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Prepublished online as a Blood First Edition Paper on June 21, 2002; DOI 10.1182/blood-2002-02-0502.
HEMATOPOIESIS
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.
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 CD45 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.
Human tissues
In vitro culture of human fetal cells
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 hematopoietic-associated 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 µg/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 photomicroscope. Omission of primary antibody or antiserum resulted in no detectable staining.Colony assays 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 (GM-SCF) 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.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.
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 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.16Human 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 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 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 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. 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% 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 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).
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 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 muscle-derived 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 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.48With the use of viability stains such as 7AAD, live cells devoid
of CD34 cell surface expression were isolated, further gated for
CD45
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
neural-derived 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 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.
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, 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.
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 Excellence Program (K.J.).
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: Mickie Bhatia, The John P. Robarts Research Institute, Stem Cell Biology and Regenerative Medicine, 100 Perth Dr, London, Ontario, N6A 5K8, Canada; e-mail: mbhatia{at}rri.ca.
1. Fuchs E, Segre JA. Stem cells: a new lease on life. Cell. 2000;100:143-155[CrossRef][Medline] [Order article via Infotrieve]. 2. Thomson JA, Marshall VS. Primate embryonic stem cells. Curr Top Dev Biol. 1998;38:133-165[Medline] [Order article via Infotrieve].
3.
French V, Bryant PJ, Bryant SV.
Pattern regulation in epimorphic fields.
Science.
1976;193:969-981
4.
McKay R.
Stem cells
5.
Bjornson CR, Rietze RL, Reynolds BA, Magli MC, Vescovi AL.
Turning brain into blood: a hematopoietic fate adopted by adult neural stem cells in vivo.
Science.
1999;283:534-537
6.
Ferrari G, Cusella-De Angelius G, Coletta M, et al.
Muscle regeneration by bone marrow-derived myogenic progenitors.
Science.
1998;279:1528-1530 7. Gussoni E, Soneoka Y, Strickland CD, et al. Dystrophin expression in the mdx mouse restored by stem cell transplantation. Nature. 1999;401:390-394[CrossRef][Medline] [Order article via Infotrieve].
8.
Weissman IL.
Translating stem and progenitor cell biology to the clinic: barriers and opportunities.
Science.
2000;287:1442-1446 9. Dick JE. Gene therapy turns the corner. Nat Med. 2000;6:624-626[CrossRef][Medline] [Order article via Infotrieve]. 10. Medvinsky A, Dzierzak E. Definitive hematopoiesis is autonomously initiated by the AGM region. Cell. 1996;86:897-906[CrossRef][Medline] [Order article via Infotrieve].
11.
Marshall CJ, Kinnon C, Thrasher AJ.
Polarized expression of bone morphogenetic protein-4 in the human aorta-gonad-mesonephros region.
Blood.
2000;96:1591-1593 12. Yoder MC, Hiatt K, Dutt P, Mukherjee P, Bodine DM, Orlic D. Characterization of definitive lymphohematopoietic stem cells in the day 9 murine yolk sac. Immunity. 1997;7:335-344[CrossRef][Medline] [Order article via Infotrieve].
13.
Yoder MC, Papaioannou VE, Breitfeld PP, Williams DA.
Murine yolk sac endoderm- and mesoderm-derived cell lines support in vitro growth and differentiation of hematopoietic cells.
Blood.
1994;83:2436-2443 14. Dzierzak E, Medvinsky A. Mouse embryonic hematopoiesis. Trends Genet. 1995;11:359-366[CrossRef][Medline] [Order article via Infotrieve]. 15. Tavian M, Hallais MF, Peault B. Emergence of intraembryonic hematopoietic precursors in the pre-liver human embryo. Development. 1999;126:793-803[Abstract].
16.
Gallacher L, Murdoch B, Wu D, Karanu F, Fellows F, Bhatia M.
Identification of novel circulating human embryonic blood stem cells.
Blood.
2000;96:1740-1747 17. Theise ND, Krause DS. Toward a new paradigm of cell plasticity. Leukemia. 2002;16:542-548[CrossRef][Medline] [Order article via Infotrieve].
18.
Jackson KA, Mi T, Goodell MA.
Hematopoietic potential of stem cells isolated from murine skeletal muscle.
Proc Natl Acad Sci U S A.
1999;96:14482-14486 19. Toma JG, Akhavan M, Fernandes KJ, et al. Isolation of multipotent adult stem cells from the dermis of mammalian skin. Nat Cell Biol. 2001;3:778-784[CrossRef][Medline] [Order article via Infotrieve]. 20. Galli R, Borello U, Gritti A, et al. Skeletal myogenic potential of human and mouse neural stem cells. Nat Neurosci. 2000;3:986-991[CrossRef][Medline] [Order article via Infotrieve]. 21. Beverley PC, Linch D, Delia D. Isolation of human haematopoietic progenitor cells using monoclonal antibodies. Nature. 1980;287:332-333[CrossRef][Medline] [Order article via Infotrieve]. 22. Larochelle A, Vormoor J, Hanenberg H, et al. Identification of primitive human hematopoietic cells capable of repopulating NOD/SCID mouse bone marrow: implications for gene therapy. Nat Med. 1996;2:1329-1337[CrossRef][Medline] [Order article via Infotrieve].
23.
Gallacher L, Murdoch B, Wu DM, Karanu FN, Keeney M, Bhatia M.
Isolation and characterization of human CD34(-)Lin(-) and CD34(+)Lin(-) hematopoietic stem cells using cell surface markers AC133 and CD7.
Blood;
2000;95:2813-2820
24.
Krause DS, Fackler MJ, Civin CI, May WS.
CD34: structure, biology, and clinical utility.
Blood.
1996;87:1-13
25.
Corbeil D, Roper K, Weigmann A, Huttner WB.
AC133 hematopoietic stem cell antigen: human homologue of mouse kidney prominin or distinct member of a novel protein family?
Blood.
1998;91:2625-2626
26.
Cashman JD, Lapidot T, Wong JC, et al.
Kinetic evidence of the regeneration of multilineage hematopoiesis from primitive cells in normal human bone marrow transplanted into immunodeficient mice.
Blood.
1997;89:4307-4316 27. Dick JE, Bhatia M, Gan O, Kapp U, Wang JC. Assay of human stem cells by repopulation of NOD/SCID mice. Stem Cells. 1997;15(suppl 1):199-207.
28.
Brazelton TR, Rossi FM, Keshet GI, Blau HM.
From marrow to brain: expression of neuronal phenotypes in adult mice.
Science.
2000;290:1775-1779
29.
Mezey E, Chandross KJ, Harta G, Maki RA, McKercher SR.
Turning blood into brain: cells bearing neuronal antigens generated in vivo from bone marrow.
Science.
2000;290:1779-1782
30.
Maeno M, Mead PE, Kelley C, et al.
The role of BMP-4 and GATA-2 in the induction and differentiation of hematopoietic mesoderm in Xenopus laevis.
Blood.
1996;88:1965-1972 31. Huber TL, Zon LI. Transcriptional regulation of blood formation during Xenopus development. Semin Immunol. 1998;10:103-109[CrossRef][Medline] [Order article via Infotrieve]. 32. Orkin SH, Zon LI. Genetics of erythropoiesis: induced mutations in mice and zebrafish. Annu Rev Genet. 1997;31:33-60[CrossRef][Medline] [Order article via Infotrieve]. 33. Kelley C, Yee K, Harland R, Zon LI. Ventral expression of GATA-1 and GATA-2 in the Xenopus embryo defines induction of hematopoietic mesoderm. Dev Biol. 1994;165:193-205[CrossRef][Medline] [Order article via Infotrieve].
34.
Bader D, Masaki T, Fischman DA.
Immunochemical analysis of myosin heavy chain during avian myogenesis in vivo and in vitro.
J Cell Biol.
1982;95:763-770
35.
Brennan TJ, Olson EN.
Myogenin resides in the nucleus and acquires high affinity for a conserved enhancer element on heterodimerization.
Genes Dev.
1990;4:582-595
36.
Clarke DL, Johansson CB, Wilbertz J, et al.
Generalized potential of adult neural stem cells.
Science.
2000;288:1660-1663 37. Lendahl U, Zimmerman LB, McKay RD. CNS stem cells express a new class of intermediate filament protein. Cell. 1990;60:585-595[CrossRef][Medline] [Order article via Infotrieve]. 38. Rosser AE, Tyers P, ter Borg M, Dunnett SB, Svendsen CN. Co-expression of MAP-2 and GFAP in cells developing from rat EGF responsive precursor cells. Brain Res Dev Brain Res. 1997;98:291-295[Medline] [Order article via Infotrieve]. 39. Eaves C, Fraser C, Udomsakdi C, et al. Manipulation of the hematopoietic stem cell in vitro. Leukemia. 1992;6:27-30. 40. Metcalf D. Haemopoietic colonies: in vitro cloning of normal and leukemic cells. Recent Results Cancer Res. 1977;61:1-227. 41. Metcalf D. The molecular control of cell division, differentiation commitment and maturation in haemopoietic cells. Nature. 1989;339:27-30[CrossRef][Medline] [Order article via Infotrieve]. 42. Stemple DL, Anderson DJ. Isolation of a stem cell for neurons and glia from the mammalian neural crest. Cell. 1992;71:973-985[CrossRef][Medline] [Order article via Infotrieve].
43.
Bhatia M, Bonnet D, Kapp U, Wang JC, Murdoch B, Dick JE.
Quantitative analysis reveals expansion of human hematopoietic repopulating cells after short-term ex vivo culture.
J Exp Med.
1997;186:619-624
44.
Conneally E, Cashman J, Petzer A, Eaves C.
Expansion in vitro of transplantable human cord blood stem cells demonstrated using a quantitative assay of their lympho-myeloid repopulating activity in nonobese diabetic-scid/scid mice.
Proc Natl Acad Sci U S A.
1997;94:9836-9841
45.
Uchida N, Buck DW, He D, et al.
Direct isolation of human central nervous system stem cells.
Proc Natl Acad Sci U S A.
2000;97:14720-14725 46. Osawa M, Hanada K, Hamada H, Nakauchi H. Long-term lymphohematopoietic reconstitution by a single CD34-low/negative hematopoietic stem cell. Science. 1996;273:242-245[Abstract]. 47. Morrison SJ, White PM, Zock C, Anderson DJ. Prospective identification, isolation by flow cytometry, and in vivo self-renewal of multipotent mammalian neural crest stem cells. Cell. 1999;96:737-749[CrossRef][Medline] [Order article via Infotrieve]. 48. Bhatia M. AC133 expression in human stem cells. Leukemia. 2001;15:1685-1688[Medline] [Order article via Infotrieve]. 49. Grinblat Y, Lane ME, Sagerstrom C, Sive H. Analysis of zebrafish development using explant culture assays. Methods Cell Biol. 1999;59:127-156[Medline] [Order article via Infotrieve]. 50. Metcalf D. Stem cells, pre-progenitor cells and lineage-committed cells: are our dogmas correct? Ann N Y Acad Sci. 1999;872:289-304[CrossRef][Medline] [Order article via Infotrieve].
51.
Watt FM, Hogan BL.
Out of Eden: stem cells and their niches.
Science.
2000;287:1427-1430 52. Rossant J, Spence A. Chimeras and mosaics in mouse mutant analysis. Trends Genet. 1998;14:358-363[CrossRef][Medline] [Order article via Infotrieve]. 53. Choi K, Kennedy M, Kazarov A, Papadimitriou JC, Keller G. A common precursor for hematopoietic and endothelial cells. Development. 1998;125:725-732[Abstract]. 54. Morrison SJ, Uchida N, Weissman IL. The biology of hematopoietic stem cells. Annu Rev Cell Dev Biol. 1995;11:35-71[CrossRef][Medline] [Order article via Infotrieve].
55.
Kaufman DS, Hanson ET, Lewis RL, Auerbach R, Thompson JA.
Hematopoietic colony-forming cells derived from human embryonic stem cells.
Proc Natl Acad Sci U S A.
2001;98:10716-10721
© 2002 by The American Society of Hematology.
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 |
|
 |
|
  Y. M. Berkmen and B. A. Zalta Case 126: Extramedullary Hematopoiesis Radiology, December 1, 2007; 245(3): 905 - 908. [Full Text] [PDF]
|
|
|
|
 |
|
 M. Rosu-Myles, E. Stewart, J. Trowbridge, C. Y. Ito, P. Zandstra, and M. Bhatia A unique population of bone marrow cells migrates to skeletal muscle via hepatocyte growth factor/c-met axis J. Cell Sci., October 1, 2005; 118(19): 4343 - 4352. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Martinovic, S. Mazic, V. Kisic, N. Basic, J. Jakic-Razumovic, F. Borovecki, D. Batinic, P. Simic, L. Grgurevic, B. Labar, et al. Expression of Bone Morphogenetic Proteins in Stromal Cells from Human Bone Marrow Long-term Culture J. Histochem. Cytochem., September 1, 2004; 52(9): 1159 - 1167. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. J. Sadlon, I. D. Lewis, and R. J. D'Andrea BMP4: Its Role in Development of the Hematopoietic System and Potential as a Hematopoietic Growth Factor Stem Cells, July 1, 2004; 22(4): 457 - 474. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. M. Bond, M. Mesuraca, E. Carbone, P. Bonelli, V. Agosti, N. Amodio, G. De Rosa, M. Di Nicola, A. M. Gianni, M. A. S. Moore, et al. Early hematopoietic zinc finger protein (EHZF), the human homolog to mouse Evi3, is highly expressed in primitive human hematopoietic cells Blood, March 15, 2004; 103(6): 2062 - 2070. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Galli, A. Gritti, L. Bonfanti, and A. L. Vescovi Neural Stem Cells: An Overview Circ. Res., April 4, 2003; 92(6): 598 - 608. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Q. Daley, M. A. Goodell, and E. Y. Snyder Realistic Prospects for Stem Cell Therapeutics Hematology, January 1, 2003; 2003(1): 398 - 418. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
CD34
), BMP-4 (
), or BMP-4 and EPO (
). (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.
hype and hope.
Nature.
2000;406:361-364