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Review article
Differentiation plasticity of hematopoietic cells
Thomas Graf
During the past few years an avalanche of papers describing the
conversion of transplanted bone marrow cells into skeletal muscle,
heart, liver, brain, and various epithelial tissues suggested a
surprising degree of hematopoietic stem cell plasticity. For the
clinician they raised the expectation that adult hematopoietic stem
cells might one day become useful for tissue replacement therapies
and in ethically less controversial ways than embryonic stem cells.
For the developmental hematologist these findings were more
incremental in nature, because evidence accumulated over the last
2 decades indicated that hematopoietic cells can be induced to
convert from one lineage into another. Although a number of
excellent reviews have covered the general topic of stem cell
plasticity1-5 (also see http://www.nih.gov/news/stemcell/scireport.html), none has primarily focused on hematopoietic cells, and
little has been written about intrahematopoietic or “lineage”
plasticity. The present review aims to fill this gap. In the first part I
will discuss the capacity of cultured blood cells to transdifferentiate
from one lineage to another and present a model of how this might
happen. In the second part I will review experiments that show that
bone marrow cells can apparently turn into almost any tissue and
discuss the question whether or not the cells that “switch” represent
bona fide hematopoietic cells.
Lineage switches within the hematopoietic
system
Current models of hematopoietic differentiation state that stem
cells become gradually restricted in their differentiation potential
by a succession of symmetric and asymmetric divisions. Each
intermediate progenitor and each monopotent precursor has a
distinct gene expression program that is established and maintained
by specific combinations of transcription factors and chromatin
remodeling components. It is generally assumed that once a cell
commits to a given lineage and acquires a defined phenotype, it can
no longer change its fate. However, as summarized in Table 1 and
Figure 1, and as will be elaborated in the following section, there
are numerous examples where seemingly committed hematopoietic
cells can be induced to convert into cells of another lineage.
Switches between lymphoid and myeloid cells
The first experiments demonstrating that differentiated hematopoietic cells are not cast in stone were reported almost 2 decades ago.
Following reports that 3T3 fibroblasts treated with the demethylating agent 5-azacytidine can differentiate into striated muscle cells,
chondrocytes, and adipocytes, Boyd and Schrader tested the effects
of the drug on Abelson virus–transformed pre–B lymphoma cell
lines. They found that a subset of the cells acquired properties of
From the Albert Einstein College of Medicine, Bronx, NY.
Submitted September 20, 2001; accepted December 28, 2001.
Supported by NIH grants 1 RO1 CA89590-01 and 1 RO1 N543881-01.
BLOOD, 1 MAY 2002 䡠 VOLUME 99, NUMBER 9
macrophages, including the ability to adhere to plastic as well as
phagocytic and esterase activity.6 Later, using pre-B and B-cell
lines immortalized with Eu-myc, Adams and colleagues showed
that v-raf oncogene overexpression had a similar effect. The
v-raf–transfected macrophagelike cells not only expressed a plethora
of myelomonocytic markers (such as the colony-stimulating factor
[CSF]-1 receptor and lysozyme), not seen before the switch, but
also retained immunoglobulin rearrangements characteristic of the
original cells.7 Similarly, a proportion of early B-cell lines ectopically expressing the v-fms oncogene (encoding a constitutively
active form of the CSF-1 receptor) switched into macrophages, as
did murine cells expressing the human CSF-1 receptor gene after
treatment with human CSF-1.8 It is difficult to assess the frequency
of the B lineage-to-macrophage transitions, but it does not appear
to be high because in the various studies only a fraction of the cells
expressing the transgene switched lineage. Most likely the outgrowth of the reprogrammed cells involved selection (see also
below), but the immunoglobulin rearrangements in the resulting
macrophages leave no doubts about their origin. These experiments
are summarized in Figure 2.
In addition to the observed transition of B-lineage cells into
macrophages, one study reported a switch of B-lymphoid cells to
neutrophil granulocytes.9 While creating transgenic mouse lines
involving the max gene, the authors obtained one line (called max
41) that contained dramatically increased numbers of granulocytes
at the expense of B lymphocytes. Although this unbalance per se
does not imply an in vivo lineage switch, the following experiments suggest that this is indeed the case. When max 41 was
crossed with the Eu myc mouse, which has a propensity to form
pre–B cell lymphomas, animals were obtained that likewise
developed pre–B cell lymphomas but, in addition, contained
elevated levels of granulocytes. These lymphomas were transplantable and generated in the recipient not only new lymphoma cells
but also granulocytes of donor origin. Transplantation experiments
with cloned pre–B-lymphoma cell lines derived from this cross
showed that its granulocytes contained the same pattern of
immunoglobulin rearrangements.9 The gene whose altered activity
in the max 41 mice facilitates the switch has not been reported so
far. It is also unclear whether or not a B lymphoid-to-granulocyte
switch occurs normally (see also below).
A surprising degree of plasticity was discovered in B-lineage
cells derived from Pax-5 knockout mice: on the one hand these
cells exhibit characteristics of pre–BI cells (B220⫹, AA4.1⫹,
c-Kitlow, SL chain⫹ but CD19⫺) but, on the other hand, have
characteristics of pluripotent stem cells. In contrast to wild-type
pre–BI cells, they are capable of continuous-to-unlimited selfrenewal on stromal cells and interleukin-7 (IL-7), home to the bone
marrow after transplantation, and can differentiate into most
hematopoietic cell types in vitro and in vivo. As summarized in
Reprints: Thomas Graf, 1300 Morris Park Ave, Bronx, NY 10461; e-mail:
[email protected].
© 2002 by The American Society of Hematology
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BLOOD, 1 MAY 2002 䡠 VOLUME 99, NUMBER 9
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Table 1. Induced hematopoietic lineage switches
Cell type of origin
Resulting cell type
Type of manipulation
Reference
Pre–B lymphoma cell line
Macrophages
5-azacytidine treatment
Eu-Myc B lymphoma lines
Macrophages
v-raf expr
6
7
Pre–B cell lines
Macrophages
v-fms, CSF1-R plus CSF1
8
Pre–B lymphoma
Neutrophil granulocytes
Transplantation
9
Pax-5⫺/⫺ pre–B cells
Mac/Gr, dendrites, NK cells
Different cytokines
11
Pax-5⫺/⫺ pre–B cells
As above, plus T cells
Transplanted into Rag2⫺/⫺
10
Lymphoid prog. (CLPs)
GM-CSF
IL-2 R␣ expr, IL-2
13
14
CD19⫹ B220⫺ cells
Macrophages
Myeloid culture conditions
Pro–T cells
Macrophages
Conditioned medium
22
Macrophage cell line (70Z/3)
B-lineage cells
E2A, EBF expr
15
MEP cells
Myeloid cells
Inducible PU.1 expr
MEP cells
Eosinophils
Inducible C/EBP expr
Myeloid cell line (HD50M)
Eosinophils
Inducible GATA-1 expr
32
Myeloid cell line (HD50M)
MEPs; erythrocytes
High GATA-1 expr; ts Ets
32
30
31,41
Eosinophil cell line (1A1)
MEP cells
FOG-1 expr
33
Erythroid cell line (MEL)
Macrophagelike cells
Inducible PU.1 expr
34
Myeloid cell line (416B)
Megakaryocytic cells
GATA-1, GATA-2 expr
35
Myeloid cell line (FDCW2)
Erythroid cells
GATA-1 expr
36
Myeloid cell line (M1)
Megakar, erythroid cells
GATA-1 expr
37
GM precursors
Ery, eosin, basoph cells
Inducible GATA-1
T. Enver pers comm.,
September 2001
Prog indicates, progenitor; expr, expression; megakary, megakaryocyte; ery, erythrocyte; eosin, eosinophil; basoph, basophil; and pers comm, personal communication.
Figure 3, they can be induced to differentiate into B cells by
restoration of Pax-5 expression and culturing on stromal cells in the
presence of IL-7. However, when transplanted into RAG2 knockout mice they differentiate into T cells10; when grown in the
presence of IL-2 they differentiate into natural killer (NK) cells,
dendritic cells with CSF-1 and granulocyte-macrophage colonystimulating factor (GM-CSF), macrophages with CSF-1, and
neutrophils with IL-3, IL-6, stem cell factor (SCF), and granulocyte
colony-stimulating factor (G-CSF).11 Subclones from immortalized
Pax-5⫺/⫺ lines retain the capacity for multilineage differentiation,
suggesting that the pluripotency of the original culture is not due to
heterogeneity. It appears that the main role of Pax-5 in the
establishment of B-cell commitment is the repression of lineage
inappropriate genes, such as the CSF-1 receptor gene (c-fms),
which is expressed in the pre–BI Pax-5 knockout cells.12 It needs to
be added that cells with a phenotype comparable to the Pax-5
knockout pre–B cells probably do not exist in wild-type animals
(eg, pre–B cells from wild-type mice cannot easily be established
in culture).
Another series of experiments demonstrated that common
lymphoid progenitors (CLPs), can be reprogrammed to become
myelomonocytic.13 These experiments involved the analysis of a
transgenic mouse line that ectopically expresses the human IL-2
receptor ␤ chain. When CLPs from this mouse were cocultured
with OP9 stromal cells plus IL-7 they differentiated into B cells and
NK cells. However, additional treatment with human IL-2 induced
the formation of granulocytes and macrophages (no such cells
developed when CLPs from control mice were grown under the
same conditions). The signaling pathways required for the establishment of the 2 myelomonocytic lineages seem to be separable:
When introduced into CLPs by retroviral infection, a mutant ␤
chain of the receptor lacking the Shc/Lck-binding domain yielded
predominantly macrophages, whereas a Stat5-binding site mutant
yielded mainly granulocytes. Because the IL-2 receptor is not
normally expressed in CLPs, how likely is it that the observed
cytokine-induced reprogramming reflects a process that goes on
during normal lineage commitment? Although the answer is not in,
2 observations suggest that this treatment mimics the effects of
GM-CSF receptor signaling: activation of the IL-2 receptor in
CLPs leads to an up-regulation of the GM-CSF receptor, and
ectopic expression of the GM-CSF receptor followed by stimulation with GM-CSF likewise leads to a lymphoid-to-myeloid
conversion.13 Together, these results indicate that common
lymphoid progenitors are highly plastic and that cytokine
receptor signaling may play a role during normal lineage
commitment.
All of the experiments discussed so far were performed with cells
that were either derived from lymphomas, transformed by an oncogene,
or altered by mutations. This raises the possibility that the observed
plasticity represents a phenomenon restricted to cells with altered
growth properties. However, recent work suggests it can also happen
with normal B-lineage cells, at least in culture. Thus, CD19 antigen, DJ
rearrangement–positive, B220⫺ cells from adult mouse bone marrow
can develop into adherent cells that resemble macrophages when
cultured in the presence of IL-3, IL-6, and GM-CSF. They can also
differentiate into B cells but not into T nor NK cells.14
If B cells can be switched to become macrophages, can such a
switch also be observed in the opposite direction? In the cell system
where this was studied the answer seems to be yes. The experiment
consisted in characterizing the pattern of gene expression of
macrophagelike variants obtained from the pre–B lymphoma cell
line 70Z/3, and restore the down-regulation of transcription factors
that are suspected to play a role in commitment. Of the 3
transcription factors known to be required for progression through
the early stages of B-cell development, E2A, EBF, and Pax5,12 E2A
and EBF were completely absent in the macrophage variant,
whereas expression of Pax-5 was reduced. Enforced expression of
the E12 encoding E2A gene caused a decreased adherence of the
cells, down-regulation of Mac-1 and of c-fms, and induction of a
number B lineage–specific genes, that included the IL7R, EBF, and
RAG1 genes, and the ability to form ␬ light chain in response to
mitogens. Similar, but less dramatic, changes were observed after
enforced expression of the EBF gene, suggesting that it acts
downstream of E2A.15 Experiments in which E2A and EBF were
expressed in an embryonic kidney cell line in the presence or
absence of RAG1 showed that combinations of these genes are
capable of inducing light-chain immunoglobulin rearrangements in
nonhematopoietic cells. The fact that the cells did not acquire a full
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BLOOD, 1 MAY 2002 䡠 VOLUME 99, NUMBER 9
Figure 1. Summary of lineage switches observed between hematopoietic cells,
placed in the context of normal hematopoietic differentiation.44,45 (See text for
details). The black lines indicate normal lineage relationships, the thick red arrows
represent induced switches. (These do not necessarily imply direct transitions.) HSC
indicates hematopoietic stem cell; CLP, common lymphoid progenitor; CMP, common
multipotent progenitor; GMP, granulocyte/macrophage progenitor (in the text also
called “myeloblasts”); MEP, megakaryocyte erythrocyte progenitor; T, T lymphocyte;
B, B lymphocyte; Mac, macrophage; G, neutrophil granulocyte; Eos, eosinophil, Eb,
erythroblast; E, erythrocyte; Meg, megakaryocyte; P, platelet. Mast cells, NK cells,
and dendritic cells have been omitted from the scheme and placement of eosinophils
is speculative. Note that most of the switches were observed with transformed cell
lines in culture and that the cell type designations (both before and after the switch)
may not accurately reflect the phenotype of the normal counterparts.
B-lineage phenotype suggests that the combination of E2A, EBF,
and RAG gene expression is not sufficient for lineage specification
and that additional genes must be involved.16
The examples of lineage plasticity discussed between B cells
and myelomonocytic cells suggest a close developmental relationship. This might reflect the fact that they arise from a common
progenitor in fetal liver (together with T cells, NK cells, and
dendritic cells)17,18 and that they both require the transcription
factor PU.1 for commitment (for reviews, see Singh et al19 and
Orkin20). In addition, lin⫺ fetal liver cells from PU.1 knockout mice
infected with a PU.1-containing retrovirus convert into B-lineage
cells when expressing low concentrations of the factor and into
macrophages PU.1 at about 4-fold higher concentrations.21 Whether
commitment to these 2 lineages depends on the graded expression
of PU.1 at the level of a common progenitor, or whether the factor
is needed to maintain lineage identity after commitment, is not
known. It will be interesting to determine whether increasing the
level of PU.1 expression in B-lineage cells reprograms them to
become myeloid and, conversely, whether reducing the levels of
the factor in myeloid cells induces them to become B lymphoid.
There is also an example of a possible developmental connection between T-lineage cells and macrophages. The thymus is
known to harbor macrophages that act as scavengers to remove
dead cell bodies generated during T-cell development. Although the
developmental origin of these macrophages is obscure, a recent
study suggests that they arise locally through the differentiation of
immature T cells. Thus, culturing of a subset of purified pro–T cells
DIFFERENTIATION PLASTICITY OF HEMATOPOIETIC CELLS
3091
Figure 2. Switch of B-lineage cells into macrophages. Ig rearranged and Ig
germline indicate immunoglobulin heavy chain gene in rearranged or germline
configuration, respectively. For explanation, see text.
in the presence of conditioned medium from a thymic stromal cell
line generated functional macrophages. This lymphoid-to-myeloid
transition could also be observed in the presence of a combination
of IL-6, IL-7, and macrophage colony-stimulating factor (M-CSF;
CSF-1), but at a lower frequency.22
Figure 3. Plasticity of Pax5ⴚ/ⴚ pre–B cells. For explanation, see text.
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Lineage switches within the myeloid/erythroid compartment
Most experiments showing lineage plasticity within the myeloid/
erythroid compartment are based on enforced transcription factor
expression. Perhaps, surprisingly, the factors capable of inducing
commitment are all activators of lineage-specific gene expression,
paralleling observations made with myogenic transcription factors.
In the following I will describe an experimental system that has
allowed investigators to direct the differentiation of cultured cell
lines from one lineage into another in reproducible and predictable
ways, fitting into a logical framework. (For the interested reader the
biology of the relevant factors in a nutshell: GATA-1 and FOG-1
are required for erythroid and megakaryocytic differentiation and
synergize in the induction of genes specific for these lineages.
GATA-1 seems to have a wider role than FOG-1 in that it is also
expressed in eosinophils and mast cells. Importantly, however, both
factors are down-regulated in myelomonocytic cells [for a review,
see Orkin20]. In contrast, PU.1 and C/EBP family members are
required for myeloid cell differentiation and synergize in the
induction of myelomonocytic genes. The inactivation of PU.1 leads
to the abrogation of myeloid [as well as lymphoid] cells; that of
C/EBP␣ to a lack of neutrophil and eosinophil granulocytes; and
ablation of C/EBP␤ to an impairment of macrophage function. All
3 factors are down-regulated during erythroid/megakaryocytic
differentiation [for a review see Tenen et al23]). The system consists
of erythroid/megakaryocytic progenitors transformed by the MybEts–encoding E26 leukemia virus, dubbed “MEPs” for Myb-Etsprogenitors (illustrated in Figure 4A). E26-MEP cells express a
number of megakaryocytic/stem cell surface markers (such as
MEP21/thrombomucin/PCLP1 and GPIIaIIIb/CD4124,25), as well
as GATA-1 and FOG-1, but no myelomonocytic cell surface
markers and no or low levels of PU.1, C/EBP␣, and C/EBP␤.
BLOOD, 1 MAY 2002 䡠 VOLUME 99, NUMBER 9
Inactivating v-Ets in these cells, either by using a temperature
sensitive (ts) mutant of E26 with a point mutation in the DNA
binding domain of Ets, or by deleting it with a FLP recombinase
approach, induces erythroid differentiation.26,27 Conversely, inactivation of v-Myb induces the differentiation of MEP cells into
thrombocytes, the avian equivalents of megakaryocytes and platelets.28 In contrast, introducing into MEP cells retroviruses encoding
oncogenes of the ras pathway, or treating them with phorbolesters,
thus activating protein kinase C (PKC), induces them to become
either eosinophils or myeloblasts, depending on the strength of the
signal (Figure 4B).24,29 These lineage conversions can be mimicked
by enforced transcription factor expression (Figure 4C): PU.1
converts MEPs into cells that resemble myeloblasts (macrophage/
granulocyte precursors),30 whereas C/EBP␣ converts them into
eosinophils.31 They can also move backwards and “sideways”:
forced expression of GATA-1 in myeloid cells induces the formation of either MEP cells or eosinophils, depending on the concentration of the factor. (The transition into MEP cells requires relatively
high levels of ectopic GATA-1, whereas eosinophils require 2- to
3-fold lower levels, reflecting the relative proportions of endogenous GATA-1 expressed in these cell types32). The myeloid cells
can be reprogrammed all the way into erythrocytes, using a 2-step
protocol. If the myeloid-to-MEP cell switch induced by GATA-1 is
performed in a background of ts E26 cells, inactivation of v-Ets
induces erythroid differentiation.32 Finally, enforced expression
of FOG-1 in eosinophils causes these cells (but not myeloblasts)
to acquire an MEP phenotype.33 Again, in all of these conversions, the phenotypic changes involve a complete downregulation of MEP markers concomitant with an up-regulation
of lineage-specific markers.
Similar observations were made in other systems and in normal,
cultured cells. PU.1 has been reported to induce the expression of
macrophage markers in a murine erythroid cell line.34 Conversely,
GATA-1 confers an erythroid/megakaryocytic phenotype in myeloid cells.32,35,36 These cells up-regulate a number of erythroid/
megakaryocytic genes, such as c-mpl, EPO receptor, a-globin,
EKLF, NF-E2, and ets-1, while down-regulating myelomonocytic
genes, such as mac-1 and c-fms.36,37 Finally, a recent study suggests
that primary myelomonocytic progenitors can be reprogrammed
into erythroid cells by enforced expression of GATA-1. As in the
MEP system, eosinophils (and basophils) were also observed (C.
Heyworth and T. Enver, personal communication, September 2001).
As can be seen from the summary in Figure 1, not all possible
transitions between lineages have been described. Whether this
simply reflects limited number of attempts or lack of appropriate
conditions, or whether certain switches are not possible, remains to
be seen. Probably, however, the facility with which cells from one
lineage can transdifferentiate into another depends on how closely
related they are to each other and how much their transcription
factor machinery overlaps.
Selection of a few or instruction of many?
Figure 4. Plasticity of MEP cells transformed by E26 leukemia virus. (A)
Differentiation potential of MEP cells, as demonstrated by the use of ts mutants of
E26 virus with point mutations in either v-ets or v-myb. (B) Differentiation of MEP cells
into eosinophils and myeloblasts (granulocyte/macrophage precursors) by activation
of the Ras or PKC pathways. Different phorbolester concentrations preferentially lead
to the differentiation of either eosinophils or myeloblasts (high and low PKC levels).
(C) Arrows indicate induction of eosinophil, myeloblast, and MEP cell differentiation
by enforced expression of transcription factors shown. GATA-1 low indicates that
lower levels of the factor are required for eosinophil differentiation than for MEP cell
differentiation.
Induced lymphoid-to-nonlymphoid conversions, because of the
irreversibility of immunoglobulin gene rearrangements, leave little
doubt about the occurrence of true reprogramming events. However, in the case of conversions involving myeloid/erythroid cells
this becomes a more difficult question. Here an alternative explanation of the observations made is the selection of pre-existing cells
in the population studied. These instruction-versus-selection alternatives (Figure 5) reflect a problem formally akin to the question
whether cytokine receptor signaling induces cell fate commitment
or merely promotes selective cell survival (for a discussion of this
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BLOOD, 1 MAY 2002 䡠 VOLUME 99, NUMBER 9
DIFFERENTIATION PLASTICITY OF HEMATOPOIETIC CELLS
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tained small amounts of donor type ␣-globin, as revealed by
polymerase chain reaction (PCR) analysis.43
Despite their apparent capacity to retrodifferentiate, there is no
evidence that any of the lineage switches described involve a
detour via an earlier progenitor. For example, if it is assumed that a
B cell-to-myeloid switch involves the formation of a lymphohematopoietic progenitor, then cells from lineages other than macrophages, such as T cells or erythroid cells, should be generated.
However, this has not been described. Likewise, the GATA-1–
induced expression of eosinophil markers in myeloid cells is not
preceded by the up-regulation of MEP cell markers.32,33 Finally, the
CSF-1–induced transition of Pax-5⫺/⫺ pre–B cells into macrophages also does not seem to involve the formation of an earlier
progenitor.11
Do intrahematopoietic lineage switches occur in vivo?
Figure 5. Instruction versus selection models of lineage switching. The cells
illustrate a hypothetical experiment where cultures were exposed to switch-promoting
conditions, and the cell phenotypes determined at different times thereafter (before,
during, and after). In the instruction model the cells gradually change phenotype,
going through a phase where they exhibit mixed traits. In the selection model a
pre-existing minor cell population becomes dominant in the “after” population.
issue, see Kaushansky,38 Metcalf,39 and Enver et al40). In the E26
leukemia virus system the selection alternative could be ruled out
by the use of inducible transcription factors. This was achieved by
fusion to the estrogen receptor (ER) hormone-binding domain,
making their activity dependent on estrogen or tamoxifen treatment. Thus, ␤-estradiol treatment of MEP cells expressing PU.1-ER
led to a change, over a period of 2 to 4 days, into cells resembling
myeloblasts. This involved the concomitant down-regulation of
MEP markers and the up-regulation of myeloid markers via cells
that express both sets of markers, although at reduced levels.30
Likewise, ␤-estradiol treatment of MEP cells containing
C/EBP␤-ER induces them to switch, in this case into eosinophils.31,41 Finally, ␤-estradiol induces myeloblasts expressing GATA1-ER to convert into eosinophils.32 In these experiments a limited
exposure to ␤-estradiol (2-3 days), activating the transcription
factor temporally, led to an irreversible commitment. This observation, together with the fact that the conversion of one population
into another occurred gradually via cells that express both markers
(as opposed to the sudden outgrowth of cells with a distinct
phenotype) strongly argues against the hypothesis of selection of
pre-existing cells.
Does transdifferentiation imply a reversal of differentiation?
The arrows in Figure 1 seem to imply that different cell types can
directly convert into one another by transdifferentiation. However,
it is also possible that these conversions involve retrodifferentiation
to an earlier progenitor. What is the evidence that blood cells can
retrodifferentiate? An example in case are chicken myelomonocytic cells transformed by a ts mutant of v-myb. These cells exhibit
an immature phenotype, resembling myeloblasts, at the permissive
temperature. If shifted to the nonpermissive temperature, they
differentiate into adherent, phagocytic, macrophagelike cells and
cease dividing. Time-lapse experiments showed that this process is
reversible, with most adherent cells acquiring a blastlike morphology and re-entering the cell cycle within 2 to 3 days after shift to the
permissive temperature.42 Another example is the possible formation of primitive erythroid cells after injection of transgenic bone
marrow cells into blastocysts. Here, the resulting embryos con-
Can lineage switches also be shown to occur in vivo, under
physiologic conditions, or are they restricted to experimental
manipulations in cell culture? The existing knowledge, accumulated over many years of research, would suggest that the answer is
no. Thus the concept that cells may “change their mind” during
differentiation flies in the face of the belief that hematopoiesis is a
linear process with multilineage progenitors becoming restricted in
a stepwise but irreversible fashion. The following examples
illustrate this point: (1) The fact that lethally irradiated mice
receiving transplants of short-term repopulating (STR) cells do not
survive suggests that these cells cannot convert into long-term
repopulating (LTR) cells in vivo. (2) The existence of progenitors
that are apparently exclusively committed to the production of
restricted progeny (CLPs, common multipotent progenitors [CMPs],
granulocyte/macrophage progenitors [GMPs], MEPs, B- and T-cell
progenitors44,45) would also argue that, if lineage switching occurs,
it must be at low frequencies. (3) A similar argument can be made
for the high degree of congruency in the phenotype of individual
colonies observed after replating single cells from colony “starts.”46
However, none of these approaches rule out the possibility that
lineage switching occurs in vivo, but at frequencies too low to be
detected with technologies used so far. Finally, even if lineage
switching does not occur in unperturbed animals, it is still possible
that it happens under conditions of stress, such as during immunization, inflammation, or pathogen infection.
How can the question of in vivo plasticity best be approached
experimentally? Lymphoid-to-myeloid cell transitions offer a seemingly straightforward approach because immunoglobulin and T-cell
receptor gene rearrangements are irreversible and nonlymphoid
cells of this origin should be irreversibly marked. For example, the
transition from CD19⫹ cells into macrophages (and B cells),14 if it
happens in vivo, should result in a fraction (if small) of resident
monocyte/macrophages that contain DJ rearrangements. However,
this has (not yet) been reported. In practice, Southern blots may not
be sensitive enough to detect minor fractions of cells with
rearrangements, and PCR techniques require cell populations that
are 100% pure, which is not always easy to achieve. Because no
DNA rearrangements occur in cells of other lineages, it is even
more difficult to demonstrate transitions between nonlymphoid
cells and other cell types. Here a recently developed approach
might be informative. It uses bacterial recombinase (Cre-LoxP or
FLP-FRT) to mark the DNA of cells that express a given lineage
marker. Briefly, mice are created in which the expression of the
recombinase gene, say Cre, is driven by a cell type–specific marker,
say the B cell–specific CD19 gene. These mice are then crossed to a
reporter mouse that contains a DNA cassette framed by LoxP sites,
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sequences that are recognized by the bacterial recombinase. In this
cross, Cre removes the cassette in all CD19-expressing B cells and
this change is retained in all non–B-cell derivatives. Conveniently,
removal of the cassette can be visualized at the single cell level by
␤-galactosidase or green fluorescent protein (GFP) expression,
using reporter strains in which the indicator is driven by the
enhancer/promoter of a ubiquitously expressed gene such as
ROSA2647-49 or ␤-actin.50 The method has successfully been used
to trace the ancestry of wnt-1 or protein O expressing neuroectodermal progenitors, using either Cre50,51 or FLP recombinase.52
However, it also has its pitfalls because any infidelity of gene
expression, if not properly controlled, can lead to incorrect lineage
assignments.
There may be an example of an in vivo lineage switch after all,
involving the formation of dendritic cells. Transplantation experiments showed that CMPs give rise to myeloid type (Mac-1⫹,
CD8␣⫺) as well as to lymphoid-type (Mac-1⫺, CD8␣⫹) dendritic
cells.53 This was also the case, to a lesser extent, for granulocyte/
macrophage precursors, whereas megakaryocyte/erythroid precursors could not be reprogrammed.54 Conversely, common lymphoid
progenitors can also generate both types of dendritic cells, with
T-cell precursors having low and B-cell precursors having no
dendritic cell forming potential.54 Although these experiments
suggest plasticity of immature myeloid and lymphoid cells, another
study indicates that functional macrophages can differentiate into
dendritic cells in vivo. In these experiments microspheres were
injected subcutaneously, attracting inflammatory monocytes, which
subsequently became phagocytic (a hallmark of macrophages).
Some of these phagocytic cells could be shown to migrate to
draining lymph nodes, where they localized to regions within the
nodes that are known to contain dendritic cells but not macrophages. The bead-containing cells also displayed cell surface and
functional markers of dendritic cells.55 A caveat in the interpretation of these data as an example of an in vivo switch is that
myelomonocytic and myeloid dendritic cells might be too closely
related as to represent separate lineages. The comparison of gene
expression profiles by microarray technologies might help to
clarify the issue.
A model of how hematopoietic cells might switch lineage
How can the process of lineage switching be explained at the
molecular level? Before speculating about possible mechanisms, it
is useful to briefly recapitulate current ideas about how blood cell
lineages become specified during the differentiation of multipotent
progenitors. One popular model proposes that hematopoietic stem
cells “sneak preview” a large range of lineage-restricted genes and
specific gene expression programs become dominant after that in a
random process of gene fluctuation.40,56 This concept is based on
the finding that hematopoietic multipotent progenitors express low
levels of genes characteristic for different lineages, such as globin
and myeloperoxidase as well as diverse cytokine receptor genes,57
and that Pax-5⫺/⫺ pre–BI cells express genes from nonlymphoid
lineages.11 However, extrinsic events such as cytokine signaling
might also play a role in commitment,39 with both intrinsic and
extrinsic processes probably reinforcing each other’s effects.
Ultimately, the phenotype of a cell is established and maintained by
combinations of transcription factors that regulate lineage-specific
gene expression programs.
How do ectopically expressed transcription factors manage to
reprogram cells in a reversible fashion? Here I will discuss a model,
derived from studies with the E26 system described earlier,
postulating that the relative excess of a key transcription factor over
BLOOD, 1 MAY 2002 䡠 VOLUME 99, NUMBER 9
another can lead to the “controlled collapse” of a cell’s phenotype.
This involves both the suppression of the cell’s gene expression
program and the activation of novel genes, in a fashion that can be
compared to a reversible chemical reaction, with different phenotypes representing “energy sinks.” As summarized in Figure 4C,
MEPs, eosinophils, and myeloblasts can be converted into one
another by inappropriate transcription factor expression. Lineage
transitions can be induced according to a simple code that defines
cell identity: high GATA-1 plus FOG-1 specifies MEPs; moderate
GATA-1 plus C/EBP␣/␤ specifies eosinophils; and PU.1 plus
C/EBP␤ specifies myeloblasts. The observed phenotypic changes
involve both the up-regulation of lineage-specific genes as well as
the extinction of lineage markers characteristic of the starting cell
population. Whereas activation occurs through the combinatorial
action of the above transcription factors on specific promoter/
enhancer elements (megakaryocyte-specific genes,58-60 myeloidspecific genes,23 and eosinophil-specific genes61,62), suppression
appears to be mediated by a completely different mechanism. Thus,
GATA-1 suppresses PU.1 activity and PU.1 suppresses GATA-1
activity through direct protein interactions.63-66 Therefore, as
illustrated in Figure 6, the stoichiometry between the 2 factors
determines the cell’s phenotype, with an excess of GATA-1
resulting in MEP cells and an excess of PU.1 resulting in myeloid
cells. (The balance between GATA-1 and PU.1 is also decisive in
determining the differentiation of murine erythroleukemia cells,
with an excess of PU.1 maintaining an immature phenotype, and an
excess of GATA-1 inducing terminal differentiation.63) The crossantagonism model in Figure 6 can also explain why the transcription factor–induced phenotypic changes become irreversible, resulting in “commitment.”20,67,68 Expression of high levels of GATA-1
in myeloid cells would interrupt the PU.1 autoregulatory loop,69
activate MEP cell-specific genes, and induce the expression of
endogenous GATA-1, thus establishing its own autoregulatory
loop.70 In turn, an excess of PU.1 in MEP cells would induce them
to acquire a myeloid phenotype, following the same principles but
in reverse order. Thus, once an exogenous transcription factor has
turned on the corresponding endogenous gene the cells become
“committed,” no longer requiring the initial stimulus.
What is the ground state of transcription factor expression in
erythroid/myeloid progenitors and how does commitment get
started? The idea is that multipotent progenitors express both
GATA-1 and PU.1 and that the latter is expressed at relatively low
levels.45 External signaling events or intrinsic fluctuations or both
may tip the balance between the 2 transcription factors, thus
allowing the progenitors to differentiate along one or another
pathway. This interpretation is supported by the observation that
MEP cell clones can differentiate spontaneously into myeloblasts at
low frequencies and that this transition can be accelerated by
treatments that mimic growth factor receptor stimulation.24,29
Is the transcription factor cross-antagonism model more generally applicable? Although a definitive answer is not yet in, a few
more examples can be cited. Within the E26 system, FOG-1
antagonizes C/EBP expression, driving an eosinophil to MEP
transition, and vice versa, although a direct interaction of the 2
factors remains to be demonstrated.20,33 The latter process may
involve the interruption by FOG-1 of an autoregulatory loop of
C/EBP␣.71,72 Another example of transcription factor crossantagonism has been described for Ets-1 and Maf-B. Here overexpression of the macrophage-specific factor Maf-B in erythroid cells
leads to a block of differentiation through a direct interaction with
Ets-1 and its subsequent inactivation, although the relevance of this
observation in the context of normal differentiation is unclear. It is
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BLOOD, 1 MAY 2002 䡠 VOLUME 99, NUMBER 9
Figure 6. Model of hematopoietic lineage switching. The scheme is based on the
reciprocal lineage switches between MEP cells and myeloblasts induced by GATA-1
and PU.1 (see text). The left part of the figure illustrates that the balance between the
2 factors determines the cell’s phenotype; MEPs are specified by an excess of
GATA-1, myeloblasts by an excess of PU.1. The right part of the figure illustrates what
might be going on in the nucleus. In MEP cells GATA-1 activates multiple target genes
(A, B, C), as well as possibly GATA-1 itself. In myeloid cells PU.1 activates another
set of target genes (X, Y, Z), including PU.1 itself. Enforced expression of PU.1 in
MEP cells leads to the inactivation of GATA-1 through direct protein interactions.
GATA-1, when overexpressed in myeloid cells, inactivates PU.1 through a similar
mechanism (the cross-antagonism between the 2 factors is illustrated by the T bars).
possible that the cross-antagonism principle is also operative
within the lymphoid compartment. Thus, a direct protein interaction between PU.1 and Pax-5 has been reported and this might play
a role in the decision of whether a cell acquires a myeloid cell or a
B-cell fate, respectively.73
The handful of transcription factors covered in this review are
not the only players involved, and readers are referred to other
reviews discussing additional factors (for myeloid/erythroid lineages, see Tenen et al,23 Yamanaka,74 Nagamura-Inoue et al75; for
lymphoid lineages, see Rothenberg et al,76 Glimcher and Singh,77
Glimcher and Murphy78). In addition, transcription factors involved in lineage specification almost certainly do not act in simple
2-way combinations but are part of cell-specific transcription factor
networks, where they participate in positive and negative crossregulations at transcriptional and posttranscriptional levels. One
can imagine that these networks have a 3-dimensional shape that
changes as the cell’s transcription factor composition is altered.
Such “conformational changes,” rather than being merely consequences of differentiation, might constitute a driving force during
commitment.79
Other mechanisms, not involving direct transcription factor
interactions, also play a role during hematopoietic lineage specification and transdifferentiation. Examples include competition for
the general coactivators CBP/p30080-82 and histone-modifying
factors that are involved in chromatin remodeling. These processes
help to establish and stabilize the differentiated state.83
Conversions between hematopoietic and
nonhematopoietic cells
Until recently it has been widely assumed that there are 2 major
classes of stem cells: embryonic stem cells derived from early
DIFFERENTIATION PLASTICITY OF HEMATOPOIETIC CELLS
3095
embryos, able to replenish all cell types in the animal, and stem
cells located in various organs of the body, dedicated to the
replenishment of specific tissues, such as blood, muscle, and brain
(for a review, see Fuchs and Segre84). This assumption was shaken
a few years ago by the observation that bone marrow cells can
differentiate into muscle cells after transplantation,85 followed by
reports that cells derived from brain and muscle can fully reconstitute the hematopoietic system of lethally irradiated mice.86-88 Are
all of these conversions due to the plasticity of hematopoietic cells?
It is particularly difficult to assess whether true conversions occur
from nonhematopoietic to hematopoietic tissues because hematopoietic stem cells (HSCs), entering these tissues via the circulation,
might act as “contaminants.” Indeed, recent experiments revealed
that skeletal muscle tissue contains multilineage hematopoietic
progenitors and LTR-HSCs. These cells were clearly derived from
bone marrow because HSCs recovered from the muscle of mice
that were previously reconstituted with marked donor cells were of
donor origin.89 In addition, cells in the skeletal muscle capable of
generating hematopoietic colonies and of hematopoietic engraftment are CD45⫹, Sca-1⫹, whereas cells that contribute to muscle
cell formation after transplantation are CD45⫺ (M. Goodell,
personal communication). These findings suggest that the muscleto-hematopoietic cell switch originally described can be attributed
to hematopoietic progenitors homing to muscle tissue. The reported
switch of neuronal precursors to blood cells has also come under
question. Thus, extensive experiments involving the transplantation of neural stem cell cultures (“neurospheres”) into irradiated
mice, following the protocol of Bjornson et al,86 failed to reconstitute the hematopoietic system (C. Morshead, personal communication, October 2001). In addition, experiments in which neurospheres were injected into mouse blastocysts revealed the presence
of donor-type cells in a wide variety of tissues in the resulting
embryos with the notable exception of hematopoietic tissues.90
The remainder of this review will focus on conversions from
hematopoietic to nonhematopoietic tissues. The different types of
conversions that have been reported are summarized in Figure 7
and Table 2. Using mice transgenic for the muscle-specific myosin
light chain 3F promoter driving LacZ (MLC3F-lacZ mice) as
donors, Ferrari and coworkers injected bone marrow cells into leg
muscles regenerating after chemical injury. When they analyzed
muscle sections 2 to 5 weeks later they detected ␤-galactosidase–
positive muscle fibers.85 These authors also described that
muscle cells can be recruited from the circulation. For this,
Figure 7. Summary of hematopoietic-to-nonhematopoietic cell conversions.
The scheme summarizes experiments in which bone marrow cells were transplanted
into mice, which were then analyzed several months later for hematopoietic and
nonhematopoietic donor type cells.
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Table 2. Bone marrow cells yielding nonhematopoietic tissues after transplantation
Donor cells, fraction
Nonhematopoietic derivative
Recipients
Reference
BM, adherent cells
Skeletal muscle
Scid/bg mice
BM, SP cells
Skeletal muscle
Mdx mice
85
88
BM
Skeletal/cardiac muscle
Mdx mice
91
Lin⫺Kit⫹ cells
Endothelial cells, cardiac muscle
Irrad, infarct mice
93
CD34lo, Sca-1⫹Kit⫹ cells
Endothelial cells, cardiac muscle
Irrad, infarct mice
94
96
CD34⫹ lin⫺ cells
Hepatocytes
Irradiated mice
Lin⫺Sca-1⫹Kit⫹ cells
Hepatocytes
FAH⫺/⫺, irrad mice
BM
Astrocytes, microglia
Irradiated mice
BM
Immature neuronal cells
PU.1⫺/⫺ mice
102
BM
Immature neuronal cells
Irrad mice
103
97
101
CD34 high, Kit⫹ cells
Various epithelial cells
Serial transplants
104
Cultured CD34⫹ cells
Vascular endothelial cells
Canine model
113
CD34⫹Kit⫹Flk1⫹AC133⫹ cells
Vascular endothelial cells
Irrad mice
114
BM
Liver oval cells, hepatocytes
Rats, liver injuries
137
BM indicates bone marrow; SP, side population; irrad, irradiated; and infarct, infarcted.
lethally irradiated mice received transplants of MLC3F-lacZ
bone marrow; a muscle injury was induced after 5 weeks and
muscles were analyzed 2 to 3 weeks later. Most animals again
contained labeled myocytes, albeit at lower frequencies than
after direct injection.85 Several groups reported that transplantation of bone marrow cells into mice with a defect in the
dystrophin-related mdx gene induced the formation of Y chromosome–positive, Mdx-expressing muscle fibers.88,91 In one of
these studies, as few as 2000 “side population (SP) cells,”
purified from bone marrow after Hoechst 3042 staining, were
able to generate muscle cells.88 However, the efficiency of the
tissue replacement was exceedingly low and no ameliorating
effects on muscular dystrophy could be observed.92 In addition
to donor-origin skeletal muscle cells, the animals that received
transplants had cardiomyocytes, particularly when previously
infarcted by arterial occlusion. The new cardiomyocytes located
preferentially to the ischemic region.91,93,94 Lin⫺Kit⫹ cells, but
not Lin⫺Kit⫺ cells, exhibited cardiomyogenic potential.93 The
observation that cardiomyocyte-generating cells, which alleviate myocardial infarction, can be generated by mobilizing
blood-borne cells by treatment with G-CSF and SCF caused
great excitement among clinicians.95 Whether the relevant cells
are of hematopoietic origin has yet to be established.
Although the reported transitions of bone marrow–derived cells
into muscle cells were unexpected, a series of papers describing
conversions into epithelial cells were even more surprising. Several
studies reported the formation of liver cells from transplanted bone
marrow in mice,96,97 rats,98 and humans.99 Others suggested that
bone marrow cells can turn into neuronal cells. Following publications describing donor-type microglia and astrocytes in mice that
received bone marrow transplants,100,101 Mezey et al102 and Brazelton et al103 reported the detection of cells with neural markers in the
brain PU.1 defective or irradiated recipients, respectively. However,
these studies failed to demonstrate the formation of mature/functional
neurons and their hematopoietic origin is also unclear. Finally, a
recent paper described the in vivo conversion of bone marrow cells
into epithelial cells of various organs in mice that underwent
transplantation.104 This paper will be discussed in more detail below.
Which of the bone marrow-to-nonblood cell conversions can
be attributed to bona fide hematopoietic cells?
If it is already difficult to rigorously establish hematopoietic cell
plasticity using cultured cell lines, it is even more difficult to
conclude that hematopoietic cells are plastic based on transplanta-
tion experiments. There is always the possibility that the bone
marrow hosts a variety of dedicated tissue-specific stem cells, such
as muscle stem cells, neuronal stem cells, and hepatic progenitors.
The following requirements have to be met before concluding that
a hematopoietic-to-nonhematopoietic transdifferentiation has been
demonstrated (“the gold standard”): (1) Show that bone marrow
cells purified on the basis of established cell surface markers cannot
only reconstitute irradiated mice but can also participate in the
formation of nonhematopoietic tissues. (2) Demonstrate that this
can be done with a single cell or else clonal reconstitution of both
types of tissues. (3) Demonstrate that the bone marrow–derived
nonhematopoietic cells are differentiated and functional.
Although there is (as yet) no evidence for the presence of
dedicated muscle, neuronal, or liver cell progenitors in the bone
marrow, this tissue is certainly heterogeneous and appears to
contain various types of hematopoietic as well as nonhematopoietic
progenitors (Figure 8). Based on their long-term or short-term
repopulation capacity of lethally irradiated hosts, 2 types of
hematopoietic stem cells can be distinguished: LTR-HSCs and
STR-HSCs (eg, see Lanzkron et al105). Both types of HSCs in the
mouse are Lin⫺, CD45⫹, Kit⫹, Sca-1⫹, but LTR-HSCs are mostly
CD34⫺CD38⫹, whereas STR-HSCs are CD34⫹CD38⫺.106 However, definition of a cell’s potential by its phenotype is complicated
by the fact that expression of a given marker does not necessarily
indicate a cell’s developmental potential. Thus, mice treated with
5-fluorouracil (to deplete cycling progenitors) develop LTR-HSCs
that transiently down-regulate c-Kit and up-regulate Mac-1107 and
reversibly modulate CD34 expression.108 In addition, CD34 expression levels of murine, and possibly human, HSCs is age dependent,
whereas the percentage of CD34⫹ HSCs is high in fetal liver and in
bone marrow of young mice it decreases dramatically in adult
animals, with only about 20% of HSCs expressing CD34.109,110
In addition to hematopoietic cells, the bone marrow cavity
probably contains angiogenic precursors (also called endothelial
progenitor cells or angioblasts), which in humans are CD34⫹,
Kithigh, Flk-1⫹, Tie-2⫹, AC133. Endothelial progenitor cells have
been found in the circulation111-113 and can be mobilized by
G-CSF.114 These cells, when transplanted, give rise to mature
endothelial cells in vessels and can be selected by adherence and
culture under endothelial conditions.114 However, some of the
selected cells might be of hematopoietic origin because human
monocytes cultured in the presence of angiogenic growth factors
have been shown to acquire properties of endothelial cells.115-117 A
third class of stem cells contained in the bone marrow are
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BLOOD, 1 MAY 2002 䡠 VOLUME 99, NUMBER 9
Figure 8. The bone marrow cavity contains different types of hematopoietic and
nonhematopoietic progenitors. HSC indicates hematopoietic stem cells; MSC,
mesenchymal stem cells; AnBl, angioblasts. The boxes represent “drawers” that
indicate differentiation potential. Colors within the drawers indicate tissue conversions that have been described or postulated (question marks). The presence of
angioblasts in the bone marrow is speculative. Additional types of precursors may
also exist.
mesenchymal stem cells (MSCs, also called stromal cells) which
are CD45⫺, CD34⫺, Mac-1⫺. Like macrophages and angiogenic
cells, they can be selected by plastic adherence.118 Cultured human
MSCs have been shown to differentiate into adipocytes, chondrocytes, osteocytes, and muscle cells.119,120 Perhaps surprisingly, they
were also found to differentiate into cardiomyocytes following in
utero transplantation in a sheep model.121 The amazing differentiation potential of MSCs does not stop here. Cultures of rat, human,
and mouse MSCs can differentiate into neuronlike cells in culture,122,123 and injection of mouse MSCs into neonatal mouse brains
gives rise to astrocytes and possibly neurons.124
Which of the reported conversions of bone marrow to nonhematopoietic cells involve bona fide hematopoietic progenitors, rather
than reflecting developmental potentials of other types of progenitors? Here 2 papers will discussed in some detail. In an elegant
study, Lagasse et al97 transplanted bone marrow cells from a mouse
ubiquitously labeled with ␤-galactosidase (ROSA26 LacZ mouse125)
into lethally irradiated mice with a congenital liver defect (the
protocol used is illustrated in Figure 9). This resulted in a rescue of
the animals from disease onset and death through the restoration of
the hematopoietic system and the formation of large ␤-galactosidase–positive colonies of parenchymal liver cells. Sorting experiments revealed that effective cell fractions were Kit⫹, Lin⫺ and
Sca1⫹, thus exhibiting a phenotype corresponding to that of HSCs.
As little as 50 Lin⫺Sca-1⫹Kit⫹ cells were able to both reconstitute
hematopoiesis and to produce donor-derived liver cells. Although it
cannot be excluded that the bone marrow contains, in addition to
HSCs, contaminating liver progenitors, this appears unlikely
because both biologic activities correlated in limiting dilution
experiments. However, liver stem cells (“oval cells”) are known to
share a number of markers with HSCs, such as CD34 and Kit (eg,
see Crosby et al126).
That a single bone marrow–derived stem cell can differentiate
not only into blood cells but also into a wide variety of epithelial
cells is indicated by work of Krause et al.104 In this study, the
authors enriched Lin⫺, quiescent cells from the bone marrow,
labeled them with a vital dye, transplanted them into a lethally
irradiated primary recipient, and recovered donor type cells from
the bone marrow 2 days later. (The dye-positive cells were 500- to
1000-fold enriched for LTR-HSC activity and contained approximately 10 times more CD34⫹Sca-1⫹ cells than the starting
DIFFERENTIATION PLASTICITY OF HEMATOPOIETIC CELLS
3097
population.) Then, single cells obtained by limited dilution were
injected into secondary recipients and the animals receiving
transplants were analyzed several months later for donor contribution, using fluorescent in situ hybridization for the identification of
Y chromosome–positive cells. A sixth of these animals were fully
reconstituted in their hematopoietic system and showed, in addition, significant levels of donor engraftment in alveoli, bronchi,
esophagus, stomach, small and large bowel, skin, and bile duct (as
well as in liver hepatocytes, D. Krause, personal communication,
July 2001). Some of these cells, such as lung type II pneumocysts,
were shown to be functional through the detection of messenger
RNA encoding a surfactant protein characteristic of these cells.
Because of the high percentages of donor-derived epithelial cells
observed (up to 20%), there must have been a considerable
expansion of the originally transplanted cell, although perhaps
oddly, no distinct y-chromosome–positive colonies were found.
These results raise a number of questions: Can donor-type epithelial cells also be observed in animals that received primary
transplantations or is the serial transplantation protocol used a
prerequisite? If the latter is true, what happens to the HSCs in the
bone marrow of the primary recipients? Do the nearly totipotent
cells described still represent classical HSCs or are they more akin
embryonic stem cells? Where does the cell expansion and the
mesenchymal-to-epithelial cells transition occur—in the bone
marrow or after the cells home to epithelial tissues? Is tissue injury
necessary for engraftment in epithelial tissues (see also below), and
what are the homing clues?
Although experiments discussed strongly indicated the potential of bona fide hematopoietic cells to convert into various types of
epithelial cells, this is less clear for the reported conversions into
muscle, endothelial cells, and brain cells. Considering that the bone
marrow contains various types of stem cells (Figure 8), it is
possible that the donor-derived muscle and endothelial cells
observed after bone marrow transplantation85,93,94 originate from
mesenchymal stem cells and angiogenic precursors, respectively.
Likewise, the neuronal cells observed in the transplantation
experiments of Mezey et al102 and Brazelton et al103 might have
arisen from mesenchymal stem cells. As an alternative to this more
Figure 9. Protocol used to show the in vivo conversion of hematopoietic cells
into parenchymal liver cells.97
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conventional interpretation, it has recently been postulated that a
universal adult stem cell exists that comes in different coats.5 In this
extreme view the various types of stem cells residing in the bone
marrow, brain, heart, and muscles are considered to represent
different states of a universal adult progenitor whose phenotype is
defined by its local environment. These stem cells can move from
one tissue into another via the circulation and are more plastic in
early than in more differentiated stages.5 Accepting this view,
which might be called the “Heisenberg principle of stem cell
biology,” the issue of stem cell plasticity becomes almost semantic.
Taking it to the limit, the only way to prove plasticity would be in
instances where already differentiated cells can be shown to
transdifferentiate into other, well-defined cell types. That this is a
possibility is illustrated by examples from the world outside Blood.
Myocytes have been shown to “transdifferentiate” into proliferating stem cells during tissue regeneration in amphibians127 or
following expression of the msx-1 gene,128; C/EBP␤ has been
shown to induce a conversion of pancreas to liver cells129; and
oligodendrocyte precursors have been induced to retrodifferentiate into stem cells that can go on to become astrocytes and
neuronal cells.130
Do hematopoietic-to-nonhematopoietic conversions occur
normally during embryonic development and in adult animals?
Almost all reported conversions between hematopoietic and nonhematopoietic cells in vivo were carried out by forced tissue
relocations in irradiated or injured animals. For example, the
experiments demonstrating a conversion from hematopoietic to
epithelial cells were performed with irradiated animals. Irradiation,
on the other hand, has been shown to destroy not only hematopoietic but also endothelial and epithelial tissues.131 It is therefore
important to know whether such tissue conversions also occur
under physiologic conditions. This question seems irrelevant if the
ultimate purpose is to use somatic stem cells in tissue replacement
therapies. However, knowledge about existing cellular processes
might well be useful in guiding the development of tissue
replacement protocols. In principle, tissue conversions might occur
during embryonic development, involving the migration of hematopoietic cells from the yolk sac, aorta/gonad/mesonephros (AGM),
or fetal liver to the anlagen of nonhematopoietic organs. For
example, it has been reported that macrophages migrate from the
yolk sac into the neural fold area,132 possibly seeding the brain with
microglia (strictly speaking, however, this would not represent a
“switch,” because microglia are phagocytic cells that express
macrophage markers). So far, there are no reports showing a
reprogramming of hematopoietic cells nonhematopoietic tissues
during development, but some evidence suggests a developmental
flow in the opposite direction, from endothelial to hematopoietic
cells. Thus, early blood cell progenitors share a number of markers
with endothelial cells133 and colony assays indicate the existence of
a common endothelial/blood cell progenitor, the “hemangioblast.”134 In addition, endothelial cells within the chick embryonic
aorta labeled with a vital stain or with a LacZ-encoding retrovirus
were shown to develop into blood cells.135,136 These experiments
were interpreted as evidence for cells with a dual hematopoietic
and endothelial differentiation potential. However, it is also
conceivable that hemangioblasts/hematopoietic cells can originate
from fully functional endothelial cells,133 such as through local
signaling events that induce their transdifferentiation. This issue
might be hard to resolve. Clearly, there is a close relationship
between endothelial cells and cells from other mesodermal lineages, a concept strengthened by the recently described “mesoangioblast” discovered in birds. This embryonic aorta-derived stem cell,
which expresses CD34, c-Kit, and Flk-1, is capable of giving rise to
vessels, blood, cartilage, bone, as well as to smooth, skeletal, and cardiac
muscle cells (G. Cossu, personal communication, February 2002).
Conclusions
The cell culture experiments reviewed here leave little doubt about
the intrinsic plasticity of hematopoietic cells, with cells from
almost any lineage being capable of switching into cells of another
lineage. The switches appear to represent true transdifferentiation
events, with cells moving backwards as well as sideways within the
differentiation tree. Reprogramming is initiated either by extrinsic
or intrinsic events that result in changes in transcription factors
active in commitment and maintenance of lineage-specific gene
expression programs. The transcription factors involved in commitment appear to have a dual role; they not only set up novel gene
expression programs through transcriptional activation but also
extinguish old programs through inactivating interactions with an
antagonistic transcription factor. Whether committed hematopoietic cells can “change their mind” during normal development and
in adult animals, perhaps in situations of stress, is an area that needs
to be explored. Knowledge of such mechanisms may shed new
light on the evolution of the hematopoietic system, its homeostasis,
and its adaptation to pathogen invasion during innate immunity.
The interpretation of the hematopoietic-to-nonhematopoietic
cell conversions described is complicated by the fact that the bone
marrow cavity not only hosts bona fide hematopoietic precursors
but 2 or more additional types of stem cells. In most studies it is
therefore difficult to conclude with certainty that a blood-tononblood cell switch has actually occurred. Thus, although it is
clear that hepatocytes and other epithelial cell types can derive
from transplanted hematopoietic stem cells, the conversion to
skeletal and cardiac muscle, neuronal cells, macroglia, and vessels
is less clear and could involve mesenchymal stem cells as well as
angiogenic precursors. And, as for the intrahematopoietic lineage
conversions, it remains to be seen whether blood-to-nonblood
conversions occur during normal embryonic development or
even as an ongoing process in adults. Another interesting area
for future investigations is to define stem cell niches, and how
different microenvironments influence the developmental potential of bone marrow–derived stem cells.
Some of the reports covered in this review have suggested that
hematopoietic stem cells can serve as an alternative to embryonic stem
cells for tissue replacement after injury or disease, such as stroke and
heart infarct, Parkinson disease, cystic fibrosis, muscular dystrophy, and
congenital liver diseases. Because of the vast amount of knowledge
accumulated in their handling for the purpose of transplantation and
their relatively easy isolation, they would indeed be an ideal source for
organ repair and reconstitution. The clinical prospects for the use of
adult hematopoietic stem cells for these purposes largely depends on
whether it will be possible to develop protocols that increase the tissue
conversion frequencies and noninvasive strategies that involve the
mobilization of bone marrow progenitors. Clearly, the field is still in its
infancy and basic research will have to go hand in hand with clinical
research to explore the suitability of hematopoietic stem cells for tissue
replacement therapies.
Acknowledgments
I would like to thank Todd Evans, Gordon Keller, Kelly McNagny,
Fabio Rossi, Gwen Randolph, and members of the Graf lab for
discussions and suggestions.
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2002 99: 3089-3101
doi:10.1182/blood.V99.9.3089
Differentiation plasticity of hematopoietic cells
Thomas Graf
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