By
From the Institut National de la Santé et de la Recherche Médicale U 362, Institut Gustave Roussy, 94800 Villejuif, France
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Transplantation of genetically marked donor cells in mice have unambiguously identified individual clones with full differentiative potential in all lymphoid and myeloid pathways. Such evidence has been lacking in humans because of limitations inherent to clonal stem cell assays. In
this work, we used single cell cultures to show that human cord blood (CB) contains totipotent
CD34+ cells capable of T, B, natural killer, and granulocytic cell differentiation. Single CD34+
CD19Thy1+ (or CD38
) cells from fresh CB were first induced to proliferate and their progeny separately studied in mouse fetal thymic organotypic cultures (FTOCs) and cocultures on
murine stromal feeder layers. 10% of the clones individually analyzed produced CD19+, CD56+,
and CD15+ cells in stromal cocultures and CD4+CD8+ T cells in FTOCs, identifying totipotent
progenitor cells. Furthermore, we showed that totipotent clones with similar lymphomyeloid
potential are detected in the bone marrow of nonobese diabetic severe combined immunodeficient (NOD-SCID) mice transplanted 4 mo earlier with human CB CD34+ cells. These results
provide the first direct demonstration that human CB contains totipotent lymphomyeloid progenitors and transplantable CD34+ cells with the ability to reconstitute, in the marrow of recipient mice, the hierarchy of hematopoietic compartments, including a compartment of functional totipotent cells. These experimental approaches can now be exploited to analyze
mechanisms controlling the decisions of such primitive human progenitors and to design conditions for their ampification that can be helpful for therapeutic purposes.
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Syngeneic transplantation approaches using genetically marked stem cells (1, 2) or limited numbers of them (3, 4) have led to the identification of murine progenitor cells with multiple differentiative potentials. These strategies have been exploited to analyze the contribution of stem cell populations to short- and long-term hematopoietic reconstitution (5) and define conditions for their amplification (or self-renewal) (6). Both issues have an obvious clinical relevance, but experimental strategies used in mice cannot be easily applied to human cells (7). Although engraftment is the rule after injection of human cells into immunodeficient mice (8), there is no definite proof as yet that the entity defined as nonobese diabetic (NOD)1-SCID-repopulating cells (13, 14) is a reliable indicator of long-term reconstituting human stem cell function. One important limitation both in vitro and in vivo has been the difficulty in obtaining efficient lymphoid differentiation from primitive human progenitors. Thus, in NOD-SCID recipients, lymphoid differentiation is restricted to the development of the B cell lineage (8, 9, 12), and mature T or NK cells are produced very rarely or not at all (9). It is presently unknown whether this defect is accounted for by the fact that regulatory controls do not operate in these chimeras or because engrafted human CD34+ cells have an impaired T and NK potential. In the case of T cells, differentiation can be rescued by the coimplantation of human thymic tissue (15), and data in favor of a migration defect have recently been provided (16). In contrast to the chimeric in vivo situation, high numbers of functional, mature NK and dendritic cells can be obtained in vitro from various cell sources if appropriate cytokines are provided (17, 18). Human B cell differentiation requires stromal feeders, and, unexpectedly, decisive progress in the production of CD19+ B cells from human primitive progenitors has resulted from the use of murine (as opposed to human) stromal cells (19, 20). The T cell potential of primitive CD34+ human cells can also be successfully identified in fetal thymic organotypic cultures (FTOCs) using embryonic thymic lobes from both normal (21) or immunodeficient mice (including NOD-SCID mice) (22). However, even though FTOC conditions can be manipulated to obtain B, NK, and myeloid cells in addition to T cells, at least in mice (25, 26), cocultures of human CD34+ populations on stromal feeders, even if they are derived from the thymus, do not reproducibly allow T cell differentiation (27), and the identification of individual cells with all possible lymphoid and myeloid potentials still requires the use of separate assays (28).
In this study, we show for the first time that in vitro
conditions exist that allow investigators to reproducibly assess all lymphoid (T, B, NK) and some myeloid potentials
from single CD34+CD19Thy1+ human cord blood (CB)
cells. The strategy first requires induction of cell proliferation with cytokines to separately assess T cell potential in
FTOCs and NK cell, B cell, and myeloid potentials in a
unique coculture assay using MS5 murine stromal feeder
layers. We also detected totipotent cells in the marrow of
NOD-SCID recipient mice 4 mo after their transplantation
with CD34+ CB cells. Taken together, our data demonstrate that human CB contains totipotent cells and in vivo-
transplantable cells with similar lymphomyeloid differentiative potentials. This strongly suggests that cells exist in CB
that have some characteristics of true stem cells.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Collection and Fractionation of Cells.
CB samples were collected with the informed consent of the mothers involved in our study. Low density CB mononuclear cells were first subjected to a standard CD34 immunomagnetic bead separation using the miniMACS® system (Miltenyi Biotec). Bead-separated CD34+ cells (purity >75%) were either injected into NOD-SCID mice (105 cells/mouse) or further fractionated in CD34+CD19
|
Assessment of Human T Cell Potential in FTOC.
Isolation of murine embryonic thymic lobes, incubation with human cells after the hanging drop procedure, and organotypic cultures were performed following standard procedures initially described to analyze mouse T lymphoid differentiation and adapted to the identification of human T cell potential (22, 29). Our slight modifications of the technique included the use of NOD-SCID embryonic (day 14) thymic lobes and the addition of recombinant human (rhu) IL-2 (5 ng/ml; Diaclone), 20 ng/ml rhu-IL-7 (Diaclone), and 50 ng/ml rhu-stem cell factor (SCF; provided by Amgen) during the hanging drop procedure. Thymic lobes were incubated onto floating filters (Isopore membrane, 25-mm diameter, 8-µm pore size; Millipore SA) at 37°C and 5% CO2 in medium without cytokines. Cells recovered from the thymic lobes after 28-35 d were labeled with anti-human CD4-PE and CD8-FITC (Becton Dickinson), TCR-Simultaneous Assessment of B, NK, and Granulomonocytic (Gr/M) Differentiation.
The different cell fractions were incubated in 24- or 96-well plates precoated with confluent murine marrow-derived MS5 cells (30) in RPMI supplemented with 10% human serum, 5% FCS, and the following six rhu cytokines: rhu-SCF (50 ng/ml; Amgen), rhu-Flt3-ligand (50 ng/ml; Diaclone), pegylated (PEG)- rhu-megakaryocyte growth and differentiation factor (MGDF) (50 ng/ml; Amgen), rhu-IL-2 (5 ng/ml; Diaclone), rhu-IL-15 (10 ng/ ml; Diaclone), and rhu-IL-7 (20 ng/ml; Diaclone). Wells with significant cell proliferation (usually >500 cells after 3-6 wk) were selected, and cells were collected and their phenotype assessed by flow cytometry after labeling with the following mAbs: CD19-PE (Becton Dickinson), CD15-FITC (Dako Corp.), and CD56-PE-Cy5, CD34-PE-Cy5, CD1a-PE, and HLA-DR-FITC (all from Immunotech). In some experiments, the intracellular expression of myeloperoxidase (MPO; Caltag Labs.) was determined using a cell permeabilization kit (Harlan, Sera-Lab Ltd.). Analysis was performed on a FACScanTM (Becton Dickinson) using CellQuest software.Analysis of the Lymphoid and Gr/M Potentials of Single Progenitors.
Individual CD34+CD19 ![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Because primitive lymphomyeloid cells most likely represent only a minor fraction of human CD34+ cells collected
from both fresh CB and NOD-SCID marrow, we performed an enrichment step. We first removed CD34+
CD19+ committed pre-B cells, which represented 13 ± 8% (n = 6) of fresh CB cells (Fig. 1 A) but 83 ± 6% (n = 11) of NOD-SCID-derived CD34+ cells (Fig. 1 C) (12).
CD34+CD19 cells were further fractionated into Thy1+
or CD38
cells, as both cell types exhibit primitive functions. Thy1+ cells represented 10 ± 4% (n = 5) of fresh
CD34+CD19
CB cells (Fig. 1 B), whereas Thy1+ (Fig.
1 D) and CD38
(Fig. 1 E) cells accounted only for 2.5 ± 1.8% and 2.3 ± 0.6% of CD34+CD19
cells from NOD-SCID mice (n = 5), respectively.
We have previously shown that murine MS5 stromal cells support myeloid (30) and B cell differentiation (20) from CD34+ CB cells when studied in separate assays but also from single bipotent B/myeloid progenitors in a switch system (20). Because MS5 cells also promote the production of NK cells from CD34+ cells in the presence of human IL-15 (31), we reasoned that slight modifications of these culture conditions should allow the simultaneous development of all three lineages (B, NK, and Gr/M). To this end, we supplemented the medium with rhu-SCF, IL-2, IL-15 (three-cytokine condition), and, in later experiments, PEG-rhu-MGDF, Flt3-ligand, and IL-7 were also added (six-cytokine condition). PEG-rhuMGDF and Flt3-ligand were chosen to trigger active proliferation of immature progenitors that may not respond to lineage-specific cytokines (32), and IL-7 was chosen to minimize the loss of B and/or T cell potential (33).
Thus, CD34+CD19 and CD34+CD19
Thy1+ fresh
CB cells (2-10,000 cells/well), when cocultured on MS5
cell feeder layers in three- or six-cytokine conditions, reproducibly generated CD19+ B, CD56+ NK, and CD15+/
MPO+ Gr/M cells in 2-3 wk (Table I). In the presence of
six cytokines, up to 70% of cells recovered after 2 wk of culture were still CD34+ (not shown). Table I and Fig. 2 A-C
also show that CD34+CD19
cells collected from the marrow of NOD-SCID mice (n = 4) and cultured for 3 wk in
the presence of six cytokines generated 3.1 ± 0.7% CD19+
B cells, 9.5 ± 4.1% CD56+ NK cells, and 22 ± 13% CD15+
Gr/M cells (mean ± SEM of nucleated cells). Interestingly,
all three cell types also exhibited almost nonoverlapping forward scatter versus side scatter profiles. Similar differentiation profiles and lineage yields were observed in three experiments initiated with CD34+CD19
CD38
cells (1 ± 0.4, 44 ± 3, and 28 ± 5% B, NK, and Gr/M cells, respectively) and CD34+CD19
Thy1+ cells (6.4 ± 6, 47 ± 9, and 20 ± 5% B, NK, and Gr/M cells, respectively) (Table I).
Cells coexpressing CD1a and HLA-DR, most likely of dendritic origin, were detected in a few experiments in
which these markers were analyzed (data not shown). As previously described, cultured CD19+ cells coexpressed CD38
and CD10 but lacked CD20, CD22, and sIgM, whereas NK
cells were mature and functional, as shown by their spontaneous cytotoxic activity against K562 and Daudi cells (reference 31; data not shown).
|
|
The culture conditions described above were not permissive to T cell
differentiation, which requires the presence of thymic stromal elements. We have recently reported the successful use of NOD-SCID embryonic thymus to identify T cell potential of CB and adult marrow CD34+, CD34+CD38+,
and CD34+CD38 cells (24). Here, we applied this strategy to demonstrate that human CD34+CD19
cells selected
from the bone marrow of NOD-SCID mice generated CD4bright and double positive (DP) CD4+CD8+ T cells in
NOD-SCID FTOC. As illustrated in Fig. 2 B-G, in 5/5 experiments, human CD4+ cells (which represented 69 ± 13%
of nucleated cells collected from thymi) were produced, and
82 ± 5% of those were CD4bright, 46 ± 12% were DP
CD4+CD8+, 31 ± 5% were CD4+TCR-
/
+ (n = 3), and
32 ± 7% were CD4+CD3+ (n = 3). As described for fresh
CB cells, very few CD4
CD8+ cells were produced (22).
Taken together, these experiments demonstrated that
CD34+CD19 human cells detected in NOD-SCID recipients 4 mo after intravenous injection of CD34+ CB
cells had retained the ability to differentiate into B, NK, and Gr/M lineages as well as T cells in vitro, even though
T and NK terminal maturation did not occur in vivo.
Before investigating the full lymphomyeloid differentiative potential of single cells, we first determined the frequency of single cells that produce high numbers of B, NK, and Gr/M cells when cocultured on MS5 cells with six cytokines.
In two experiments, 180 single CD34+CD19 cells from
fresh CB were cultured in the presence of six cytokines and
MS5 cell feeder layers. Cells from 55 wells were phenotyped and found to contain multiple combinations of B,
NK, and Gr/M cells (Table II). Nine clones (16%) contained cells from all three lineages, and 54% of the clones
yielded cells of only one lineage. When CD34+CD19
Thy1+ cells were used, the frequency of the three-lineage
clones increased to 28% (28 clones analyzed) and the frequency of one-lineage clones decreased (21%), in agreement with the preferential expression of Thy1 by immature progenitors. Lineages were similarly distributed in
clones grown from single CD34+CD19
Thy1+ or CD38
cells isolated from the marrow of transplanted NOD-SCID
mice. Analysis, for instance, of cells from 240 wells, each
seeded with a single CD34+CD19
Thy1+ (120 wells) or
CD34+CD19
CD38
(120 wells) cell, indicated that >50%
of the clones contained cells of two lineages and 15-33% cells
of three lineages but only 13-23% cells of one lineage (Table
II). In contrast and as noted above, 69% of the more mature
CD34+CD19
cells generated cells of only one lineage.
|
Importantly, individual clones grown as described above
in six cytokines on stromal feeders contained high numbers
of nucleated cells (from 10,000 to 400,000 total cells per
well at week 2-3), which made it possible to remove part
of the clone to separately assay T cell differentiation in
FTOC. Thus, in a second set of experiments, multiple
wells were seeded with CD34+CD19Thy1+ from fresh
CB (420 wells) and with CD34+CD19
Thy1+ and CD34+
CD19
CD38
cells from the marrow of transplanted NOD-SCID mice (each population, 240 wells). After 2-3 wk,
clones that had proliferated (150, 76, and 92, respectively)
were divided to initiate NK-B-Gr/M and FTOC assays
(Table III).
|
The most remarkable result was that 10/150 clones
grown from fresh CD34+CD19Thy1+ CB cells and 14/
68 clones grown from human CD34+CD19
Thy1+ (or
CD38
) cells from NOD-SCID chimeras generated not
only B, NK, and Gr/M cells but also T cells (CD4bright cells
usually associated with DP CD4+CD8+ cells) in FTOC assays (Table III). As illustrated in Fig. 3, the proportions of
lymphoid (CD19+, CD56+) and Gr/M (CD15+) cells varied between clones. However, the distribution of clones containing one, two, three, or four lineages in experiments
initiated with either fresh CB cells or cells from NOD-SCID mice was almost identical (Table III). Interestingly,
the distribution of lineages was not strictly identical in
clones grown from CD34+CD19
Thy1+ and CD34+
CD19
CD38
NOD-SCID-derived human cells. The biological significance of this observation is unclear, and this
result must be confirmed in experiments initiated with
fresh CB cells of both phenotypes. In addition to totipotent
clones, multiple combinations of lineages were detected:
30% of the clones (45/150 wells seeded with CD34+
CD19
Thy1+ fresh CB cells and 27/92 wells seeded with
CD34+CD19
CD38
cells from transplanted NOD-SCID
mice) generated combinations of three lineages. Among
these three-lineage clones, 30% included T cells, and 4%
combined B, NK, and T cells but no CD15+ cells and were
most likely derived from lymphoid-restricted progenitors (Fig. 4 A). None of the clones contained B cells alone and
<2% exclusively T cells (Table III). Both T (52/54) and B
(151/154) cells were almost always combined with NK
cells. This was the predominant phenotype in our culture
conditions: 128/150 clones from fresh CB and 145/168
clones from marrow of transplanted NOD-SCID mice
contained human NK cells.
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In this study we provide, for the first time in humans,
direct evidence that a significant proportion of CD34+
CD19Thy1+ (and/or CD38
) fresh CB cells are totipotent and that similar totipotent cells can be isolated from the
marrow of NOD-SCID mice transplanted several months
earlier with fresh human CD34+ CB cells. The ability of a
single cell to generate T, B, and NK lymphocytes and Gr/M
cells defines totipotency and was demonstrated in vitro by
combining FTOCs and cocultures on competent murine stromal cell feeder layers.
A prerequisite for the success of these experiments was to first overcome previous limitations in the assay system for B and T lymphoid potential of human cells in vitro, and a decisive step was to use the murine stromal cell line MS5 (34). Feeder layers from MS5 and other cell stromal lines (17, 19, 35), in contrast to those from human stromal cells, support the differentiation of CD34+ cells into pro-B cells; although spontaneous terminal B cell maturation remains compromised, probably because T helper function is lacking, it can be induced (36). However, despite recent suggestions that extra-thymic differentiation may occur in the marrow environment (37) or in the gut and the unique finding of CD3+ human cells in the marrow of bgnuXid mice engrafted with CD34+ marrow cells (7), human T cells never developed in vitro in MS5 cocultures. The presence of an intact thymic structure remains essential, although recent results indicate that thymic stromal feeders can replace the intact thymus (27, 38). The observation that cultures of embryonic thymic lobes, initially used to follow mouse T cell development, could be successfully applied to the study of human T cell differentiation represents an improvement over the use of human embryonic thymus. Thus, we (24) and others (22, 23) recently described that FTOC initiated with thymic lobes from immunodeficient mice efficiently supported the first steps of human T cell differentiation up to the DP CD4+CD8+ stage and are sensitive enough to detect the production of CD4+CD8+ T cells from as few as 100 CD34+ CB cells (24).
To prove cell totipotency, it was essential to work at the
clonal level, which imposed the need to initiate FTOC and
cocultures on MS5 feeders with cells issued from the same
clone. This required induction of the proliferation of input
single cells to allow subdivision of the clone without loss of
potentials. This was accomplished by adding early-acting
proliferative cytokines, including IL-7, to avoid the loss of
early T cell progenitors (33) and by plating the cells directly
on MS5 cells, because we have shown that these stromal
cells are important to retain the primitive potential of actively proliferating adult CD34+CD38 marrow long-term
culture-initiating cells (39).
Our results showed that 6.7% of CD34+CD19Thy1+
CB cells were totipotent, which leads to an estimate of one
totipotent progenitor per 200 CD34+ CB cells. This number is surprisingly high and may even be underestimated, as
we selected only actively proliferating clones and also because the hanging drop procedure that we used may limit
the homing of T cell progenitors into thymic lobes. Although it is hazardous to draw any firm conclusion about
stem cell hierarchy based on the distribution and combination of lineages observed in single clones grown in vitro,
both were very similar to those reported from the analysis
of lymphomyeloid clones grown in vitro from murine fetal
liver (26) or embryonic splanchnopleura (40) and in vivo
from recipients of genetically marked donor cells (1). Particularly interesting was the observation in our study that
some clones, derived from fresh CB cells and the marrow
of transplanted NOD-SCID mice, produced T, B, and NK
but no Gr/M cells. Although we cannot exclude that other
conditions could have unmasked additional myeloid potentials, erythroid or megakaryocytic, the total lack of CD15+
cells makes this hypothesis very unlikely. The parental progenitors most probably represented the human counterpart
of the murine lymphoid-restricted progenitors identified
among IL-7R
+c-kit+Lin
Sca+ adult mouse marrow cells
(28). As reported in the mouse (26), we also failed to detect
bipotent T/B progenitors, an observation that contrasted
with the high frequency, in our study, of clones with both
NK and B cells. It is important to stress that NK cells were
the most frequent cell type in our cultures and that their outgrowth might have interfered (in a positive or negative
way) with the development/survival of other lineages. Indeed, there were very few if any clones containing only B
or T cells. A possible explanation for the lack of B cell
clones was the lack of signals released by T cells, which was
not compensated for by the action of MS5 cells.
A second major observation was that human totipotent cells were detected in the marrow of NOD-SCID recipients several months after transplantation of CD34+ CB cells. Tracing individual stem cell fate and function in vivo is complicated in the NOD-SCID chimeras, as opposed to the syngenic murine situation, as terminal T and NK cell differentiation, in contrast to B cell differentiation, does not take place in vivo. However, human CD34+ cells isolated from NOD-SCID chimeras reproducibly produced CD4+ CD8+ T cells and NK cells in vitro, indicating that human cells have not lost their intrinsic T and NK potential after their engraftment in the NOD-SCID environment. A more likely hypothesis is that regulatory steps involved in the development of T and NK pathways do not take place in this xenogenic model. For T cells, the defect most likely involves molecules regulating cell trafficking, whereas the maturation of human NK cells was blocked because human cells do not respond to murine IL-2 and IL-15.
A close frequency of totipotent clones (7%) was found in
experiments initiated with fresh CD34+CD19 Thy1+ CB
cells and CD34+CD19
CD38
cells from transplanted
NOD-SCID mice (14%). Calculations yielded an absolute
number of ~50 totipotent cells in four long bones of a
NOD-SCID 4-mo posttransplant. Considering that 500 totipotent cells were present among the 105 CD34+ cells
injected, this suggests loss rather than amplification of totipotent cells. However, totipotent clones may in fact actively proliferate in vivo. A strong argument in favor of this
hypothesis is that, in our experience as well as in others' (8),
<1% of the injected CD34+ human cells are detected in the
marrow of NOD-SCID mice 48 h after their injection
(range 0.1 to 4% in six experiments; our unpublished data).
Therefore, it is conceivable that <10 totipotent cells eventually colonize the marrow of NOD-SCID mice and contribute to the reconstitution of all compartments, a hypothesis
that is in agreement with murine studies (1, 4, 41, 42). Definite determination of the number of human clones contributing to the reconstitution of hematopoiesis in NOD-SCID
mice will await results from transplantation experiments using genetically marked human cells. These studies will be facilitated by the availability of conditions that allow the expression of the full differentiative potentials of individual
clones, as demonstrated in this study, but also by improved
procedures that will allow efficient transduction of NOD-SCID-CRU competitive repopulating unit (43, 44).
![]() |
Footnotes |
---|
Address correspondence to Laure Coulombel, INSERM U 362, Institut Gustave Roussy, 39 Av Camille Desmoulins, 94800 Villejuif, France. Phone: 33-1-42-11-42-33; Fax: 33-1-42-11-52-40; E-mail: laurec{at}igr.fr
Received for publication 11 December 1998 and in revised form 9 March 1999.
C. Robin and F. Pflumio contributed equally to this work.We thank Annie Rouches and Patrice Ardouin for their invaluable contribution to the breeding and care of the NOD-SCID mice; Brigitte Izac for her excellent technical assistance; Philippe Rameau and André Katz for cell sorting; John Dick for providing the original NOD-SCID breeding pairs and for his constant support; Marina Cavazzana-Calvo, Jean Plum, and Magda de Smedt for their advice on the initial device of the FTOC system; and Anne-Lise Bennaceur-Griscelli and Cristina Tourino for stimulating discussions. We also thank Amgen-USA and Amgen-France for their gift of PEG-rhu-MGDF and rhu-SCF.
This work was supported by grants from Institut National de la Santé et de la Recherche Médicale (INSERM), Electricité de France, Ligue Nationale Contre le Cancer, Association de la Recherche contre le Cancer (ARC 6532 to L. Coulombel), Institut Gustave Roussy, and Ministère de la Recherche et de la Technologie (to L. Coulombel). C. Robin was funded by fellowships from the French Ministère de la Recherche et de la Technologie and French Society of Hematology.
Abbreviations used in this paper CB, cord blood; DP, double positive; FTOCs, fetal thymic organotypic cultures; Gr/M, granulomonocytic; MGDF, megakaryocyte growth and differentiation factor; NOD, nonobese diabetic; PEG, pegylated; rhu, recombinant human; SCF, stem cell factor.
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Jordan, C.T., and I.R. Lemischka. 1990. Clonal and systemic analysis of long-term hematopoiesis in the mouse. Genes Dev. 4: 220-232 [Abstract]. |
2. | Keller, G., and R. Snodgrass. 1990. Life span of multipotential hematopoietic stem cells in vivo. J. Exp. Med. 171: 1407-1418 [Abstract]. |
3. | Osawa, M., K.I. Hanada, H. Hamada, and H. Nakauchi. 1996. Long-term lymphohematopoietic reconstitution by a single CD34-low/negative hematopoietic stem cell. Science. 273: 242-245 [Abstract]. |
4. | Szilvassy, S.J., R.K. Humphries, P.M. Lansdorp, A.C. Eaves, and C.J. Eaves. 1990. Quantitative assay for totipotent reconstituting hematopoietic stem cells by a competitive repopulation strategy. Proc. Natl. Acad. Sci. USA. 87: 8736-8740 [Abstract]. |
5. |
Zijlmans, J.M.,
J.W. Visser,
L. Laterveer,
K. Kleiverda,
D.P. Heemskerk,
P.M. Kluin,
R. Willemze, and
W.E. Fibbe.
1998.
The early phase of engraftment after murine blood cell
transplantation is mediated by hematopoietic stem cells.
Proc.
Natl. Acad. Sci. USA.
95:
725-729
|
6. |
Miller, C.L., and
C.J. Eaves.
1997.
Expansion in vitro of
adult murine hematopoietic stem cells with transplantable
lympho-myeloid reconstituting ability.
Proc. Natl. Acad. Sci.
USA.
94:
13648-13653
|
7. |
Nolta, J.A.,
M.A. Dao,
S. Wells,
E.M. Smorgorzewska, and
D.B. Kohn.
1996.
Transduction of pluripotent human hematopoietic stem cells demonstrated by clonal analysis after
engraftment in immune-deficient mice.
Proc. Natl. Acad. Sci.
USA.
93:
2414-2419
|
8. | Cashman, J., K. Bockhold, D.E. Hogge, A.C. Eaves, and C.J. Eaves. 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: 1026-1036 [Medline]. |
9. |
Hogan, C.J.,
E.J. Shpall,
O. McNulty,
I. McNiece,
J.E. Dick,
L.D. Shultz, and
G. Keller.
1997.
Engraftment and development of human CD34(+)-enriched cells from umbilical cord
blood in NOD/LtSz-scid/scid mice.
Blood.
90:
85-96
|
10. | Lapidot, T., F. Pflumio, M. Doedens, B. Murdoch, D.E. Williams, and J.E. Dick. 1992. Cytokine stimulation of multilineage hematopoiesis from immature human cells engrafted in SCID mice. Science. 255: 1137-1141 [Medline]. |
11. |
Nolta, J.,
M. Hanley, and
D. Kohn.
1994.
Sustained human
hematopoiesis in immunodeficient mice by cotransplantation
of marrow stroma expressing human interleukin-3: analysis of
gene transduction of long-lived progenitors.
Blood.
83:
3041-3051
|
12. |
Pflumio, F.,
B. Izac,
A. Katz,
L.D. Shultz,
W. Vainchenker, and
L. Coulombel.
1996.
Phenotype and function of human
hematopoietic cells engrafting immune-deficient CB17-
severe combined immunodeficiency mice and nonobese diabetic-severe combined immunodeficiency mice after transplantation of human cord blood mononuclear cells.
Blood.
88:
3731-3740
|
13. |
Conneally, E.,
J. Cashman,
A. Petzer, and
C. Eaves.
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. USA.
94:
9836-9841
|
14. |
Wang, J.C.,
M. Doedens, and
J. Dick.
1997.
Primitive human hematopoietic cells are enriched in cord blood compared with adult bone marrow or mobilized peripheral blood
as measured by the quantitative in vivo SCID-repopulating
cell assay.
Blood.
89:
3919-3924
|
15. |
Yurasov, S.,
T.R. Kollman,
A. Kim,
C.A. Raker,
M. Hachamovitch,
F. Wong-Staal, and
H. Goldstein.
1997.
Severe
combined immunodeficiency mice engrafted with human T
cells, B cells and myeloid cells after transplantation with human fetal bone marrow or liver cells and implanted with human fetal thymus: a model for studying human gene therapy.
Blood.
89:
1800-1810
|
16. |
van der Loo, J.C.,
H. Hanenberg,
R.J. Cooper,
F.Y. Luo,
E.N. Lazaridis, and
D.A. Williams.
1998.
Nonobese diabetic/severe combined immunodeficiency (NOD/SCID)
mouse as a model system to study the engraftment and mobilization of human peripheral blood stem cells.
Blood.
92:
2556-2570
|
17. | Galy, A., M. Travis, D. Cen, and B. Chen. 1995. Human T, B, natural killer and dendritic cells arise from a common bone marrow progenitor cell subset. Immunity. 3: 459-473 [Medline]. |
18. |
Mrozek, E.,
P. Anderson, and
M. Caligiuri.
1996.
Role of
interleukin 15 in the development of human CD56+ natural
killer cells from CD34+ hematopoietic progenitor cells.
Blood.
87:
2632-2640
|
19. |
Rawlings, D.J.,
S. Quan,
Q.L. Hao,
F.T. Thiemann,
M. Smogorzewska,
O.N. Witte, and
G.M. Crooks.
1997.
Differentiation of human CD34+CD38![]() |
20. |
Berardi, A.C.,
E. Meffre,
F. Pflumio,
A. Katz,
W. Vainchenker,
C. Schiff, and
L. Coulombel.
1997.
Individual CD34+
CD38lowCD19![]() ![]() |
21. |
Merkenschlager, M., and
A. Fisher.
1992.
Human postnatal thymocytes generate phenotypically immature CD3dim,
CD5dim, CD1abright progeny in organ culture.
J. Immunol.
148:
1012-1015
|
22. |
Plum, J.,
M. De Smedt,
M.-P. Defresne,
G. Leclercq, and
B. Vandekerckhove.
1994.
Human CD34+ fetal liver stem cells
differentiate to T cells in a mouse thymic microenvironment.
Blood.
84:
1587-1593
|
23. |
Verhasselt, B.,
M. De Smedt,
R. Verhelst,
E. Naessens, and
J. Plum.
1998.
Retrovirally transduced CD34++ human cord
blood cells generate T cells expressing high levels of the retroviral encoded green fluorescent protein marker in vitro.
Blood.
91:
431-440
|
24. | Robin, C., A. Bennaceur-Griscelli, F. Louache, W. Vainchenker, and L. Coulombel. 1999. Identification of T lymphoid progenitor cells in CD34+CD38low and CD34+ CD38+ subsets of human cord blood and bone marrow cells using NOD-SCID fetal thymus organ cultures. Br. J. Haematol. 104: 809-819 [Medline]. |
25. | Barcena, A., A. Galy, J. Punnonen, M. Muench, D. Schols, M. Grazia, Roncarolo, J. de Vries, and H. Spits. 1994. Lymphoid and myeloid differentiation of fetal liver CD34+ lineage cells in human thymic organ culture. J. Exp. Med. 180: 123-132 [Abstract]. |
26. | Kawamoto, H., K. Ohmura, and Y. Katsura. 1997. Direct evidence for the commitment of hematopoietic stem cells to T, B and myeloid lineages in murine fetal liver. Int. Immunol. 9: 1011-1019 [Abstract]. |
27. |
Rosenzweig, M.,
D.F. Marks,
H. Zhu,
D. Hempel,
K.G. Mansfield,
P.K. Sehgal,
S. Kalams,
D.T. Scadden, and
R.P. Johnson.
1996.
In vitro T lymphopoiesis of human and
rhesus CD34+ progenitor cells.
Blood.
87:
4040-4048
|
28. | Kondo, M., I.L. Weissman, and K. Akashi. 1997. Identification of clonogenic common lymphoid progenitors in mouse bone marrow. Cell. 91: 661-672 [Medline]. |
29. | Jenkinson, E.J., and G. Anderson. 1994. Fetal thymic organ cultures. Curr. Opin. Immunol. 6: 293-297 [Medline]. |
30. |
Croisille, L.,
I. Auffray,
A. Katz,
B. Izac,
W. Vainchenker, and
L. Coulombel.
1994.
Hydrocortisone differentially affects the ability of murine stromal cells and human marrow-derived adherent cells to promote the differentiation of
CD34++/CD38![]() |
31. | Carayol, G., C. Robin, J.-H. Bourhis, S. Chouaib, L. Coulombel, and A. Caignard. 1998. NK cells differentiated from bone marrow, cord blood and peripheral blood stem cells exhibit similar phenotype and functions. Eur. J. Immunol. 28: 1991-2002 [Medline]. |
32. |
Petzer, A.L.,
P.W. Zandstra,
J.M. Piret, and
C.J. Eaves.
1996.
Differential cytokine effects on primitive (CD34+CD38![]() |
33. | Akashi, K., M. Kondo, U. von Freeden-Jeffry, R. Murray, and I.L. Weissman. 1997. Bcl-2 rescues T lymphopoiesis in Interleukin-7 receptor-deficient mice. Cell. 89: 1033-1041 [Medline]. |
34. | Itoh, K., H. Tezuka, H. Sakoda, M. Konno, K. Nagata, T. Uchiyama, H. Uchino, and K.J. Mori. 1989. Reproducible establishment of hemopoietic supportive stromal cell lines from murine bone marrow. Exp. Hematol. 17: 145-153 [Medline]. |
35. |
Hao, Q.L.,
E.M. Smogorzewska,
L.W. Barsky, and
G.M. Crooks.
1998.
In vitro identification of single CD34+CD38![]() |
36. |
Fluckiger, A.,
E. Sanz,
M. Garcia-Lioret,
T. Su,
Q.L. Hao,
R. Kato,
S. Quan,
A. de la Hera,
G. Crooks,
O.N. Witte, et al
.
1998.
In vitro reconstitution of human B-cell ontogeny:
from CD34+ multipotent progenitors to Ig-secreting cells.
Blood.
92:
4509-4520
|
37. |
García-Ojeda, M.E.,
S. Dejbakhsh-Jones,
I.L. Weissman, and
S. Strober.
1998.
An alternate pathway for T cell development supported by the bone marrow microenvironment: recapitulation of thymic maturation.
J. Exp. Med.
187:
1813-1823
|
38. | Gardner, J.P., M. Rosenzweig, D.F. Marks, D. Harper, K. Gaynor, R.J. Fallon, D.A. Wall, R.P. Johnson, and D.T. Scadden. 1998. T-lymphopoietic capacity of cord blood- derived CD34+ progenitor cells. Exp. Hematol. 56: 991-999 . |
39. | Bennaceur-Griscelli, A.L., C. Tourino, B. Izac, W. Vainchenker, and L. Coulombel. 1999. Murine stromal cells counteract the loss of LTC-IC potential induced by cytokines in CD34+CD38low/neg human bone marrow cells. Blood. In press. |
40. | Cumano, A., F. Dieterlen-Lievre, and I. Godin. 1996. Lymphoid potential, probed before circulation in mouse, is restricted to caudal intraembryonic splanchnopleura. Cell. 86: 907-916 [Medline]. |
41. | Snodgrass, R., and G. Keller. 1987. Clonal fluctuation within the haematopoietic system of mice reconstituted with retrovirus-infected stem cells. EMBO (Eur. Mol. Biol. Organ.) J. 6: 3955-3960 [Abstract]. |
42. |
Pawliuk, R.,
C. Eaves, and
R.K. Humphries.
1996.
Evidence
of both ontogeny and transplant dose-regulated expansion of
hematopoietic stem cells in vivo.
Blood.
88:
2852-2858
|
43. | Marandin, A., A. Dubart, F. Pflumio, F.L. Cosset, V. Cordette, S. Chapel-Fernandes, L. Coulombel, W. Vainchenker, and F. Louache. 1998. Retrovirus-mediated gene transfer into human CD34+/CD38low primitive cells capable of reconstituting long-term cultures in vitro and non obese diabetic severe combined immunodeficient mice in vivo. Hum. Gene Ther. 9: 1497-1511 [Medline]. |
44. |
Conneally, E.,
C.J. Eaves, and
R.K. Humphries.
1998.
Efficient retroviral-mediated gene transfer to human cord blood
stem cells with in vivo repopulating potential.
Blood.
91:
3487-3493
|