ll

Previews
and is an inventor on multiple patients related to
CD47 cancer immunotherapy licensed to Gilead.
REFERENCES
DiNardo, C.D., Pratz, K.W., Letai, A., Jonas, B.A.,
Wei, A.H., Thirman, M., Arellano, M., Frattini,
M.G., Kantarjian, H., Popovic, R., et al. (2018).
Safety and preliminary efficacy of venetoclax with
decitabine or azacitidine in elderly patients with
previously untreated acute myeloid leukaemia: a
non-randomised, open-label, phase 1b study.
Lancet Oncol. 19, 216–228.
Jones, C.L., Stevens, B.M., D’Alessandro, A.,
Reisz, J.A., Culp-Hill, R., Nemkov, T., Pei, S.,
Khan, N., Adane, B., Ye, H., et al. (2018).
Inhibition of Amino Acid Metabolism Selectively
Targets Human Leukemia Stem Cells. Cancer
Cell 34, 724–740.e4.
Jones, C.L., Stevens, B.M., Pollyea, D.A., Culp-
Hill, R., Reisz, J.A., Nemkov, T., Gehrke, S.,
Gamboni, F., Krug, A., Winters, A., et al. (2020).
Nicotinamide Metabolism Mediates Resistance to
Venetoclax in Relapsed Acute Myeloid Leukemia
Stem Cells. Cell Stem Cell 27, this issue, 748–764.
Lagadinou, E.D., Sach, A., Callahan, K., Rossi,
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(2020). Integrated analysis of patient samples iden-
tifies biomarkers for venetoclax efficacy and com-
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Can. 1, 826–839.

A ‘‘Switch’’ in Time through Genes Aligned:

Unraveling the Genomic Landscape
of HSC Development
April C. Apostol,1 Diego A. López,2,* and Anna E. Beaudin3,*
1Quantitative and Systems Biology Graduate Program, University of California-Merced, Merced, CA, USA
2Division of Microbiology and Immunology, Department of Pathology, University of Utah School of Medicine, Salt Lake City, UT, USA
3Division of Hematology and Hematologic Malignancies, Department of Internal Medicine, University of Utah School of Medicine, Salt Lake

City, UT, USA

*Correspondence: diego.lopez@path.utah.edu (D.A.L.), anna.beaudin@hsc.utah.edu (A.E.B.)
https://doi.org/10.1016/j.stem.2020.10.011

Seeking to define the ‘‘switch’’ from fetal to adult hematopoiesis, Li et al. (2020) performed extensive genomic
and epigenomic profiling of hematopoietic stem and progenitor cells across ontogeny (as explored in this
issue of Cell Stem Cell). Gradual and stochastic changes in genomic and epigenomic regulation suggest
the absence of any specific regulator.

Hematopoiesis is driven by HSCs (he-
matopoietic stem cells) and HPCs (he-
matopoietic progenitor cells). The first
definitive HSCs arise in the early embryo,
and are capable, like adult HSCs, of re-
constituting adult hematopoiesis (Mu €ller
et al., 1994). Fetal HSCs, however, are
still phenotypically and functionally
disparate from adult bone marrow (BM)
HSCs (Copley and Eaves, 2013). They
are less quiescent and more proliferative,
yet display higher self-renewal potential
(Bowie et al., 2007). They also generate
unique lineages including distinct sub-
sets of innate-like lymphocytes, such as
peritoneal B-1a cells (Ikuta et al., 1990;
Kikuchi and Kondo, 2006). These differ-
ences can also be observed in
comparing the distinct transcriptional
profiles and dependencies of fetal and
adult HSCs (Copley and Eaves, 2013)
but the mechanisms that drive the transi-
tion from fetal to adult phenotypes are
under investigation. While previous
studies have described the ‘‘switch’’ in
HSC phenotype from fetal to adulthood
as the downregulation of fetal genes
and upregulation of adult genes during
the neonatal period (Bowie et al., 2007),
neither the origins nor the temporal
mechanisms that govern this switch
have been ascertained. In this issue of
Cell Stem Cell, Li et al. profile the tran-
scriptional and epigenomic landscape
of HSCs and HPCs across ontogeny to
gain insight into the genetic drivers of
this transition (Li et al., 2020).
To approach the question of how the
transition from fetal to adult identity oc-
curs, the authors performed single-cell
sequencing (scRNAseq) of sorted HSCs/
HPCs across ontogeny, at E16.5, P7,

Cell Stem Cell 27, November 5, 2020 ª 2020 Published by Elsevier Inc. 695
 ll
P14, and 8 weeks of age. Perhaps surpris-
ingly, neonatal HSCs/HPCs clustered
independently from either fetal or adult
HSCs/HPCs, suggesting that the transi-
tion between the two identities is not
bimodal, but rather gradual. Using a
quadratic programming approach, the au-
thors determined identity scores for fetal,
neonatal, and adult HSCs/HPCs by inte-
grating previously published datasets en-
riched for both fetal and adult identity
genes. In agreement with the concept of
a gradual transition, there was a progres-
sive shift in expression of adult identity
genes over time in fetal to adult clusters.
Furthermore, these results were recapitu-
lated using an unsupervised approach
that calculated identity scores of all ex-
pressed genes, demonstrating that glob-
ally, fetal and neonatal HSC and HPC
populations were becoming more adult-
like with age.
The authors proposed three possible
models to explain the gradual changes in
HSC and HPC identity: (1) individual cells
may coordinate global changes in gene
expression through strictly regulated net-
works; (2) environmental cues may direct
coordinated changes in gene expression;
and (3) HSCs/HPCs may upregulate adult,
and downregulate fetal, identity genes in
an uncoordinated manner. Analysis of
fetal and adult identity gene expression
within HSCs/HPCs across all three stages
revealed that, although most cells identi-
fied by higher adult identity scores aligned
with adult identity gene expression, they
also expressed fetal identity genes
(Igf2bp2, Hmga2, and Arid3a). In a similar
fashion, cells with greater fetal identity
scores also displayed low levels of adult
identity gene expression (Cpne2, H2-Q7,
and Sdsl). This observation is consistent
with a model in which fetal identity genes
are gradually switched off as adult identity
genes are upregulated in an independent,
stochastic manner.
To gain more clarity on the processes
that govern gradual changes in HSC iden-
tity, the authors probed epigenetic regula-
tion in the transition to adult identity.
Similar to transcriptional analysis, ATAC
sequencing revealed a steady shift
from fetal to adult-like chromatin accessi-
bility across ontogeny. Although adult
enhancer regions became more open as
the HSCs/HPCs matured, fetal identity
genes remained accessible. Through
ChIPmentation, they determined that
696 Cell Stem Cell 27, November 5, 2020
while repressive histones (H3K27me3)
were present on adult promoters and en-
hancers within fetal HSC/HPCs, there
were no differences in H3K27me3 levels
on fetal promoters and enhancers within
adult HSCs/HPCs, indicating that fetal
identity is overwritten by adult program-
ming. These epigenetic changes mirror
observations in transcriptomic data,
further emphasizing the successive and
uncoordinated changes between fetal
and adult HSC/HPC identity.
To probe the potential role of environ-
mental cues in the regulation of fetal-to-
adult transition, the investigators posited
that gradual changes to transcription
and chromatin accessibility during the
neonatal period may be initiated by the
migration of fetal HSCs from fetal liver
(FL) to BM (Mu €ller et al., 1994). To test
this, they compared single-cell transcripts
of HSCs in P0 FL and BM to transcripts of
HSCs in E16.5 FL and P7 BM. P0 HSCs
clustered together regardless of location,
rather than with E16.5 or P7 HSCs, indi-
cating that BM migration alone does not
regulate establishment of adult identity.
Coinciding with the perinatal FL to BM
transition, the robust and sudden expres-
sion of type I interferon (IFN) target genes,
such as Irf7, yielded another possible
driver of the switch between fetal to adult
identity. Bulk-sequencing pinpointed the
induction of IFN activity beginning at
E16.5, with the highest expression occur-
ring between E18.5 and P0, just prior to
and after birth. IFNa expression in E18.5
FL was 3-fold higher relative to earlier
time points. The spike in IFN could not
be explained by exogenous maternal cy-
tokines or a drop in progesterone in late
gestation. Normal fetal gut flora, which
expands between E16 and E18, was
also not the source, as evidenced by
normal levels of IFN expression in HSCs/
HPCs at E18 in germ-free mice. RT-PCR
of IFNa expression within fetal organs re-
vealed high expression in E18 skin
compared to other fetal organs such as
placenta and thymus. This suggests that
the skin may be a primary source of
IFNa, although further investigation is
needed. Analysis of Ifnar / mice re-
vealed that this perinatal spike in IFN pro-
moted HSC expansion, while lack of IFNa
receptor significantly depleted HPC pop-
ulations, particularly the lymphoid biased
Flk2+ HPC-1 (MPP4). Not surprisingly,
the onset of adult identity in Ifnar /
Previews
HPCs (and to a lesser extent, HSCs) was
delayed in both scRNA and ATAC-seq ex-
periments compared to control, due to
Ifnar dependence of several adult identity
genes. Despite this delay, however, HSCs
otherwise retained normal function. These
observations suggest that IFN signaling
coincides with this transition, but whether
it directly regulates the transition from
fetal to adult hematopoiesis remains to
be determined.
Although the authors showcased exten-
sive profiling of HSCs across ontogeny us-
ing rich datasets that contribute greatly to
our current understanding of hematopoi-
etic development, the mechanisms that
drive these changes are still not entirely
clarified. It is possible that gradual shifts
in gene expression and chromatin acces-
sibility reflect a fundamental shift in the
makeup of HSCs and HPCs at different
time points, i.e., the changes observed
are not within the same population of cells
but are within temporally distinct cell pop-
ulations that arise in ‘‘waves’’ across
development. This view is supported by
the observation that the Ifnar / pheno-
type delayed the emergence of specific
Flk2+ progenitors. Because FL hemato-
poiesis is marked by rapid expansion
and differentiation, and HSCs with devel-
opmentally restricted activity exist along-
side canonical HSCs (Beaudin et al.,
2016), alterations to sterile signaling may
impinge upon these normal dynamics.
Furthermore, the phenotypic markers
used to characterize fetal HSC and HPC
populations (Pietras et al., 2015) are not
as well-defined as their adult counterparts
and may not capture the true depth of HSC
heterogeneity. It may therefore be difficult
to parse out mechanistic differences from
phenotype-sorted populations alone; a
broader look at the entire hematopoietic
stem and progenitor compartment may
be insightful. Together, these data provide
elegant insight into how HSCs transition
into adulthood and also reveal how much
still remains to be understood about the
complex nature and dynamics of hemato-
poietic development.
REFERENCES
Beaudin, A.E., Boyer, S.W., Perez-Cunningham,
J., Hernandez, G.E., Derderian, S.C., Jujjavarapu,
C., Aaserude, E., MacKenzie, T., and Forsberg,
E.C. (2016). A Transient Developmental
Hematopoietic Stem Cell Gives Rise to Innate-
like B and T Cells. Cell Stem Cell 19, 768–783.

Previews
Bowie, M.B., Kent, D.G., Dykstra, B., McKnight,
K.D., McCaffrey, L., Hoodless, P.A., and Eaves,
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timed developmental checkpoint that reprograms
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Dynamic Hydrogels
for Investigating Vascularization
Gordana Vunjak-Novakovic1,*
1Columbia University, New York, NY 10032, USA
*Correspondence: gv2131@columbia.edu
https://doi.org/10.1016/j.stem.2020.10.009
Extracellular matrix is known to regulate vascularization by a sequence of multiple factors including mechan-
ical forces. In this issue of Cell Stem Cell, Wei et al. (2020) investigate the roles of matrix biomechanics on
early stages of vasculogenesis by using hydrogels with tunable stiffness and stress relaxation.
Cell fate and function, in vivo and in vitro,
are regulated by the entire context of the
cellular environment, through cascades
of interacting molecular, structural, and
physical signals that change in space
and time. The cells in turn regulate their
environment as they secrete cytokines,
build and degrade the extracellular ma-
trix, influence the surrounding cells, and
generate physical forces. These two-way
interactions between the cells and their
environment are instrumental for native
tissue development, phenotypic changes
associated with diseases, and the assem-
bly and function of engineered tissues. In
recent years, in vitro models of increas-
ingly high biological fidelity have been
developed to enable controllable studies
under conditions more closely emulating
the in vivo milieu (Paek et al., 2019; Ro-
naldson-Bouchard and Vunjak-Nova-
kovic, 2018; Skylar-Scott et al., 2019).
In this issue of Cell Stem Cell, the Wei
et al. (2020) article reports a study of
vascularization, which remains one of
the universal challenges in cell and
potential occurs at the level of hematopoietic
stem cells. Cell 62, 863–874.
Kikuchi, K., and Kondo, M. (2006). Developmental
switch of mouse hematopoietic stem cells from
fetal to adult type occurs in bone marrow after
birth. Proc. Natl. Acad. Sci. USA 103,
17852–17857.
Li, Y., Kong, W., Yang, W., Patel, R.M., Casey,
E.B., Okeyo-Owuor, T., White, J.M., Porter, S.N.,
Morris, S.A., and Magee, J.A. (2020). Single-Cell
Analysis of Neonatal HSC Ontogeny Reveals
Gradual and Uncoordinated Transcriptional
tissue culture. While vascular phenotypes
change with the developmental stage,
parent tissue type, and the state of health
or disease, achieving angiogenesis and
vasculogenesis is critically important
both for the survival of implanted tissues
and for designing tissue models for bio-
logical research. In this study, the focus
is on the effects of the mechanical envi-
ronment, established by a hydrogel used
to encapsulate cells, on the initiation, pro-
gression, and outcomes of early stages of
vasculogenesis.
It is well known that the extracellular
matrix provides not only a three-dimen-
sional setting for the cultured cells but
also serves as a major source of regulato-
ry signals that determine vascular
development and function, including
physical forces (Discher et al., 2009).
With this understanding, the field has
gradually moved from generic, inert,
and nondegradable biomaterials toward
designing those with tunable properties
that can be tailored to control cell differen-
tiation and assembly. In recent years, the
Cell Stem Cell 27, November 5, 2020 ª 2020 Elsevier Inc. 697
ll
Reprogramming that Begins before Birth. Cell
Stem Cell 27, this issue, 732–747.
Mu€ller, A.M., Medvinsky, A., Strouboulis, J.,
Grosveld, F., and Dzierzak, E. (1994).
Development of hematopoietic stem cell activity
in the mouse embryo. Immunity 1, 291–301.
Pietras, E.M., Reynaud, D., Kang, Y.A., Carlin, D.,
Calero-Nieto, F.J., Leavitt, A.D., Stuart, J.M.,
Göttgens, B., and Passegué, E. (2015).
Functionally Distinct Subsets of Lineage-Biased
Multipotent Progenitors Control Blood Production
in Normal and Regenerative Conditions. Cell
Stem Cell 17, 35–46.
field has also started to incorporate
another facet of control where materials
that change their properties in response
to cell-generated signals are being
explored. The present study is an impor-
tant step toward establishing and utilizing
dynamic cellular environments. The au-
thors explored if the activation of specific
mechanosensing mechanisms in cultured
vascular cells, in conjunction with matrix
remodeling, can be utilized to induce the
assembly of vascular networks. To this
end, they developed a viscoelastic hydro-
gel with dynamic crosslinks that pro-
moted cellular contractility and enabled
formation of large focal adhesions and
robust vascular networks. This sequence
of events could not be initiated using
otherwise identical hydrogels with static
covalent crosslinks.
To decouple the effects of stiffness and
matrix dynamics, the authors conducted
experiments with dynamic and static
matrices that had the same initial stiffness
at two different levels: low (soft matrix)
and high (stiff matrix). Cultivation of