HEMATOPOIESIS
Emergence of muscle and neural hematopoiesis in humans
Karen E. Jay, Lisa Gallacher, and Mickie Bhatia
During human development, hematopoiesis is thought to be compartmentalized
to the fetal circulation, liver, and bone
marrow. Here, we show that combinations of cytokines together with bone
morphogenetic protein-4 and erythropoietin could induce multiple blood lineages
from human skeletal muscle or neural
tissue. Under defined serum-free conditions, the growth factors requirements,
proliferation, and differentiation capacity
of muscle and neural hematopoiesis were
distinct to that derived from committed
hematopoietic sites and were uniquely
restricted to CD45ⴚCD34ⴚ cells expressing the prominin AC133. Our study de-
fines epigenetic factors required for the
emergence of hematopoiesis from unexpected tissue origins and illustrates that
embyronically specified microenvironments do not limit cell fate in humans.
(Blood. 2002;100:3193-3202)
© 2002 by The American Society of Hematology
Introduction
Mammalian development involves a series of complex cellular
events that originates from a single totipotent cell.1,2 As these
cells proliferate and differentiate, groups of daughter cells are
organized by morphogenetic movements to pattern and specify
tissues necessary for the formation and function of the developing animal.3 During this process, lineage potential becomes
increasingly limited and is restricted to the majority of cell types
found at a given anatomical site.2 However, the precise epigenetic signals required for lineage specification or whether
commitment encompasses all cells comprising a given tissue
remains unknown. This limited understanding of cell fate
commitment during mammalian development is exemplified by
observations in the mouse that suggest that the differentiation
potential of cells is not restricted to the tissue source from which
they reside, eg, the ability to generate neural cell types from
cells harvested from bone marrow (BM).4-7 Although there are
several interpretations of the cellular basis of these observations, it is clear that anatomical tissue/organ sites that have been
specified during development are not exclusively restricted and
are capable of unexpected cell fates. Therefore, similar to
specific lineage induction during embryonic development, the
molecular signals governing development of unique lineages
from unrelated tissue sites has yet to be characterized.
Because most of these fundamental observations are predicated from experimental evidence in the mouse, it remains
unknown if human tissue displays similar properties. Distinctions between mouse and human progenitor behavior is epitomized by the successful expansion and gene transfer into
hematopoietic repopulating cells in the mouse, which has
enjoyed limited success in the human scenario.8,9 Human
hematopoiesis represents one of the most rigorously studied and
characterized processes of proliferation, differentiation, and
lineage commitment. In the mouse, hematopoietic cells are first
detected in the yolk sac, followed by definitive hematopoiesis
originating in the aorta-gonad-mesonephros (AGM) region10,11
and is sustained in committed hematopoietic tissue sites such as
fetal liver and BM. These established sites of hematopoiesis
contain primitive blood stem/progenitors capable of sustained
development into multiple blood lineages.10,12,13 In humans,
characterizing the initiation of hematopoiesis has been limited;
however, studies indicate that hematopoietic progenitors can be
detected in similar regions to the mouse AGM and later on in the
fetal circulation, liver, and BM.14-16
Observations of unexpected cell fate from a given tissue have
been demonstrated in complex in vivo environments,5,17 thereby
preventing further elucidation of the factors governing this cellular
process.5,7,18 In vitro, most observations of unexpected cell fate
have been documented by using uncharacterized culture conditions
containing either conditioned media or serum extracts.17,19,20 These
previous approaches have provided pioneering observations but
limit our ability to understand cell fate progression because of the
lack of defined systems required to elucidate the specific factors
and cell types responsible for unexpected cell fate emergence. By
using serum-free culture systems, we show that hematopoietic
lineage development can be induced from human muscle and
neural tissue sites under the control of hematopoietic-associated
cytokines and combinations of bone morphogenetic protein-4
(BMP-4) and erythropoietin (EPO). Both qualitative and quantitative analyses indicate that emergence from either muscle or neural
tissue is distinct to that demonstrated previously in accepted sites of
hematopoiesis. Our study indicates that epigenetic signals are
capable of inducing muscle and neural hematopoiesis and that
tissue microenvironments do not limit cell fate potential during
human development.
From the John P. Robarts Research Institute, Developmental Stem Cell
Biology, The University of Western Ontario, and the Fetal Medicine Division, St
Joseph’s Hospital and London Health Sciences Center, London, ON, Canada.
Excellence Program (K.J.).
Submitted February 14, 2002; accepted June 11, 2002. Prepublished online as
Blood First Edition Paper, June 21, 2002; DOI 10.1182/blood-2002-02-0502.
Supported by a grant (MT-15063) from the Canadian Institutes of Health
Research, Ontario, Canada, and a scholarship award (MSH-35681) from the
Canadian Institutes of Health Research, Ontario, Canada, (M.B.) and
studentship from the Stem Cell Network, Canadian National Centre of
BLOOD, 1 NOVEMBER 2002 䡠 VOLUME 100, NUMBER 9
Reprints: Mickie Bhatia, The John P. Robarts Research Institute, Stem Cell
Biology and Regenerative Medicine, 100 Perth Dr, London, Ontario, N6A 5K8,
Canada; e-mail: [email protected].
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.
© 2002 by The American Society of Hematology
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JAY et al
Materials and methods
photomicroscope. Omission of primary antibody or antiserum resulted in no
detectable staining.
Human tissues
Colony assays
Samples of 16- to 22-week gestational age human brain and muscle tissues
were obtained in conjunction with local ethical and biohazard authorities of
the University of Western Ontario and London Health Sciences Centre.
Skeletal muscle was removed from the femur bone and cut into fine pieces
in Hanks buffered salt solution (HBSS; Gibco, Burlington, Ontario,
Canada). Collagen was added to a final concentration of 0.2%, and tissue
was incubated for 20 minutes at 37°C followed by 10 minutes in 0.25%
Trypsin (Gibco). Neural tissue was triturated in HBSS, and both tissues
were forced through needles of increasing gauge.18,21,22 Cell suspensions
were filtered through sterile 70-␮m cell strainers and plated in tissue culture
plates. After 1 hour, nonadherent cells were removed, counted, and replated
in fibronectin-coated plastic. Fetal blood was washed twice with HBSS and
filtered through a 70-␮m cell strainer, counted, and plated.
Human clonogenic progenitor assays were performed by plating 500 000 to
1 000 000 de novo–isolated muscle and neural cells into serum-free
Methocult H4236 (Stem Cell Technologies) containing 50 ng/mL rhu-SCF,
10 ng/mL rhu-granulocyte macrophage colony-stimulating factor (GMSCF) and rhu–IL-3, and 3 U/mL rhu-EPO, or with or without BMP-4 (25
ng/mL). Cultured cells were harvested, washed with PBS, and counted;
5000 cells were plated in serum containing Methocult. Differential colony
counts were assessed by morphology following incubations for 10 to 14
days at 37°C and 5% CO2 in a humidified atmosphere.
In vitro culture of human fetal cells
Counted cells were cultured in 5 different conditions. A medium contained
epidermal growth factor (EGF), fibroblast growth factor (FGF), and nerve
growth factor (NGF) with added 10% chick embryo extract and retinoic
acid. Base serum-free essential media contained bovine serum albumin
(BSA), insulin, and transferrin in Iscoves modified Dulbecco medium
(IMDM) (Stem Cell Technologies). Hematopoietic growth factors (HGFs)
added to BIT (BSA, insulin, transferrin) media included 300 ng/mL stem
cell factor (SCF), FLT-3 ligand, 50 ng/mL granulocyte-colony-stimulating
factor (G-CSF), and 10 ng/mL interleukin-3 (IL-3), IL-6. To assess
contribution of candidate extrinsic factors, 25 ng/mL BMP-4 (R & D
Systems) and/or 3 U/mL EPO were added to essential media containing
growth factors. Cells were incubated at 37°C and 5% CO2, and media and
growth factors were replenished every 2 to 3 days.
Flow cytometric analysis of human muscle and neural cells
Fetal tissues were prepared as mentioned above and resuspended at
10 ⫻ 106 cells/mL in phosphate-buffered saline (PBS). The following
fluorochrome antibodies were used for the detection of hematopoieticassociated surface proteins: human CD45-fluorescein isothiocyanate (FITC;
Becton Dickinson Immunocytometry Systems [BDIS], San Jose, CA),
human AC133-phycoerythrin (PE; Miltenyi Biotech), and CD34-allophycocyanin (APC; BDIS), vascular endothelial (VE)–Cadherin-FITC (Alexis
Biochemicals, CA), and mouse immunoglobulin G1 (IgG1) tails conjugated
to each fluorochrome for isotype controls (BDIS). Cells were then stained
with AC133-PE–conjugated antibody, and 5 ␮g/mL propidium iodide
(Sigma) for detection of viable overlapping populations. Human neural
cells expressing surface AC133 were isolated and used for colony-forming
cell assays.
Immunohistochemistry of human muscle and neural cells
Cultured cells were washed several times with PBS and fixed with 70%
EtOH and 0.15 M NaCl for 10 minutes at room temperature and washed 3
times with PBS. Fixed cells were blocked with 10% goat serum for 30
minutes at room temperature and washed 3 times with PBS. Neural cells
were incubated with a rabbit polyclonal antibody specific for nestin and
monoclonal microtubule-associated protein-2 (MAP-2) antibody, whereas
muscle cells were stained with monoclonal antiserum directed against
mysoin heavy chain and a monoclonal specific for myogenin. Antibody
staining was done in PBS with 10% BSA for 1 hour at room temperature.
Cells were rinsed with PBS 3 times and incubated for 1 hour at room
temperature with FITC-conjugated goat antibody to rabbit IgG (nestin),
Cy5-conjugated goat antibody to mouse IgG (MAP-2, myogenin), or
FITC-conjugated goat antibody to mouse IgG (myosin) (1:100; Jackson
Immunochemicals). After 3 final washes in PBS, coverslips were mounted
with mounting medium and viewed and photographed with a Zeiss
Transplantation of human muscle and neural cells and analysis
of NOD/SCID mice
Cells were intravenously injected into mice that were sublethally irradiated
at 335 cGy by using a 137 Cs ␥-irradiator at cell doses indicated. Mice were
killed 4 to 8 weeks after transplantation, and BM cells were recovered from
each mouse. Other tissues were collected at time of death and kept frozen
for DNA extraction and subsequent analysis. Analysis of human cell
engraftment was evaluated by analyzing genomic DNA extracted from all
murine tissues collected. Extracted DNA was used for polymerase chain
reaction (PCR) amplification by using human Cart-1–specific primers
(Gibco). PCR products were resolved on 1.4% agarose and transferred onto
nylon membrane (Amersham). Membranes were used for southern hybridization with a 32dCTP–labeled Cart-1 DNA fragment.
Flow cytometric analysis of murine BM
BM cells were harvested by flushing removed femur, tibia, and iliac crests
of mice that underwent transplantation. Cells were stained with monoclonal
antibodies at 4°C for 30 minutes and washed several times prior to analysis
by flow cytometry using a FACSCalibur (BDIS) and Cell Quest software
(BDIS). Antibody specific to human CD45 was conjugated to FITC for
detection of human hematopoietic cells.
Results
Phenotypic characterization of human muscle and neural
tissues using hematopoietic-associated cell surface markers
To phenotypically characterize the nature of human muscle or
neural cells in the context of hematopoietic potential, we examined
surface markers associated with the hematopoietic lineage. Little to
no de novo–isolated human muscle (Figure 1Aii) or neural (Figure
1Bii) tissue demonstrated expression of CD45, the pan-leukocyte
marker expressed on all committed hematopoietic tissue.21 Because
of the absence of appreciable CD45-expressing cells in either
muscle or neural tissue, substantial numbers of AC133⫹ or CD34⫹
muscle (Figure 1Aii) or neural (Figure 1Bii) cells coexpressing
CD45 could not be detected in any samples (n ⫽ 12). However,
both muscle and neural human tissues were shown to exhibit
expression for cell surface markers AC133 and CD34, previously
associated with primitive human hematopoietic cells23-25 (Figure
1Aiv and Figure 1Biv). Interestingly, the percentage and extent of
expression of AC133 and CD34 was substantially higher in human
muscle tissue, although both tissue types contained double-positive
(AC133⫹CD34⫹) cells (Figure 1A-B, iv). These analyses indicate
that human markers considered to be restricted to hematopoietic
tissue are capable of more ubiquitous tissue expression and are
shared among both muscle and neural tissues in humans, suggesting that phenotype alone is inefficient at making predictions of
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Figure 1. Flow cytometric analysis of cell surface
phenotype of cells derived from human muscle and
neural tissues. Flow cytometry was used to evaluate the
cell surface expression of the human-specific proteins
associated with hematopoietic tissue: AC133, and cell
differentiation markers CD34 and CD45. A representative
FACS analysis of de novo–isolated muscle (A) and
neural (B) cells is shown, and the percentage of subpopulations expressing single or coexpressing human hematopoietic markers is shown as the average mean ⫾ SEM
(n ⫽ 4) in each quadrant. Specificity of hematopoietic
antibody staining and background signal was determined
by comparing muscle and neural cells stained with
mouse IgG1 (Ai and Bi) to establish positive quadrant
levels to omit fluorescence because of nonspecific binding and auto fluorescence properties of the cells. Results
are based on data from 4 independent samples of muscle
and neural tissues analyzed in duplicate.
cell fate commitment and/or restriction without comparative
functional analysis.
Human muscle and neural tissues are devoid of
hematopoietic-reconstituting function
To functionally examine if human muscle or neural tissue was
capable of pluripotent hematopoietic reconstituting capacity, immune-deficient nonobese diabetic (NOD)/severe combined immunodeficiency disease (SCID) mice received transplants intravenously of de novo–isolated fetal cells in single cell suspensions
derived from neural and muscle tissues and were compared with
cells obtained from previously accepted sites of human hematopoiesis such as liver, blood, and BM. Because more mature human
hematopoietic progenitors cannot be detected in this model,22,26
this biologic assay discriminates between human hematopoietic
stem cells from hematopoietic progenitors devoid of repopulating
function. This human-mouse xeno-transplantation approach has
previously allowed for the development of an assay for candidate
human hematopoietic stem cells,27 including fetal hematopoietic
stem cells from liver, blood, and BM.16
Human chimerism was evaluated in the BM of mice that
underwent transplantation between 4 to 8 weeks after transplantation (n ⫽ 46). A representative analysis of the BM of NOD/SCID
mice that underwent transplantation stained for the human hematopoietic specific marker CD45 is shown in Figure 2. Compared with
isotype control (Figure 2A), fetal liver (2B), blood (2C), and BM
(2D) demonstrated human hematopoietic engraftment in vivo,
whereas both skeletal muscle (2E) and neural (2F) tissues were
unable to repopulate recipient BM to similar levels, using the same
range of cell doses (2 ⫻ 106 to 5 ⫻ 106 cells). From 2.5 ⫻ 106 to
6.5 ⫻ 106 cells derived from muscle or neural tissue were cultured
for 5 days and then transplanted into NOD/SCID without any
human hematopoietic chimerism detected in recipient BM. Therefore, in contrast to fetal liver, blood, and BM tissue at the same
stage of human development, cells derived from human muscle or
neural tissue were devoid of repopulating function by using this in
vivo transplantation model.
At lower limits of detection (0.1%-0.01%), microchimerism of
human cells was detected in several organs after 4 to 8 weeks after
transplantation of fetal muscle and neural cells in NOD/SCID mice.
This range of detection is indicative of approximately 1 human cell
in 10 to 50 000 mouse cells at a given site and is similar to levels of
neural engraftment from mouse BM.28,29 Although it is possible
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Figure 2. Human chimerism in the tissue of immunedeficient mice that received intravenous transplants
of human fetal tissues. Comparison of human hematopoietic chimerism from hematopoietic and nonhematopoietic sources. Scatterplots show FACS analysis of mouse
BM stained with the pan-leukocyte marker CD45 for the
detection of human hematopoietic cells (gated box) after
transplantation with isotype control (A), fetal blood (B),
fetal liver (C), fetal BM (D), fetal muscle (E), and fetal
neural cells (F). Cell doses ranged between 2 ⫻ 106 and
5 ⫻ 106 to up to 15 ⫻ 106 for muscle and neural transplants (n ⫽ 43).
that human cells engrafting these murine tissue sites have undergone cell fate alteration, the microchimerism of human cells
detectable (1 in 10 to 50 000 cells) suggests that the cells present
are insufficient to be physiologically relevant to the recipient
animal. Similar levels of microchimerism detected by this PCR
method did not detect human chimerism in multiple organs of
NOD/SCID mice that received transplants of purified CD34⫺Lin⫺
cells derived from fetal blood (data not shown), suggesting that this
level of organ chimerism is unique to transplantation of muscle and
neural cells. Furthermore, the presence of cells derived from human
muscle and neural tissues expressing VE-cadherin suggests the
endothelial cells present in the transplanted products derived from
muscle or neural tissue could account for the low level of human
chimerism detected by PCR. On the basis of our results and
previous murine studies, alternative methods other than complex in
vivo systems are required to study the potential of hematopoiesis
arising from unexpected origins of human muscle or neural tissue.
Induction of muscle and neural hematopoiesis
Primary samples of cells derived from muscle and neural tissues
were harvested from human fetal skeletal muscle and brain. Single
cell suspensions of human muscle and neural cells were cultured
under a variety of conditions. As shown in Figure 3A-B, cells
derived from muscle and neural tissues were cultured in serum-free
media containing essential amino acids, albumin, insulin, and
transferrin, together with specific hematopoietic-associated cytokines SCF, FLT-3L, IL-3, IL-6, and G-CSF (HGF, where HGF
refers to the combination of these hematopoietic growth factors),
and addition of EPO and BMP-4. Because factors such as BMP-4
or EPO have been shown to be involved in the induction of a
hematopoietic cell fate during embryogenesis of invertebrates and
mammals30-32 and initiation of primitive hematopoiesis,32,33 we
hypothesized that these proteins may serve as candidate regulators
of hematopoietic specification from nonhematopoietic tissue. After
4 days of culture, cells were examined by light microscopy and in
situ immunohistochemical analysis for specific lineage markers.
Human muscle tissue (Figure 3Ai) expressed both structural
myosin heavy chain protein34 (Figure 3Aii) and myogenin DNA
binding protein (Figure 3Aiii) that are specific to the muscle
lineage,35,36 whereas human neural tissue (Figure 3Bi) showed
expression of both the neural progenitor marker, nestin37 (Figure
3Bii), and MAP-238 (Figure 3Aiii).
Although cultured muscle and neural cells showed morphologic
and phenotypic commitment, we examined whether these cells
possessed hematopoietic potential. Cultured cells from muscle and
neural tissues were evaluated for functional hematopoietic progenitor capacity by using hematopoietic colony-forming unit (CFU)
assays.23,39-41 Cells from respective liquid cultures were harvested
and seeded into methylcellulose containing hematopoietic growth
factors under serum-free conditions and examined for clonal
hematopoietic potential after 14 days. Both muscle (Figure 3Aiv)
and neural (Figure 3Biv) cells cultured in serum-free media with
specific growth factors were able to give rise to multiple hematopoietic progenitors of erythroid (BFU-E), granulocytic (CFU-G),
macrophage (CFU-M), and hematopoietic clones with tetrapotent
lineage capacity (CFU-GEMM [granulocyte, erythroid, macrophage, megakaryocyte]). On the basis of unexpected hematopoietic
potential arising from human muscle and neural tissues, we further
examined this observation by using quantitative analysis to better
understand the basis of hematopoietic lineage emergence.
Single-cell suspensions derived from muscle and neural tissues were
further examined for de novo hematopoietic potential and after in vitro
culture with and without specific extrinsic factors as shown (Figure 4).
To determine if cells within human muscle or neural tissue possessed
hematopoietic potential on de novo isolation, individual samples were
seeded into clonal hematopoietic progenitor assays. Several initial
experiments indicated that de novo–isolated muscle or neural tissue
were completely devoid of clonal hematopoietic potential (data not
shown), suggesting that cells with hematopoietic progenitor function are
absent in human muscle or neural tissue. To ensure that seeding of
muscle and neural cells into methylcellulose hematopoietic clonal
assays was in the linear range of response, dilutions of input cells were
carried out, ranging from 10 000 to 1 000 000 cells. When human
muscle or neural cell input was increased to more than 500 000
cells/well, a small number of hematopoietic progenitors could be
detected from de novo–isolated muscle (Figure 4A) and neural tissues
(Figure 4B). The total number of hematopoietic progenitor detectable
among 500 000 cells derived from muscle or neural tissue was 8 ⫾ 2
and 1 ⫾ 1, respectively (Table 1). To confirm that muscle or neural cells
did not inhibit CFU formation, we seeded bona fide hematopoietic cells
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Figure 3. Immunohistochemical and morphologic analysis of human fetal muscle and neural cells using tissue-specific markers and emergence of functional
hematopoietic progenitors. Light microscopy was used to visualize cells comprising human muscle (Ai) and neural (Bi) tissues, showing morphologic features associated
with these tissue types. Human muscle tissue specificity was analyzed by immunohistochemistry for the expression of muscle-specific markers recognizing heavy chain of
myosin (Aii) and the nuclear DNA binding factor, myogenin (Aiii), whereas human neural tissue demonstrated expression of neural progenitor-specific marker, nestin (Bii) and
MAP-2 (Biii), respectively. A representative panel of multilineage hematopoietic colonies derived from muscle (Aiv) and neural (Biv) tissues cultured in the presence of HGF
together with BMP-4 and EPO is shown. Human hematopoietic colony types generated from muscle and neural tissues include erythroid burst-forming unit (BFU-E),
CFU-granulocyte (G), -macrophage (M), and tetrapotent mixed colonies (granulocyte, erythroid, macrophage, megakaryocyte [GEMM]). Similar results were obtained from 9
independent samples of muscle and neural tissue samples. Magnification ⫻ 200.
enriched for a known frequency of blood progenitors with or without
muscle and neural cells. There was no difference in the number of CFU
progenitors in hematopoietic progenitor assays with or without the
addition of muscle or neural cells, suggesting the large number of input
neural or muscle cells does not effect the detection of hematopoietic
progenitor in this assay system (data not shown). Our results suggest that
hematopoietic potential of muscle and neural tissues during human
development is nearly absent and optimally only represents 0.0016%
and 0.0002%, respectively.
To provide an extrinsic environment conducive of the embryonic
development and promotion of intrinsic cell fate of cells derived from
muscle and neural tissues, 10% chick embryonic extract (10% CEE)
was added to defined serum-free media cultures.18,42 The addition of
10% CEE media, shown previously in murine studies to promote
hematopoietic cell fate induction,18 had no affect on hematopoietic
potential of either muscle or neural cells in humans (Figure 4A-B).
However, exposure of cells derived from muscle and neural tissues
cultured in serum-free media with combinations of HGFs was capable
of inducing hematopoietic progenitors (Figure 4A-B). The addition of
BMP-4 and EPO induced hematopoietic progenitors from cells derived
from muscle and neural tissues by an additional 4- to 5-fold, with
development into multiple hematopoietic lineages (Figure 3Aiv and
Biv). Our results demonstrate that neither muscle nor neural tissue are
capable of hematopoiesis de novo, but development into multiple
hematopoietic lineages can be induced under the control of specific
epigenetic signaling factors.
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CEE, hematopoietic progenitors were nearly undetectable from
these cultures. CD45, a marker of hematopoietic lineage commitment, or double-positive CD34⫹CD45⫹ cells considered to be
enriched for hematopoietic progenitors were completely absent in
muscle cultures treated with HGFs, whereas hematopoietic progenitor potential of an average of 182 progenitors was detectable. The
inability to correlate presence of specific subsets of CD34 or
CD34⫹CD45⫹ cells within the neural-treated cultures was consistent. The detection of an average of 630 and 2475 hematopoietic
progenitors in cultures of muscle and neural cells containing HGFs
and BMP-4 ⫹ EPO corresponded to as few as 59 and 132
CD34⫹CD45⫹ cells. On the basis of comparative quantitative
analysis among de novo–isolated and treated cultures, our data
indicate that surrogate markers for hematopoietic progenitors do
not apply to hematopoietic potential induced from muscle and
neural tissues and suggest that emergence of hematopoiesis from
these unexpected sources is unique to other forms of hematopoietic
lineage progression that have been characterized to date.
Epigenetic signals required for emergence of muscle and
neural hematopoiesis are distinct from circulating fetal
hematopoietic progenitors
Figure 4. Evaluation of human hematopoietic cell fate potential of de novo and
cultured muscle and neural cells. Primary human muscle (A) and neural (B)
tissues were examined for hematopoietic progenitor capacity (CFU) at isolation (day
0) or after 5 days of in vitro culture in essential media containing 10% CEE, or human
hematopoietic growth factors (HGF) alone or with BMP-4 or EPO, as single additions
or in combination as indicated. In all culture conditions, media and cytokines were
replenished every other day. Single, double, and triple asterisks indicate substantial
differences within measured groups of P ⬍ .01. Data shown is based on 6 to 13
independent samples analyzed in culture conditions indicated.
Known phenotypic characteristics of hematopoietic
progenitors do not correlate with muscle and neural
hematopoietic potential
On the basis of the ability of HGFs and BMP-4 ⫹ EPO treatment in
serum-free media to induce hematopoietic progenitors, we examined changes in cell number and absolute number of phenotypic
subsets previously associated with hematopoietic progenitor function. By using a starting population of 500 000 cells derived from
muscle and neural tissues (de novo), an average number of AC133,
CD34, CD45, and double-positive CD34/CD45 cells were calculated from 13 independent human fetal samples and compared with
totals after in vitro culture in essential serum-free media with 10%
CEE, HGF,43,44 or HGF treatment in combination with BMP-4 and
EPO (Table 1). Human muscle or neural cells cultured in 10% CEE
lose AC133 expression, whereas in the presence of HGF combinations a larger number of AC133⫹ cells were detectable in both
cultures of muscle and neural cells. AC133 expression was further
augmented in the presence of HGF together with BMP-4 and EPO
by more than 2-fold compared with HGF-treated muscle cultures
but had little effect on neural-derived cells (Table 1). Changes in
total AC133 cells within treated cultures do not correlate with
increases in hematopoietic progenitors for either muscle or neural
tissue (Table 1). Cell surface expression of CD34 or CD45 on either
muscle- or neural-derived cells did not correlate with hematopoietic progenitor function. Despite the presence of greater numbers of
total CD34 cells in muscle and neural cultures treated with 10%
During human fetal development, hematopoietic progenitors can
be isolated from the circulation between 16 and 22 weeks of
gestation.16 On the basis of our observations that hematopoietic
progenitors can arise from cells derived from muscle and neural
tissues in response to HGFs together with BMP-4 and EPO (Figure
4 and Table 1), we performed side-by-side comparisons of the
proliferative and differentiation potential of hematopoietic progenitors isolated from fetal blood (FB), fetal muscle, and fetal neural
tissues obtained from the same human donors and cultured under
identical defined culture conditions (Figure 5).
Table 1. Comparison of total hematopoietic progenitors emerging from
muscle and neural tissue to changes in total number of specific
phenotypically characterized subsets
De novo
10% CEE
HGF
HGF ⫹ BMP-4
⫹ EPO
Total cells
500 000
920 000
330 000
320 000
AC133⫹
190 000
0
11 612
28 139
CD34⫹
23 500
87 400
46 054
41 195
CD45⫹
1 025
0
0
5 165
600
0
0
59
8
4
182
630
82 000
Muscle tissue
CD34⫹CD45⫹
Total hematopoietic
progenitors
Neural tissue
Total cells
500 000
120 000
110 000
AC133⫹
97 450
291
1 116
658
CD34⫹
21 650
1 087
283
789
CD45⫹
4 850
2 388
9 843
9 574
95
669
541
132
1
5
507
2 475
CD34⫹CD45⫹
Total hematopoietic
progenitors
Flow cytometry was used to assess the expression of human-specific cell surface
proteins AC133, CD34, CD45, and double-positive CD34/CD45 before and after in
vitro culture in serum-free media containing either 10% chicken embryonic extract
(CEE) or human hematopoietic growth factors (HGFs) alone or with bone morphogenetic protein-4 (BMP-4) and erythropoietin (EPO) for a total of 500 000 human
muscle– and human neural–derived cells. Values show average absolute number of
cells of each phenotype. Absolute number of detected hematopoietic progenitors
after culture in each medium is given. Treated human muscle and neural cells
maintained normal expression of tissue-specific markers (myosin for muscle and
nestin for neural tissue) irrespective of changes in hematopoietic cell surface
phenotype as shown in Figure 3.
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Figure 5. Comparative analysis of human hematopoietic-, muscle-, and neural-derived hematopoietic progenitors. Human tissues indicated were harvested under
identical conditions, and functional hematopoietic progenitor capacity was evaluated at day 0 (de novo–isolated tissues) and again at day 5 of culture in HGF conditions with or
without BMP-4 or BMP-4 together with EPO. (A) Developmental potential of hematopoietic progenitors was compared among various human tissues by enumerating colony
types as a measure of hematopoietic lineage commitment that include erythroid (BFU-E), monocytic (CFU-M), granulocytic (CFU-G), and myelocytic (CFU-GM). Composition
of CFU types generated for each tissue is expressed as mean percentage ⫾ SEM (n ⫽ 5) of the total colonies for muscle (gray), neural (white), and blood (black) cells cultured
in essential media containing HGF (i), BMP-4 (ii), or BMP-4 and EPO in combination (iii). Single and double asterisks indicate substantial differences within measured groups of
P ⬍ .01, (n ⫽ 5). (B) Fold changes in total hematopoietic progenitor expansion was compared in response to culture conditions indicated and compared with day 0 of cultured
fetal blood (i), muscle (ii), and neural (iii) cells. Each graph shows the increase in progenitors after culture in essential media containing HGF (F), BMP-4 (⽧), or BMP-4 and
EPO (f). (C) Fold expansion of fetal blood cultured for 5 days in the absence or presence of cells derived from fetal muscle (i) or fetal neural (ii) tissue using cytokine
combinations indicated. Cells were cocultured using 5.0 ⫻ 105 fetal blood cells to equal numbers of fetal muscle or neural cells to maintain the same cell density used in
previous experiments shown in panel B. Insets show the relative fold expansion to fetal blood cells cultured alone compared with the various coculture treatments. (D)
Hematopoietic progenitors assayed from single cells suspensions of muscle (i), neural (ii), and blood (iii) in the absence or presence of BMP-4 directly added to methylcellulose
used in this clonal assay. Graphs show number of clonogenic progenitor of de novo–isolated tissues in the absence of BMP-4 (control) shown standardized as 100% and in the
presence of BMP-4 as a percentage relative to control ⫾ SEM. Results are based on a total of 5 independent samples.
Biologic differences between FB, muscle, and neural hematopoietic lineage potential were examined by comparing the relative
frequencies of erythroid, granulocytic, monocytic, and myelocytic
hematopoietic progenitors detected. Relative frequencies of hematopoietic progenitors from each human tissue displayed their own
distinct profiles (Figure 5Ai-iii). Most strikingly, cells derived from
muscle and neural tissues possessed less erythroid hematopoietic
potential (BFU-E) but demonstrated enhanced granulocytic
(CFU-G) development than FB. To compare proliferative expansion of hematopoietic progenitors derived from FB versus muscle
and neural tissues, single-cell suspensions of each tissue were
harvested and seeded at identical concentrations into essential
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JAY et al
media containing HGFs alone and compared HGF with BMP-4 or
BMP-4 and EPO (Figure 5B). Hematopoietic progenitor expansion
from FB averaged 18-fold when cultured in the presence of HGF
(Figure 5Bi), whereas HGF induced an 8-fold increase in musclederived hematopoietic progenitors (Figure 5Bii) and a 200-fold
expansion of neural-derived hematopoietic progenitors (Figure
5Biii). Addition of BMP-4 or BMP-4 and EPO in combination had
no effect on changes in the expansion of hematopoietic progenitors
derived from FB (Figure 5Bi). In contrast, addition of BMP-4 and
EPO resulted in a 6-fold and 8-fold augmentation of hematopoietic
progenitor expansion induced from muscle (Figure 5Bii) and
neural (Figure 5Biii) tissues, respectively. These results demonstrate that muscle- and neural-derived hematopoietic progenitors
are responsive to BMP-4 and EPO, whereas BMP-4 and EPO are
unable to affect hematopoietic progenitor expansion from circulating FB. To further examine whether differential responsiveness of
muscle and neural hematopoietic progenitor expansion was not due
to progenitors from fetal circulation uniquely responding via an
indirect effect of muscle and neural cells in the culture, we
performed direct coculture experiments shown in Figure 5C. Equal
numbers of neural and muscle cells were cocultured with FB cells
and treated with or without BMP-4 or BMP-4 and EPO. In the
presence of either muscle (Figure 5Ci) or neural (Figure 5Cii)
coculture, blood cells derived from the fetal circulation remained
unresponsive to either BMP-4, or BMP-4 and EPO and did not
demonstrate enhanced progenitor expansion as compared with
treatment with HGFs alone. These results suggest that muscle and
neural cells are unable to indirectly affect fold expansion of
hematopoietic progenitors that originate from fetal blood sources,
decreasing the likelihood of differential cytokine response shown
among muscle or neural hematopoietic progenitors to be arising
from contaminating fetal blood cells.
To address whether BMP-4 was able to affect the hematopoietic
lineage induction from individual muscle or neural clones, muscle
and neural cells were plated into defined, serum-free methylcellulose assays for hematopoietic progenitor detection, in the absence
and presence of BMP-4, and were compared with progenitors
derived from circulating fetal blood. Direct addition of BMP-4 to
clonal assay systems was able to increase the total number of
hematopoietic progenitors detectable from both muscle and neural
tissues by 2-fold, whereas the frequency of fetal blood progenitors
was unaffected (Figure 5D). These results indicate that hematopoietic progenitors can be induced from de novo–isolated muscle and
neural cells at the single cell level.
Taken together, our findings illustrate biologic differences in
human hematopoiesis arising from nonhematopoietic tissue compared with committed blood sources in (1) developmental program
of hematopoietic lineages, (2) hematopoietic progenitor expansion,
and (3) responsiveness to instructive factors such as BMP-4 and
EPO. Quantitative and qualitative differences between fetal blood
and hematopoietic emergence from human muscle and neural
tissues indicate that the origin and biologic nature of hematopoiesis
from muscle and neural tissues is distinct to embryonically
specified hematopoietic sites.
Purification of cells with human hematopoietic potential
The nature of candidate cells responsible for progenitor function
from various tissues has been prospectively isolated by using the
presence or absence of cell surface markers.45-47 Human blood cells
with the ability to reconstitute the BM of NOD/SCID mice were
shown to be restricted to a subfraction expressing the prominin
AC133, similar to sustained chimerism of the brain of neonatal
BLOOD, 1 NOVEMBER 2002 䡠 VOLUME 100, NUMBER 9
NOD/SCID mice implanted with human neural cells.45 These
combined studies suggest that AC133 expression serves as a
marker for the identification of human hematopoietic and neural
stem/progenitor cells.48
With the use of viability stains such as 7AAD, live cells devoid of
CD34 cell surface expression were isolated, further gated for CD45⫺
cells, and divided into AC133⫹ and AC133⫺ subfractions. A representative gating strategy is shown in Figure 6A. AC133⫹CD34⫺CD45⫺ and
AC133⫺CD34⫺CD45⫺ cells were cultured in essential media containing HGFs, combined with BMP-4 and EPO for 5 days. The proliferation
of AC133⫹ cells was greater than the AC133⫺ subset over the culture
period. In addition, phase contrast light microscopy indicated that
AC133⫹ cells developed into large spheric clusters and established
projections, whereas poor growth of AC133⫺ cells was accompanied
with lack of notable projections or cluster formation (Figure 6Ai-ii).
After 5 days, cells resulting from AC133⫹CD34⫺CD45⫺ and
AC133⫺CD34⫺CD45⫺ cultures were harvested and placed into serumfree hematopoietic progenitor assays. Functional hematopoietic potential of producing progenitors of multiple blood lineages, similar to that
Figure 6. Isolation of AC133ⴙ and AC133ⴚ subsets from human embryonic
tissues. Human fetal neural cells were stained with human-specific antibodies raised
to the prominin AC133, CD34, and CD45. AC133⫹ and AC133⫺ cells were isolated
according to sorting gates shown in Figure 1, from the population of cells devoid of
CD34 and the hematopoietic marker CD45. (A) Purified subpopulations of
AC133⫹CD34⫺CD45⫺ (Bi) and AC133⫺CD45⫺CD34⫺ (Bii) cells from human neural
tissue were cultured under serum-free conditions shown to induce neural hematopoiesis. Magnification ⫻ 200 (Bi) and ⫻ 400 (Bii). Hematopoietic colonies of multiple
lineages were detected from AC133⫹CD34⫺CD45⫺ cells. The composition of
colonies was similar to that shown in Figure 4. (C) Quantitative analysis of
hematopoietic colonies arising from either AC133⫹ or AC133⫺ subsets from the
CD34⫺CD45⫺ population. Cells were cultured in HGF with BMP-4 and EPO and were
then collected and plated into colony-forming assays. Colonies were scored after 12
to 14 days and shown as the average number of colonies per 50 000 cell input ⫾
SEM. Averages shown are based on 4 independent samples.
BLOOD, 1 NOVEMBER 2002 䡠 VOLUME 100, NUMBER 9
shown from unpurified samples (Figure 3), was restricted to the
AC133⫹CD45⫺CD34⫺ subset (Figure 6C). No hematopoietic colonies
could be detected among AC133⫺CD45⫺CD34⫺ cells in any experiments using 4 independent neural samples (Figure 6C). The frequency
of AC133⫹CD45⫺CD34⫺ cells originating from neural tissue with
primitive hematopoietic progenitor potential was 1 in 787 in contrast to
the AC133⫺CD45⫺CD34⫺ subset in which no hematopoietic progenitors were detectable using as many as 500 000 purified cells. Our results
indicate that the population of human AC133-expressing cells possess
greater lineage potential than their AC133⫺ counterpart. In addition to
AC133 expression providing a marker for human hematopoietic and
neural stem cells,23,45 our study indicates this prominin family member
enriches for a population cells derived from human neural tissue that is
capable of hematopoietic progenitor function.
Discussion
Here we report that de novo–isolated cells from human muscle
and neural tissues show nearly undetectable hematopoietic
properties, suggesting that these localized tissue environments
are not permissive to hematopoiesis. However, cells derived
from muscle and neural tissues were capable of inducible
hematopoietic progenitor function into multiple lineages once
treated with specific extrinsic factors under serum-free conditions. These epigenetic signals and the phenotypic nature of
cells capable of hematopoiesis were distinct from hematopoiesis
arising from expected, bona fide hematopoietic tissue such as
circulating fetal blood. Observations from historical experiments in which whole explants of tissue were removed from one
site of a developing embryo and reimplanted into an alternative
site have established concepts of lineage commitment.49 The
inability of these removed tissues to be respecified to adopt the
fate of the new environment while retaining its original cell fate
potential describes the paradigm of tissue specification during
embryonic development.50 In contrast to classical embryonic
explant experiments using intact sections, our approach liberates the individual cells from the extrinsic influences of
neighboring cells or soluble factors in the original parent tissue.
On the basis of our results, we suggest that parent microenvironment imposes cell fate restriction and/or prevents cells from
responding to atypical hematopoietic factors; therefore, a 2-step
process of both removal and treatment of single-cell suspensions
in serum-free media allows emergence of hematopoietic progenitors from muscle and neural tissues.
Human cells derived from muscle and neural tissues displayed distinct responsiveness to the combinations of EPO and
BMP-4. Hematopoietic differentiation from muscle-derived
cells was greatest in the presence of EPO, whereas the addition
of BMP-4 was less effective. In contrast, human neural tissue
showed the greatest response in the presence of both BMP-4 and
EPO. Treatment of committed fetal blood cells with hematopoietic-inducing factors such as BMP-4 and EPO had no effect,
unlike primitive hematopoiesis arising from muscle- and neuralderived cells. These functional differences in cellular response
suggest that emergence of hematopoietic potential from muscle
and neural tissues are physiologic distinct and represent unique
origins of hematopoiesis. This is further supported by the
distinct nature of hematopoietic progenitors arising from muscle
and neural tissues that are devoid of CD45 or CD34 cell surface
expression. Previous studies from our laboratory indicate that
primitive CD34⫺ cells from cord blood (CB) are capable of
EMERGENCE OF MUSCLE/NEURAL HEMATOPOIESIS IN HUMANS
3201
hematopoietic cell fate.23 In contrast to CD34⫺ cells derived
from muscle or neural human tissue, de novo–isolated CD34⫺
CB-derived cells that coexpress AC133 are (1) highly enriched
for hematopoietic progenitor function and do not require further
ex vivo culture, (2) capable of engrafting the BM site of
NOD/SCID mice that underwent transplantation, and (3) express cell surface CD45.23 Our laboratory is currently optimizing methods to enrich similar subpopulation from nonhematopoietic fetal tissues so that adequate numbers of these cells can be
assayed for SCID-repopulating cells (SRC) function in vivo.
Our current study in humans supports the notion that preconceived relationships between tissue restriction and anatomical
position are more complex than previously appreciated. Our
results indicate that regulatory mechanisms responsible for
emergence of hematopoietic progenitors from unexpected tissue
origins can be controlled by extrinsic influences that are
sufficient to induce neogenic blood formation.
We suggest that the cellular origin of hematopoiesis from
muscle or neural tissue arises from novel precursors within
muscle or neural tissue capable of hematopoietic progenitor
function. Subpopulations of cells within specific tissues may
remain unrestricted and possess pluripotent potential that is
reminiscent of embryonic stem cells.4,36,51 The inability to
generate reconstituting hematopoietic stem cells from murine
embryonic stem (ES) cell parents in vitro, in contrast to the ease
in inducing hematopoietic progenitors, provides biologic precedence for this property in pluripotent cells.52,53 Therefore, unlike
bona fide hematopoietic tissue that has been committed during
development of the embryo, hematopoiesis induced from nonhematopoietic tissue does not give rise to detectable repopulating
stem cells. This notion is further supported by previous mouse
studies in which hematopoiesis from muscle or neural sites
could be demonstrated in vivo but failed to meet several criteria
used to define bona fide murine hematopoietic stem cells such as
long-term reconstitution, self-renewal properties, and protection
after lethal irradiation.54 Similar to muscle- and neural-derived
hematopoietic progenitors shown in our current study, recent
studies by Kaufman et al55 demonstrate that hematopoietic
progenitors arising from human ES cell lines are devoid of cell
surface CD45 expression, further supporting a common functionality among human ES cells and blood progenitors emerging
from human muscle and neural tissues. Multifunctional signals
supplied by parent tissue sites limits differentiation potential of
these cells; therefore, removal of these restrictions, combined
with the introduction of unique growth factor combinations
found in alternative tissue microenvironments, is sufficient in
instructing unique developmental programs. Defined serum-free
in vitro systems used in this study has allowed us to identify
critical factors that include BMP-4 and EPO, that are central to
the emergence of hematopoietic lineages from muscle and
neural tissues, and that do not affect committed blood cells. The
utility of newly identified inducing factors capable of instructing hematopoiesis from unexpected tissue origins provides a
system to further characterize cell fate restriction of primary
human cells.
Acknowledgments
We thank Amgen (Thousand Oaks, CA) for cytokines and the
staff of the labor and delivery departments of St Joseph’s
Hospital and London Health Sciences, London, ON, Canada,
3202
BLOOD, 1 NOVEMBER 2002 䡠 VOLUME 100, NUMBER 9
JAY et al
and especially the assistance of Dr Fraser Fellows. In addition,
we would like to thank Dr M. Underhill for critically reviewing
this manuscript and Drs J. M. Verdi, G. F. Pickering, R.
Hammond, M. Bani-Yahoub, and I. Skerjanc for assistance in
developing immunohistochemical protocols and providing
muscle- and neural-specific antibodies.
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