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HEMATOPOIESIS
From The Jackson Laboratory, Bar Harbor, ME;
and the Sidney Kimmel Cancer Center, San Diego, CA.
Bone marrow cells (BMCs) from CXB-12/HiaJ (CXB-12) mice had 14 times the total long-term repopulating ability found in the best of 11 other CXB recombinant inbred (RI) lines. BMCs from each RI line donor
were mixed with genetically marked standard competitor BMCs from the
BALB/cBy×C57BL/6 F1 (CByB6F1) hybrid, the mice used to produce the RI
lines, and the mixtures repopulated lethally irradiated CByB6F1
recipients. Percentages of donor-type erythrocytes and lymphocytes
measured the actual long-term repopulating functions of the donor RI
lines relative to the standard competitor. CXB-12 BMCs repopulated
better after 3 or 6 months than after 1 month, suggesting that the most
primitive precursors were involved. Compared to CByB6F1 standard
competitor cells, CXB-12 cells repopulated 3 to 12 times as well, with
their advantage increasing when higher doses of cells were
transplanted, probably because of hybrid resistance of the recipient
against low doses. This was far better than expected, because F1 cells
normally function 2 to 3 times as well as cells from an inbred strain.
In competitive dilution, the advantage resulted from 2 factors: more
precursor cells and more function per precursor. In the model that best
fit the data, CXB-12 donors had 2.4 times the concentration of
hematopoietic stem cells (HSCs) as the CByB6F1 standard, and each HSC
repopulated 1.4 times as well. CXB-12 mice did not have elevated
erythrocyte and lymphocyte numbers in blood and marrow and did not have
unusually elevated concentrations of colony-forming unit spleen,
cobblestone colonies, and long-term colony-initiating cells in marrow.
(Blood. 2000;96:4124-4131) Hematopoietic stem cells (HSCs) function long term
and proliferate to self-renew and differentiate to produce progenitors of all hematopoietic lineages.1-5 Cells obtained from bone
marrow, cord blood, or mobilized peripheral blood of healthy donors are clinically useful as sources of transplantable HSCs. After
transplantation, donor HSCs compete for engraftment with residual host
HSCs.6 High levels of function from donor HSCs are
extremely valuable for competing effectively with host HSCs in many
clinical conditions.7-10 Unfortunately, donor HSCs are
often damaged by clinical manipulations so that they repopulate less
well than residual host HSCs.11-13 Analysis of the
genetically superior HSCs reported here may suggest how functional
abilities of donor HSCs can be improved.14-17
A broad range of genetic backgrounds and defined genetic combinations
are available in mouse models,18,19 including recombinant inbred (RI) lines.20-22 The set of CXB RI lines used in
the current study were derived from inbred BALB/cBy (BALB) and
C57BL/6By (B6) strains by intercrossing individuals from the F2
generation and then inbreeding for at least 20 generations of
brother-sister mating. All mice of a particular RI line are
genetically identical and homozygous at all loci, with approximately
half the loci derived from B6 and half from BALB.20
The competitive repopulation assay directly measures the ability of a
population of donor HSCs to engraft recipients relative to the
repopulating ability of a population of standard competitor HSCs.23-25 When genetically distinguishable donor and
standard competitor cells are mixed in a specific ratio and injected
into lethally irradiated recipients, the fraction of donor-type blood cells is proportional to the fraction of donor cells injected if
neither type of HSC has a repopulating advantage. If a mixture of such
donor-competitor cells were engrafted in a 2:1 ratio, two thirds of
the blood cells would be donor type. If the observed proportion of
donor-type blood cells is significantly different from the proportion
of donor cells transplanted, a repopulating advantage or disadvantage
in the donor HSCs is detected. The percentage of donor type blood cells
in the recipient circulation is used to calculate total repopulating
units (RUs) in a donor cell population relative to the standard
competitor,25 where 1 donor RU has the same repopulating
ability as 105 standard competitor bone marrow cells (BMCs).
We compared the relative long-term repopulating abilities of 12 CXB RI lines in vivo in the current study, using the CByB6F1 hybrid of
the BALB and B6 parent strains as recipients and standard competitors
because they carry all B6 and BALB alleles. Therefore, they provide the
best system available in which long-term functional ability of HSCs
from multiple CXB RI lines can be studied in a constant environment,
though F1 hybrid resistance in the recipients must be taken into
consideration. The advantage of this technique is the direct
measurement of long-term function in vivo relative to a CByB6F1
standard. BMCs from CXB-12 donors had the highest long-term
repopulating advantage ever reported. The new competitive dilution
assay shows that CXB-12 BMCs have both an increased concentration of
HSCs and an increased functional ability per HSC.
Mice
Bone marrow transplantation and competitive repopulation
CXB RI lines 2, 4, 5, 6, 10, and 12 are Gpi1a/Gpi1a type, whereas CXB RI lines 1, 3, 7, 8, 9, and 11 are Gpi1b/Gpi1b type; each type of donor BMCs was mixed with BMCs from CByB6F1 competitors specially bred to have a different Gpi1 allele. Because recipients were normal CByB6F1 mice, their cells would have been detected easily by their heterozygous Gpi1a/Gpi1b electrophoretic bands; however, this was not found. From blood collected 1, 3, and 6 months after reconstitution by the mixtures of marrow, percentages of donor-derived erythrocytes and lymphocytes were measured after separating Gpi1 types by cellulose acetate gel electrophoresis, as reported earlier.25 Repopulating units Relative repopulating abilities are most accurately compared when cells from each of the different donors are mixed with cells from the same standard competitor pool in the same experiment. Each RU is the relative repopulating ability of 105 fresh marrow cells from the same standard competitor pool. So that independent experiments could be compared, fresh marrow from standard, young congenic CByB6F1 hybrid competitors was used with a variety of donors in each experiment. Numbers of RUs from each donor were calculated from the observed percentage donor cells, where the number of fresh competitor marrow cells used per 105 equalled C. Then by definition, the percentage is 100 (RU/RU + C). By algebraic rearrangement, this gives RU = % (C)/(100 %). Thus, if a donor population contains
20 RUs, it repopulates as well as 20 × 105 standard
competitor marrow cells, and mixtures of 20 donor RUs plus
20 × 105 competitor cells produce an average of 50%
donor-type erythrocytes and lymphocytes in the recipients. Total RUs
per donor were estimated, assuming that the BMCs in both femurs and
tibias were 25% of the total marrow cells in an adult mouse.
Blood and bone marrow composition and colony-forming unit spleen Blood samples were taken from young male and female B6, BALB, CByB6F1, and CXB-12 mice through the orbital sinus. Concentrations of white blood cells (WBCs) and red blood cells (RBCs) were counted using the ZBI Coulter Counter described earlier. Portions of peripheral blood samples were incubated in Gey solution to lyse RBCs and then were stained with B220, CD4, and CD8 antibodies and analyzed through fluorescence activated cell staining (FACS) using a FACScan II (Becton Dickinson, Mountain View, CA) flow cytometer to determine percentages of B220, CD4, and CD8 lymphocytes. BMCs from young B6, BALB, CByB6F1, and CXB-12 mice were also tested for lymphocyte composition through FACS analysis.To measure colony-forming unit spleen (CFU-S), 105 BMCs were injected into each of 4 strain-matched, lethally irradiated recipients. All recipients were killed at day 12. The spleen of each recipient was removed, fixed in Bouin fixative, and examined for the numbers of macroscopic colonies. Short-term production of CFU-S was tested as above, except in lethally irradiated CByB6F1 recipients, using 4 × 105 BMCs from lethally irradiated CByB6F1 carriers given 106 BMCs 7 days earlier or using 2 × 105 BMCs from lethally irradiated CByB6F1 carriers given 106 BMCs 14 days earlier. Competitive dilution using Poisson modeling Portions containing 5 × 104 CXB-12 donor BMCs (Gpi1a/Gpi1a) and 5 × 105 CByB6F1 standard competitor BMCs (Gpi1b/Gpi1b) were injected into each of 38 NK cell-depleted CByB6F1 recipients. The Poisson function, Pi = e N × (Ni/i!), gives the
probability (Pi) that a recipient gets a certain number (i)
of HSCs, where N is the average number of HSCs injected into each
recipient.27,28 The proportion of recipients with 0%
CXB-12-type cells 6 months after reconstitution was used to compute
the number of HSCs (N) in CXB-12 BMCs using the i = 0 term of the
Poisson function: P0 = eN or
n = lnP0. This gave n = 1.2 for
5 × 104 CXB-12 donor BMCs. Then the probabilities
(Pi) of having 0, 1, 2, 3, or 4 donor HSCs in a recipient
were, according to the Poisson function: 0.3012, 0.3614, 0.2169, 0.0867, or 0.0260, respectively. Because 5 × 105
competitor cells were used, n = 5.0 for the competitor, giving probabilities of having 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9 competitor HSCs
in a recipient as: 0.0067, 0.0337, 0.0842, 0.1404, 0.1755, 0.1755, 0.1462, 0.0653, 0.0363, or 0.0181, respectively. In practice, we
calculated 2 arrays of probabilities, one for the donor, and one for
the competitor, with i = 0 to 15 for 16 probabilities in each case.
These form a 16 × 16 matrix of 256 combined probabilities, and each
is associated with a donor:competitor HSC ratio that can be used to
predict a value representing donor contribution. In the current study
we used the highest 37 probability values because there were 37 recipients available 6 months after transplantation.
We used the 37 corresponding donor:competitor ratios in the Poisson modeling to estimate repopulating ability per cell for CXB-12 HSCs by comparing 3 hypothesized levels of repopulating abilities per cell for CXB-12 HSCs: F = 1, F = 1.4, and F = 2, each relative to a repopulating ability per CByB6F1 standard HSC of F = 1. From the value of F, a predicted percentage donor value was calculated for each of the 37 donor:competitor ratios associated with the 37 highest probabilities. This produced 3 sets of predicted donor percentage values, each ranked from low to high and compared to the 37 ranked observed data in a paired t test. The hypothesized F values that generated predictions significantly different from observed data were rejected. The F value whose predictions were not significantly different from the observed data were accepted to represent repopulating ability per cell for CXB-12 donor HSCs relative to those of the CByB6F1 standard. Long-term colony-initiating cells The long-term colony-initiating cell (LTC-IC) assay was performed as described by Müller-Sieburg and Riblet.14 Briefly, bone marrow cells were seeded onto confluent layers of the stromal cell line S17 in 96-well plates. At least 48 wells were seeded per cell dilution, and the dilations covered the range of 1 × 104 to 625 cells/well. Wells that contained colonies of small granulocytic cells were enumerated at the indicated time points, and LTC-IC frequencies were calculated from maximum likelihood statistics. The B6-Ly5 congenic mouse was used because, as described previously, it has the same frequency of LTC-IC as the parental C57BL/6 mouse.Cobblestone area-forming cell assay Procedures for cobblestone area-forming cell assay (CAFC) were adopted as described by Ploemacher et al,29 with a feeder layer from an S17 stroma cell line in 96-well microplates.14,27,30 At confluence, the S17 feeder cells were irradiated with 50 Gy (5000 rads) and overlaid with BMCs from individual donors. Each well contained 0.2 mL cobblestone media (10% fetal calf serum, 10% horse serum, 80% -MEM, 50 IU/mL penicillin,
50 µg/mL streptomycin, 50 µmol/L  -mercaptoethanol, 10 µmol/L
hydrocortisone sodium succinate, and 2 mmol/L L-glutamine). On each
plate, 20 wells were used for each of 4 cell doses (1644, 3780, 8695, or 20 000 donor BMCs), and 16 wells with no cells were
controls.31 All plates were cultured at 33°C with 5%
CO2 and fed weekly by changing half the media in each well.
At 3 and 5 weeks of incubation, wells were scored positive if they had
one or more colonies of at least 6 cells within the stromal layer with
a flat cobblestone shape under a phase-contrast microscope. CAFC
frequencies (N) in the cell dose were calculated from the fractions of
negative wells (P0) where n = lnP0.
Statistical analyses During Poisson modeling, differences between ranked predicted values and ranked observed data were analyzed through paired t test. Strain differences were tested through variance analyses using the JMP statistical discovery software (SAS Institute, Cary, NC) on "Fit Y by X" and "Fit Model" platforms, respectively.32
CXB-12 BMCs repopulate far better than BMCs from 11 other CXB lines HSC function was directly compared in 12 different CXB RI lines (Figure 1) by mixing BMCs from 2 donors of each line with a portion from 1 of 2 pools of standard competitor CByB6F1 marrow. Recipients were NK cell-depleted and lethally irradiated, and each was given 4 × 106 donor BMCs mixed with 2 × 106 CByB6F1 competitor BMCs of the opposite Gpi1 type. After 1, 3, and 6 months, numbers of RUs were calculated as detailed in "Materials and methods." The Gpi1 marker used to distinguish donor and competitor standard cells in competitive repopulation does not affect repopulating abilities25,28,33 and thus could not cause the CXB-12 advantage.
Figure 1 shows an enormous (P < .0001) strain effect as CXB-12 donors repopulated far better than donors of the other 11 CXB RI lines. CXB-12 donors had 14 times more RUs at 6 months, representing the most primitive HSCs, than did CXB-4 donors, the next highest, and 36 times more than the average (764 RUs per donor) for the other 11 RI lines. The repopulating advantage of CXB-12 BMCs increased greatly between 1 month and 3 months. This increase with time did not occur with the other RI lines. The major histocompatibility locus (H2) does not explain why CXB-12 mice have such a high repopulating ability because the CXB-1, 4, 9, and 12 lines are all H2d/H2d, but CXB-1 and CXB-4 repopulate only slightly better than the other CXB RI lines (all H2b/H2b homozygotes), whereas CXB-9 does not repopulate any better than the others. Repopulating advantage of CXB-12 BMCs increases with time and with increasing numbers of BMCs Repopulating abilities of CXB-12 BMCs were tested in a separate study, using 10 × 105 or 30 × 105 CXB-12 BMCs mixed with 100 × 105 standard competitor BMCs from CByB6F1. This study is displayed with 2 independent studies in Table 1. Although some CXB-12 repopulating advantage was present in the short-term HSCs responsible for repopulation after 1 month, by far the strongest advantage occurred in the more primitive HSCs, which repopulate after 3 and 6 months (Table 1). In all cases, the repopulating advantage of CXB-12 BMCs increased greatly between 1 month and 3-6 months after transplantation.
Percentages of CXB-12 type erythrocytes and lymphocytes in recipients after 3 and 6 months were far higher than expected if CXB-12 donor and CByB6 F1 competitor cells repopulated equally (Table 1). However, the advantage of CXB-12 BMCs increased with total numbers of donor cells. It was only 2.4- to 2.8-fold using the lowest cell numbers, 5 × 104 donor cells plus 5 × 105 standard F1 cells. When cell numbers were increased, using 106 donor cells plus 107 standard F1 cells, the CXB-12 advantage increased to 6.4- to 6.6-fold. It further increased to a 12- to 16-fold advantage using 3 × 106 donor plus 107 standard F1 cells (Table 1), probably because some hybrid resistance against CXB-12 cells remained in the recipients and was saturated when higher numbers of CXB-12 BMCs were used. The 11- to 31-fold advantage with 4 × 106 donor plus 2 × 106 standard F1 cells showed the same effect; the wide range resulted from inaccuracies when donor percentages were greater than 90%, so that a difference of a few percentage points greatly affected RU calculations. CXB-12 mice have normal myeloid, lymphoid, and 12-day CFU-S values CXB-12 mice have similar peripheral blood WBC and RBC concentrations in comparison to B6, BALB, and CByB6F1 mice (Table 2). Although there were significant strain differences in peripheral blood B220 and CD4 lymphocyte percentages, total BMC numbers, and marrow percentages of B220 or CD8 cells, in all cases values for CXB-12 mice were similar to those of at least one of the controls (Table 2). Furthermore, day 12 CFU-S from fresh marrow or pre-CFU-S from carriers after 7 or 14 days was not increased in CXB-12 mice (Table 2). Despite high primitive HSC function, regulatory mechanisms in CXB-12 mice maintain blood, marrow, and short-term precursor cell concentrations at normal levels.
Both HSC concentration and repopulating potential per HSC are increased in CXB-12 mice We used competitive dilution to test whether the CXB-12 advantage is caused by increased HSC numbers, functional ability per HSC, or both. Each of 38 CByB6F1 recipients was given 5 × 104 CXB-12 donor BMCs mixed with 5 × 105 CByB6F1 competitor BMCs. Six months after reconstitution, 11 of 37 recipients (1 died after 2 months) had 0% CXB-12 type (Gpi1a/Gpi1a) blood cells, giving a Poisson (n = 1.2) for 5 × 104 CXB-12 donor BMCs or 2.4 HSCs in 105 CXB-12 BMCs (Table 3).
In Figure 2, F values (repopulating
function relative to competitor HSCs) of 1.0, 1.4, and 2.0 in donor
HSCs were compared. Of the 3 sets of predictions, the F = 1.4 model
best fit observed data, whereas models using F = 1 or F = 2
generated predictions significantly below or above the observed data.
Thus, each CXB-12 HSC repopulated 1.4 times as well as each CByB6F1 HSC
(Table 3). Because the dose of CXB-12 BMCs used in this study was very
low, the hybrid resistance that remained in the CByB6F1 recipients probably reduced the CXB-12 HSC functional advantage. Nevertheless, the
concentration of HSCs was 2.4 times that of the F1 hybrid standard, and
the functional ability was 1.4 times the standard.
Repopulating advantage of CXB-12 cells in recipients with full hybrid resistance Hybrid resistance reduced but did not remove the CXB-12 advantage in recipients that were not treated to remove NK cells. Figure 3 shows percentages, and RU values are given in the legend. Compared to the CByB6F1 standard competitor, CXB-12 BMCs had a 2-fold repopulating advantage after 1 month, despite the presence of recipient NK cells. After 3 and 6 months, the advantage averaged 3.9- and 5.4-fold. Although the increase with time was still obvious, the increased repopulation with donor cell numbers was only proportional to their 16-fold increase in donor BMC numbers, from 0.25 to 4.0 million BMCs per recipient, whereas the amount of standard F1 cells was constant at 2.0 million BMCs. There was no longer an increase in donor cell efficiency that would have been shown by an increase in RU concentration. Because the CByB6F1 recipients were not treated to remove hybrid resistance, its effect may have been constant, not saturated by increasing doses of donor cells, as in Table 1.
In separate studies, repopulating abilities of 2 million BALB or B6 donor BMCs in CByB6F1 recipients with intact hybrid resistance were tested with 2 million CByB6F1 competitor standard BMCs. BALB cells contained between 39% and 48% as many RU as found in CByB6F1 cells, approximately 10-fold less than the relative RU concentrations of CXB-12 donors in Figure 3. BMCs from B6 donors contained only 10% to 15% as many RU as found in CByB6F1 cells because of the strong resistance in H-2d/H-2b F1 hybrids against H-2b/H-2b donors. LTC-IC frequency in CXB-12 bone marrow In the long-term LTC-IC assay, the LTC-IC is detected by its ability to repopulate a stromal layer in limiting-dilution cultures by forming colonies of myeloid cells. Concentrations of LTC-ICs in vitro from BMCs of B6, BALB, CXB-12, and CXB-4 BMCs were compared at 4, 5, and 6 weeks (Table 4). As previously reported, B6 values were approximately 10% of those found in BALB mice.14,27,30 The CXB-12 strain had values similar to those of BALB, and the CXB-4 values were intermediate. However, in competitive repopulation, BALB cells repopulate approximately 10 times less well than CXB-12 cells, as noted above. Thus, the CXB-12 advantage is not detected by the LTC-IC assay.
CAFC frequency in CXB-12 bone marrow The ability of B6, BALB, CXB-12, and CByB6F1 BMCs to form long-term CAFC colonies in vitro were compared at 5 weeks using S17 feeder cells in limiting-dilution analysis. Results, given as mean ± SD per 105 BMCs for the following strains, were: B6 = 1.6 ± 0.7; BALB = 1.8 ± 0.8; CXB-12 = 5.3 ± 1.8, and CByB6F1 = 3.6 ± 1.2. CXB-12 donors had significantly higher CAFC frequencies than B6 and BALB donors showing nonoverlapping 95% confidence limits. However, though CXB-12 BMCs had higher CFAC concentrations than CByB6F1 BMCs, the differences were not significant. Thus, the CXB-12 advantage was not detected by the CAFC assay.CXB-12 HSC repopulating abilities in old donors There was a substantial decline with age in HSC function in CXB-12 mice, with old/young RU ratios of 0.21 after 3 months and 0.17 after 6 months (comparing Figures 1 and 4). Still, old CXB-12 BMCs had uniquely high functional ability. Figure 4 shows 24-month-old CXB-12 donors tested in the same experiment presented in Figure 1. When comparing levels of cell production by old CXB-12 donors with average values for young donors in the other 11 CXB RI lines, the old CXB-12 donors still repopulate at least 6 times better. Repopulating abilities of young donors from CXB RI lines 1 to 11 declined relative to the CByB6F1 competitor as the time after transplantation increased from 1 to 6 months, probably because of a repopulating advantage in the F1 hybrid competitor cells. This also occurred for old CXB-12 donors. Nevertheless, even after 6 months, old CXB-12 BMCs still contained approximately twice the repopulating ability of the young CByB6F1 competitors (Figure 4).
Competitive repopulation directly measures how well donor HSCs engraft recipients relative to standard competitor HSCs.23 Among all the assays for HSCs, it best models reconstitution during a therapeutic transplantation in which donor HSCs are delivered into the recipient bloodstream, home to functional sites, and compete with residual host HSCs.6 A population of donor HSCs, with the large repopulating advantage seen in CXB-12 mice (Figure 1), would dramatically enhance the effectiveness of HSC transplantation as a therapeutic procedure. Hybrid effects F1 HSCs probably benefit from heterosis, and even after treatments to remove NK cells, recipients probably still have residual hybrid resistance. These factors explain why BMCs from CXB RI lines 1 to 11 all contain less long-term repopulating ability than BMCs from the CByB6F1 competitor (Figure 1). Hybrid resistance explains why the advantage of CXB-12 BMCs increases with higher doses of cells, even though the competitor cell numbers are increased proportionally (Table 1). The residual resistance is likely saturated by using donor cell doses of 3 to 4 million.26 When NK cells were not removed, hybrid resistance reduced the CXB-12 advantage to approximately 3-5 fold; there was no effect of cell number because the resistance in untreated recipients was not saturated by the donor cell doses used (Figure 3).Individual HSCs The CXB-12 advantage was due to both a 2.4-fold increased HSC concentration and a 1.4-fold proliferative advantage (Figure 2, Table 3). Either increased numbers of HSCs in CXB-12 marrow or increased ability to home to sites supporting long-term HSC function, or both, can explain the increase in HSC concentration. The CXB-12 advantage in this competitive dilution experiment was greatly reduced by residual hybrid resistance because the dose of donor cells was only 50 000 BMCs, and the total repopulating advantage at this dose was approximately 5 times less than at higher doses (Table 1). Hybrid resistance in CByB6F1 recipients reduced BALB HSC concentrations from 10 to 4 per million BMCs27; if hybrid resistance had the same type of effect on CXB-12 HSC, their concentration of 24 per million is several times too low.Donor reactions against competitors The extremely good fit between the F = 1.4 model predictions and the observed data in Figure 2 suggests that there is no killing of standard competitor CByB6F1 HSCs by CXB-12 marrow. The substantial degree of killing required to produce the CXB-12 repopulating advantage would have reduced the observed concentration of competitor HSCs and increased the number of recipients that had no competitor HSC engraftment. This did not occur. In fact, the HSC concentration of 1 per 105 competitor BMCs gave an excellent fit (Figure 2). Furthermore, marrow cells in adult mice from the other 11 CXB RI lines did not repopulate as well as the F1 hybrid competitor (Figure 1). Each of the 12 CXB RI lines tested in the current study is homozygous for either the b or the d allele at the major histocompatibility locus, so each could recognize the alternate allele carried by the CByB6F1 hybrid as foreign. If the repopulating advantage resulted from killing antigenically disparate competitor cells, all the CXB RI lines would have shown this advantage, not just the CXB-12. Finally, mouse marrow has only weak graft-versus-host activity because of low concentrations of T cells; percentages of CD4- and CD8-bearing marrow cells were low in CXB-12 marrow (Table 2), so it would not react significantly against the F1 hybrid cells.Other assays for hematopoietic precursor cells A particularly interesting aspect of CXB-12 marrow is that its long-term repopulating advantage does not cause higher numbers of 12 day CFU-S (Table 2) than found in control inbred or F1 hybrid mice. Apparently, precursor differentiation is regulated to produce normal numbers of CFU-S and normal numbers of myeloid and lymphoid cells (Table 2).Neither CAFC or LTC-IC (Table 4) frequencies are significantly higher in CXB-12 mice than in controls. LTC-IC populations contain precursors capable of long term repopulation in vivo30; perhaps those in CXB-12 mice have an inferior homing ability in vitro, or a superior homing ability in vivo, relative to those in controls. This is not the first case in which LTC-IC and competitive repopulation assays differ. Both here and in prior work,14 LTC-IC concentrations in BALB BMCs were approximately 10-fold higher than those in B6 BMCs. Yet competitive dilution in congenic recipients with congenic competitors gave similar HSC concentrations of approximately 10 per million BMCs in BALB and B6.34,35 Proliferative exhaustion Total RUs per mouse were reduced approximately 5-fold with age in CXB-12 donors (Figures 1, 4). One possibility for this reduction is that increasing age alters HSC homing during transplantation, as in B6 mice.28 Another possibility is that accelerated HSC proliferation in young CXB-12 mice leads to early exhaustion. In the latter case, the functional ability of old CXB-12 HSCs should be lower than the average for old donors from the other 11 CXB RI lines. In fact, old CXB-12 BMCs repopulated better than those of young CByB6F1 competitors or those of young donors from CXB RI lines 1 to 11 (Figure 4). We have reported previously that 9 of 11 other CXB RI lines had low old/young RU ratios.27 The loss with age in CXB-12 is similar to the reduction with age seen in these strains and in the BALB strain, but not in the other 2 CXB RI lines or in the B6 strain.27,35 The high rate of loss with age segregated in the RI lines with the BALB locus at the D12Nyu17 marker on chromosome 12.27Genetic implications Earlier reports, based on LTC-IC and CAFC frequency measurements in vitro, pointed to a few chromosome regions that may contain genes regulating HSC concentration in the mouse.14-16 It will be interesting to test whether the CXB-12 HSC hyperfunction phenotype is related to these chromosome regions. However, it is important to distinguish the phenotypes tested in prior studies from the phenotype in the current report. CXB-12 BMCs show far more effective repopulation and function than young F1 BMCs in F1 recipients for 6 months, approximately a quarter of the murine life span. It is the unprecedented degree of engraftment advantage relative to high doses of healthy competitor BMC that makes the CXB-12 hyper-HSC phenotype novel.The phenotypes in Figure 1 were tested in a genome-wide QTL mapping analyses using the Map Manager QTX-06 program with the CXB RI mice genotype data, both available online (http://www.jax.org). This analysis did not produce any linkage that is considered significant (lod score, greater than 3.0). Only insignificant linkages to D6Mit15 (lod score, 0.8) and D17Mit22 (lod score, 1.0) were found, probably by random chance, because more than 500 markers were tested. The CXB-12 hyper-HSC phenotype is not controlled by a single Mendelian locus differing between the BALB or the B6 parent strains because the hyper phenotype does not occur in either. It is found in only 1 of the 12 RI lines, so the simplest genetic explanations are either a new mutation or a unique combination of BALB and B6 genes. Testing HSC functional phenotypes in hybrid and backcross mice between CXB-12 and its parental strains will distinguish these 2 possibilities. Based on preliminary data, it could be a codominant mutation or a combination in which a single locus has a significant effect in approximately 40% of the backcross mice, though the effect is reduced from that in the CXB-12. Future studies using B6-H-2d × BALB F1 hybrid recipients and competitors to remove the strong hybrid resistance associated with a difference at the major histocompatibility locus may give clearer results. If high stem cell function results from a unique combination of 2 alleles, at least 1 must be recessive Primitive precursors are affected Regulation of the hyper-HSC phenotype appears to be focused on more primitive HSCs because CXB-12 mice have normal numbers of myeloid cells, lymphoid cells, and less primitive precursors (Tables 2, 4). In competitive repopulation, the phenotype is expressed most strongly after 3 and 6 months in the recipients (Table 1; Figures 1, 3), which is the time required for primitive HSCs to repopulate. The high number and function of CXB-12 primitive HSCs may be maintained by alterations in a receptor that regulates primitive HSCs or in its ligand. Increases in numbers of HSCs could result from changes in either ligand or receptor; however, the increased proliferative capacity of CXB-12 HSCs after transplantation in normal recipients suggests that the change is intrinsic to the HSCs, perhaps an altered receptor. Understanding the CXB-12 phenotype may eventually lead to improved clinical transplantation procedures to enhance the effectiveness of primitive HSCs.
We thank Avis Silva, Bee Stork, and Karen Davis for their excellent technical assistance. We thank Dr Ben Taylor for helpful discussions about RI line analyses and Dean Campagna, an academic year student, and Benjamin H. Schmidt, a summer student, for performing the CAFC assays.
Submitted February 28, 2000; accepted August 15, 2000.
Supported by National Institutes of Health grants HL-58820, HL-58705, and DK-48015. J.C. was an NRSA postdoctoral fellow and was supported by grant AG-05754 from the National Institute on Aging.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: David E. Harrison, The Jackson Laboratory, Campus Box 41, 600 Main St, Bar Harbor, ME 04609; e-mail: deh{at}jax.org.
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© 2000 by The American Society of Hematology.
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E. Henckaerts, J. C. Langer, and H.-W. Snoeck Quantitative genetic variation in the hematopoietic stem cell and progenitor cell compartment and in lifespan are closely linked at multiple loci in BXD recombinant inbred mice Blood, July 15, 2004; 104(2): 374 - 379. [Abstract] [Full Text] [PDF]
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E. Henckaerts, H. Geiger, J. C. Langer, P. Rebollo, G. Van Zant, and H.-W. Snoeck Genetically determined variation in the number of phenotypically defined hematopoietic progenitor and stem cells and in their response to early-acting cytokines Blood, May 13, 2002; 99(11): 3947 - 3954. [Abstract] [Full Text] [PDF]
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H. Geiger, J. M. True, G. de Haan, and G. Van Zant Age- and stage-specific regulation patterns in the hematopoietic stem cell hierarchy Blood, November 15, 2001; 98(10): 2966 - 2972. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
). We
hypothesized 3 levels of repopulating potential
"F" for
each CXB-12 donor HSC relative to F = 1 for each CByB6F1 competitor
HSC; F = 1 (
), F = 2 (
), and F = 1.4 (
)
),
3 (
), and 6 (
) months after reconstitution, percentage donor
erythrocytes and percentage donor lymphocytes were averaged for each
recipient and presented as means with standard error bars. If there
were no repopulating advantage in CXB-12 HSCs, the expected percentages
from CXB-12 donors were 11%, 33%, 50%, and 68% for the 4 donor:competitor ratios, respectively. The differences between observed
and expected CXB-12 contributions translate to 2.3-, 1.6-, 1.8-, and
2.4- (average, 2.0)-fold CXB-12 HSC repopulating advantages after 1 month; 4.2-, 2.9-, 4.0-, and 4.4- (average, 3.9)-fold advantages after
3 months; and 6.6-, 3.2-, 5.3-, and 6.7- (average, 5.4)-fold advantages
after 6 months.
