./Experimental Hematology 27 (1999) 1418–1427
with cross-species activity on hematopoietic stem cells have been identified [29–31]. More recent in vivo studies have confirmed that the major barrier to the xenotransplantation of human hematopoietic stem cells is immunologic, as shown by improvements using naive (fetal) [1,2] or other genetically compromised recipients [4–8]. The reduced or absent B, T, NK, macrophage. and complement activities of the NOD/SCID mouse makes it the most receptive host thus far described. When these mice are additionally treated with a sublethal dose of irradiation (325–400 cGy) prior to the transplantation of normal human hematopoietic cells, both human lymphoid and myeloid progeny typically can be found when the total number of human cells regenerated per mouse 6 to 8 weeks posttransplant is $105 [9]. In the present studies we show that the human stem cells (CRU) capable of producing this multilineage engraftment can be enumerated by limiting dilution analysis of mice defined as repopulated using an appropriate set of phenotypic markers to identify specific subpopulations of human B-lineage and myeloid cells in the marrow cells harvested from the mice. However, by increasing the sensitivity of human cell detection by a factor of 4 over that previously reported [8,9] (i.e., from z0.1% to z0.02%, which allows the detection of transplantable human cells with reduced proliferative potential, i.e., z 2 3 104 vs 105 cells), we also obtained evidence of a cell type that appears to exhibit a lymphoid-restricted program in this assay. Such cells were demonstrable in adult human marrow, cord blood, and fetal liver, and at frequencies slightly higher than those measured for cells with both lymphoid and myeloid reconstituting activities. These findings underscore the importance of adopting specific endpoints to measure the differentiation potential of the cells being assayed, particularly after exposure to factors that may influence their proliferative and/or differentiation responses. A less specific, but much easier and less expensive, method to assess effects on transplantable human stem cells is to infer quantitative changes in the initial innoculum from the average total level of NOD/SCID marrow engraftment obtained 6 to 8 weeks posttransplant. The validity of this approach is supported by the demonstration of a linear relationship between the number of human CRU injected and the average level of engraftment seen, at least for transplants containing #10 human CRU. This was true for all three sources of human CRU studied. A simple modification to improve the specificity of the readout for lymphomyeloid repopulating cells might then be to simply prolong the time posttransplant prior to assessing the level of engraftment, as validated by previous in vivo studies in mice [32] and sheep [33], and by in vitro studies using the long-term culture system [34,35]. In the present experiments, the development of a relatively simple procedure for quantitating transplantable human cells with lymphomyeloid repopulating potential made it possible to identify intrinsic differences imposed by ontogeny on the self-renewal and differentiation activity of
1425
human hematopoietic cells and their progeny. Interestingly, CRU from human fetal liver, cord blood, and adult marrow all produced a similarly sized population of intermediate progenitors as indicated by measurements of human CD341 cells and in vitro clonogenic cells. However, marked differences in the output of terminally differentiating erythroid and developing B cells were noted in mice that received fetal vs adult transplants. This disparity in the production of various differentiated cell populations is likely to reflect the stable transmission through successive generations of cells of previously documented ontogeny-determined differences in the cytokine responsiveness or requirements of lineagerestricted progenitor cells and their terminally differentiating progeny [36–38]. Significant differences also were noted in the output of very primitive cells by transplanted CRU derived from different ontologic sources. This was evident from an examination of the size of the human CD341CD382 population regenerated as well as in the number of human cells present that could initiate long-term hematopoiesis in stromal feeders in vitro (LTC-IC) or engraft secondary irradiated NOD/ SCID recipients (CRU). The magnitude of these differences increased progressively with ontogeny and were .100-fold for all three endpoints when fetal and adult CRU were compared. As a result, a several-fold net expansion of human CRU in vivo could be demonstrated for the first time with transplants of fetal liver cells, and these could be serially transplanted two more times. The greater self-renewal activity of fetal human hematopoietic stem cells mimics previous data from studies of murine stem cells [39,40]. The finding that the proliferation and/or self-renewal of fetal human stem cells were not further enhanced by exogenous human growth factors (in contrast to cord blood CRU where 10fold increases in CRU yields were stimulated by such treatments [22]) reinforces the concept of an altered state that persists throughout the differentiation of fetal hematopoietic cells and exaggerates the type of response elicited by certain cytokines. It also should be noted that failure to detect regeneration of CRU from injected adult or pediatric human sources does not preclude the occurrence of this activity, as implied by long-term xenotransplant studies in sheep [33] or retroviral marking studies [41]. However, in the present experiments, the reduced sensitivity of the serial transplantation procedure may have precluded detection of the smaller numbers of CRU regenerated by adult marrow cells. This necessarily involves an underestimation of the magnitude of CRU regeneration posttransplant, because the seeding efficiency of human CRU in NOD/SCID mice (which is likely to be low [6,8]) has not been factored into the present studies. That NOD/SCID mice transplanted with similar numbers of human stem cells at the peak of the initial engraftment phase [6,8] contain similar numbers of cells at intermediate stages of differentiation, despite large differences in the size of the stem cell population from which they would be con-
1426
T.L. Holyoake et al./Experimental Hematology 27 (1999) 1418–1427
tinuously originating, suggests the operation in the mice of mechanisms that also must differentially regulate this process. In other studies, we have shown that the later decline of adult human marrow-derived clonogenic progenitors may represent a response of these cells (or their immediate precursors) to endogenously produced chemokines such as MCP-1 and MIP-1 [42]. It is possible that the relatively reduced output of clonogenic cells from fetal liver or cord blood stem cells may reflect a greater sensitivity of these developmentally earlier cells to factors with this type of activity. It is interesting to note that evidence of analogous regulatory mechanisms responsible for normalizing intermediate populations of murine progenitors from differentially regenerated stem cell compartments has been obtained [43,44]. In summary, we have described a simpler and more rapid methodology for quantifying transplantable human stem cells with lymphomyeloid repopulating activity and have used it to demonstrate both ontogeny and stage-specific differences in the regulation of human hematopoiesis in an in vivo, but xenogeneic, model of engraftment. These findings set the stage for future investigation of both extrinsic and intrinsic molecular mechanisms responsible for these differences and how they may be altered in disease states. Acknowledgments We thank the staff of the Stem Cell Assay Service for their assistance in the initial preparation of primary human cell samples, Jessyca Maltman and Maya Sinclaire for their assistance in the animal studies, Pamela Austin for additional technical assistance, Gayle Thornbury and Giovanna Cameron for operating the FACS, and Tara Palmater for preparation of the manuscript. We are also grateful to P. Lansdorp (Terry Fox Laboratory), and Cangene, Novartis, and StemCell Technologies, Inc., for valuable gifts of reagents. This work was supported by grants from Novartis (Basel, Switzerland), the National Cancer Institute of Canada (NCIC) with funds from the Terry Fox Run, and the NIH (POI HL 55435). T.L. Holyoake holds a United Kingdom Leukemia Research Fund Senior Lecturership, and C.J. Eaves is a Terry Fox Cancer Research Scientist of the NCIC.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16. 17.
18.
References 1. Zanjani ED, Flake AW, Rice H, Hedrick M, Tavassoli M (1994) Longterm repopulating ability of xenogeneic transplanted human fetal liver hematopoietic stem cells in sheep. J Clin Invest 93:1051 2. Zanjani ED, Pallavicini MG, Ascensao JL, Flake AW, Langlois RG, Reitsma M, MacKintosh FR, Stutes D, Harrison MR, Tavassoli M (1992) Engraftment and long-term expression of human fetal hemopoietic stem cells in sheep following transplantation in utero. J Clin Invest 89:1178 3. Kamel-Reid S, Dick JE (1988) Engraftment of immune-deficient mice with human hematopoietic stem cells. Science 242:1706 4. Lapidot T, Pflumio F, Doedens M, Murdoch B, Williams DE, Dick JE (1992) Cytokine stimulation of multilineage hematopoiesis from immature human cells engrafted in SCID mice. Science 255:1137 5. Pflumio F, Izac B, Katz A, Shultz LD, Vainchenker W, Coulombel L (1996) Phenotype and function of human hematopoietic cells engraft-
19.
20.
21. 22.
23.
ing immune-deficient CB17-severe combined immunodeficiency mice and nonobese diabetic-severe combined immunodeficiency mice after transplantation of human cord blood mononuclear cells. Blood 88:3731 Cashman JD, Lapidot T, Wang JCY, Doedens M, Shultz LD, Lansdorp P, Dick JE, Eaves CJ (1997) Kinetic evidence of the regeneration of multilineage hematopoiesis from primitive cells in normal human bone marrow transplanted into immunodeficient mice. Blood 89:4307 Larochelle A, Vormoor J, Hanenberg H, Wang JCY, Bhatia M, Lapidot T, Moritz T, Murdoch B, Xiao XL, Kato I, Williams DA, Dick JE (1996) Identification of primitive human hematopoietic cells capable of repopulating NOD/SCID mouse bone marrow: implications for gene therapy. Nature Med 2:1329 Cashman J, Bockhold K, Hogge DE, Eaves AC, Eaves CJ (1997) Sustained proliferation, multi-lineage differentiation and maintenance of primitive human haematopoietic cells in NOD/SCID mice transplanted with human cord blood. Br J Haematol 98:1026 Conneally E, Cashman J, Petzer A, Eaves C (1997) Expansion in vitro of transplantable human cord blood stem cells demonstrated using a quantitative assay of their lympho-myeloid repopulating activity in nonobese diabetic-scid/scid mice. Proc Natl Acad Sci U S A 94:9836 Szilvassy SJ, Humphries RK, Lansdorp PM, Eaves AC, Eaves CJ (1990) Quantitative assay for totipotent reconstituting hematopoietic stem cells by a competitive repopulation strategy. Proc Natl Acad Sci U S A 87:8736 Miller CL, Eaves CJ (1997) Expansion in vitro of adult murine hematopoietic stem cells with transplantable lympho-myeloid reconstituting ability. Proc Natl Acad Sci U S A 94:13648 Bhatia M, Wang JCY, Kapp U, Bonnet D, Dick JE (1997) Purification of primitive human hematopoietic cells capable of repopulating immune-deficient mice. Proc Natl Acad Sci U S A 94:5320 Hogan CJ, Shpall EJ, McNulty O, McNiece I, Dick JE, Shultz LD, Keller G (1997) Engraftment and development of human CD341-enriched cells from umbilical cord blood in NOD/LtSz-scid/scid mice. Blood 90:85 Tjonnfjord GE, Steen R, Veiby OP, Egeland T (1996) Lineage commitment of CD341 human hematopoietic progenitor cells. Exp Hematol 24:875 Unkeless JC (1979) Characterization of a monoclonal antibody directed against mouse macrophage and lymphocyte Fc receptors. J Exp Med 150:580 Schmitt C, Eaves CJ, Lansdorp PM (1991) Expression of CD34 on human B cell precursors. Clin Exp Immunol 85:168 Hogge D, Fanning S, Bockhold K, Petzer A, Lambie K, Lansdorp P, Eaves A, Eaves C (1997) Quantitation and characterization of human megakaryocyte colony-forming cells using a standardized serum-free agarose assay. Br J Haematol 96:790 Holyoake TL, Cameron G, Horrocks C, Thomas T, Eaves C, Eaves A (1998) Development of immunomagnetic negative depletion procedures for obtaining enriched populations of primitive cells from CML blood samples or their progeny regenerated in NOD/SCID mice. Exp Hematol 26: 757 (abstr) Hogge DE, Lansdorp PM, Reid D, Gerhard B, Eaves CJ (1996) Enhanced detection, maintenance and differentiation of primitive human hematopoietic cells in cultures containing murine fibroblasts engineered to produce human Steel factor, interleukin-3 and granulocyte colony-stimulating factor. Blood 88:3765 Nicolini FE, Holyoake TL, Cashman JD, Chu PPY, Lambie K, Eaves CJ. Unique differentiation programs of human fetal liver stem cells revealed both in vitro and in vivo in NOD/SCID mice. Blood (in press) Boggs DR (1984) The total marrow mass of the mouse: a simplified method of measurement. Am J Hematol 16:277 Cashman JD, Eaves CJ (1999) Human growth factor-enhanced regeneration of transplantable human hematopoietic stem cells in nonobese diabetic/severe combined immunodeficient mice. Blood 93:481 Vormoor J, Lapidot T, Pflumio F, Risdon G, Patterson B, Broxmeyer
T.L. Holyoake et al./Experimental Hematology 27 (1999) 1418–1427
24.
25.
26.
27.
28.
29.
30.
31.
32. 33.
HE, Dick JE (1994) Immature human cord blood progenitors engraft and proliferate to high levels in severe combined immunodeficient mice. Blood 83:2489 Xu M, Tsuji K, Ueda T, Mukouyama Y, Hara T, Yang F-C, Ebihara Y, Matsuoka S, Manabe A, Kikuchi A, Ito M, Miyajima A, Nakahata T (1998) Stimulation of mouse and human primitive hematopoiesis by murine embryonic aorta-gonad-mesonephros-derived stromal cell lines. Blood 92:2032 Baum CM, Weissman IL, Tsukamoto AS, Buckle AM, Péault B (1992) Isolation of a candidate human hematopoietic stem-cell population. Proc Natl Acad Sci U S A 89:2804 Sutherland HJ, Eaves CJ, Lansdorp PM, Thacker JD, Hogge DE (1991) Differential regulation of primitive human hematopoietic cells in long-term cultures maintained on genetically engineered murine stromal cells. Blood 78:666 Issaad C, Croisille L, Katz A, Vainchenker W, Coulombel L (1993) A murine stromal cell line allows the proliferation of very primitive human CD3411/CD38 progenitor cells in long-term cultures and semisolid assays. Blood 81:2916 Thiemann FT, Moore KA, Smogorzewska EM, Lemischka IR, Crooks GM (1998) The murine stromal cell line AFT024 acts specifically on human CD341CD38 progenitors to maintain primitive function and immunophenotype in vitro. Exp Hematol 26:612 Martin FH, Suggs SV, Langley KE, Lu HS, Ting J, Okino KH, Morris CF, McNiece IK, Jacobsen FW, Mendiaz EA, Birkett NC, Smith KA, et al (1990) Primary structure and functional expression of rat and human stem cell factor DNAs. Cell 63:203 Lyman SD, James L, Vanden Bos T, de Vries P, Brasel K, Gliniak B, Hollingsworth LT, Picha KS, McKenna HJ, Splett RR, Fletcher FA, Maraskovsky E, et al (1993) Molecular cloning of a ligand for the flt3/ flk-2 tyrosine kinase receptor: a proliferative factor for primitive hematopoietic cells. Cell 75:1157 Wendling F, Maraskovsky E, Debili N, Florindo C, Teepe M, Titeux M, Methia N, Breton-Gorius J, Cosman D, Vainchenker W (1994) c-Mpl ligand is a humoral regulator of megakaryocytopoiesis. Nature 369:571 Jordan CT, Lemischka IR (1990) Clonal and systemic analysis of longterm hematopoiesis in the mouse. Genes Dev 4:220 Civin CI, Almeida-Porada G, Lee M, Olweus J, Terstappen LWMM, Zanjani ED (1996) Sustained, retransplantable, multilineage engraftment of highly purified adult human bone marrow stem cells in vivo. Blood 88:4102
1427
34. Prosper F, Stroncek D, Verfaillie CM (1996) Phenotypic and functional characterization of long-term culture-initiating cells present in peripheral blood progenitor collections of normal donors treated with granulocyte colony-stimulating factor. Blood 88:2033 35. Hao QL, Thiemann FT, Petersen D, Smogorzewska EM, Crooks GM (1996) Extended long-term culture reveals a highly quiescent and primitive human hematopoietic progentior population. Blood 88:3306 36. Rich IN, Kubanek B (1976) Erythroid colony formation (CFU-e) in fetal liver and adult bone marrow and spleen from the mouse. Blut 33:171 37. Peschle C, Migliaccio AR, Migliaccio G, Ciccariello R, Lettieri F, Quattrin S, Russo G, Mastroberardino G (1981) Identification and characterization of three classes of erythroid progenitors in human fetal liver. Blood 58:565 38. Weekx SFA, van Bockstaele DR, Plum J, Moulijn A, Rodrigus I, Lardon F, De Smedt M, Nijs G, Lenjou M, Loquet P, Berneman ZN, Snoeck H-W (1998) CD3411CD38 and CD341CD381 human hematopoietic progenitors from fetal liver, cord blood, and adult bone marrow respond differently to hematopoietic cytokines depending on the ontogenic source. Exp Hematol 26:1034 39. Micklem HS, Ford CE, Evans EP, Ogden DA, Papworth DS (1972) Competitive in vivo proliferation of foetal and adult haematopoietic cells in lethally irradiated mice. J Cell Physiol 79:293 40. Rebel VI, Miller CL, Eaves CJ, Lansdorp PM (1996) The repopulation potential of fetal liver hematopoietic stem cells in mice exceeds that of their adult bone marrow counterparts. Blood 87:3500 41. Brenner MK, Rill DR, Holladay MS, Heslop HE, Moen RC, Buschle M, Krance RA, Santana VM, Anderson WF, Ihle JN (1993) Gene marking to determine whether autologous marrow infusion restores long-term haemopoiesis in cancer patients. Lancet 342:1134 42. Cashman JD, Clark-Lewis I, Eaves A, Eaves CJ (1998) Differentiation stage-specific regulation of primitive human hematopoietic progenitor cycling by exogenous and endogenous inhibitors in an in vivo model. Blood (in press) 43. Sauvageau G, Thorsteinsdottir U, Eaves CJ, Lawrence HJ, Largman C, Lansdorp PM, Humphries RK (1995) Overexpression of HOXB4 in hematopoietic cells causes the selective expansion of more primitive populations in vitro and in vivo. Genes Dev 9:1753 44. Pawliuk R, Eaves C, Humphries RK (1996) Evidence of both ontogeny and transplant dose-regulated expansion of hematopoietic stem cells in vivo. Blood 88:2852