SPOTLIGHT

Technical challenges of studying early human development
Peter J. Rugg-Gunn1,2,3,*, Naomi Moris4 and Patrick P. L. Tam5,6

ABSTRACT

Recent years have seen exciting progress across human embryo
research, including new methods for culturing embryos,
transcriptional profiling of embryogenesis and gastrulation, mapping
lineage trajectories, and experimenting on stem cell-based embryo
models. These advances are beginning to define the dynamical
principles of development across stages, tissues and organs,
enabling a better understanding of human development before birth
in health and disease, and potentially leading to improved treatments
for infertility and developmental disorders. However, there are still
significant roadblocks en route to this goal. Here, we highlight
technical challenges to studying early human development and
propose ways and means to overcome some of these constraints.

KEY WORDS: Embryo, Stem cell models, In vitro development,
Fidelity, Reproducibility

Introduction
Early human development is defined as the first 8 weeks after
conception: a period that covers major landmarks in embryogenesis
towards the establishment of a healthy pregnancy. Weeks 1 to 3
incorporate embryo implantation, specification of the germ layers
and the germ line, and establishment of the early body plan. Weeks
3 to 5 represent early organogenesis stages, where progenitors and
their derived cell types in the major organ systems are formed,
which is accompanied by morphogenetic patterning, such as the
closure of the neural tube and looping of the heart. From weeks 5 to
8, organogenesis continues to establish more-mature organs, such as
skeletogenesis in the limb buds and formation of the spine,
alongside overall body growth. After this point, the embryo, which
has acquired species-specific anatomy and functional attributes, is
considered a fetus for the rest of its in utero development.
An informed understanding of the development of the embryo at

this formative phase is important because the patterning events and
cellular interactions that occur during this period are crucial for the
structural organisation of tissues and organ systems that ultimately
lead to viable progeny. Knowledge of normal development is also
informative of the potentially catastrophic effects of perturbations of

development, which can lead to pregnancy loss and congenital
malformations.

Towards these goals, recent technological breakthroughs have
enabled discovery research that was not previously feasible, such as
measuring and modelling tissue patterning and morphogenetic
events in increasingly rich cellular and molecular detail. And yet
significant challenges remain and will need to be addressed in order
to glean a deeper insight into this window in development.
Although this Spotlight focuses only on technical aspects (Fig. 1),
these are inextricably linked to the ethical and legal challenges of
human developmental research, and we direct the reader towards
several excellent reviews on these topics (Ismaili M’hamdi et al.,
2022; Matthews et al., 2021; Pereira Daoud et al., 2020).

Access to embryonic materials
The best way to understand the development of human embryos
would, of course, be to study the human embryo itself. This research
is enabled by generous donation of material specifically for the
purposes of research, which permits researchers to perform high-
quality characterisation of human embryos across different
developmental stages. However, this valuable opportunity can also
represent the biggest technical hurdle in studying early human
development. There is still a barrier to accessing of human
embryonic and fetal material, and, despite the frequent willingness
of individuals to donate surplus embryos to research (Samorinha
et al., 2014), clinical-research frameworks are often lacking, which
then limits opportunities for embryo research. For example, although
it is possible to obtain preimplantation embryos through the donation
of embryonic material generated by in vitro fertilization (IVF),
significant regulatory considerations, including differences across
jurisdictions, can create high entry barriers for research (Lovell-
Badge et al., 2021). Furthermore, the reliance on donated IVF
embryos raises issues of embryo quality, as embryos of assumed
high quality (based mainly on morphological measurements) are
saved for reproductive purposes, leaving those of lesser quality,
deemed not suitable for reproduction purposes, for donation to
research (Dennis et al., 2006). There is also a need to access a
sustainable source of experimental materials that is crucial for long-
term continuing research, so supporting the pathways that enable
embryo donation are vital. Setting up and maintaining the material-
transfer processes requires significant investment by the research
teams and their collaborating fertility clinics. This can also lead to the
underuse, and in some cases loss, of embryonic materials for
research. As potential solutions, more efficient processes could be
imagined, such as the establishment of strategic, collaborative
interactions and simplified processes to allow sharing of donated
materials between laboratories.

Biobanks for archiving fetal tissues from pregnancy terminations,
typically available between 4 and 20 weeks post-conception, provide
a resource of experimental materials underpinning scientific
investigation of embryos at postimplantation stages. An example is
the Human Developmental Biology Resource, which collects and
stores embryonic and fetal tissues through appropriate ethical and

1Epigenetics Programme, Babraham Institute, Cambridge CB22 3AT, UK. 2Centre
for Trophoblast Research, University of Cambridge, Cambridge CB2 3EG, UK.
3Wellcome-MRC Cambridge Stem Cell Institute, Cambridge CB2 0AW, UK. 4The
Francis Crick Institute, London NW1 1AT, UK. 5Embryology Research Unit,
Children’s Medical Research Institute, The University of Sydney, Westmead NSW
2145, Australia. 6School of Medical Sciences, Faculty of Medicine and Health, The
University of Sydney, Sydney NSW 2006, Australia.

*Author for correspondence (peter.rugg-gunn@babraham.ac.uk)

P.J.R.-G., 0000-0002-9601-5949; N.M., 0000-0003-1910-5454; P.P.L.T., 0000-
0001-6950-8388

This is an Open Access article distributed under the terms of the Creative Commons Attribution
License (https://creativecommons.org/licenses/by/4.0), which permits unrestricted use,
distribution and reproduction in any medium provided that the original work is properly attributed.

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© 2023. Published by The Company of Biologists Ltd | Development (2023) 150, dev201797. doi:10.1242/dev.201797

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mailto:peter.rugg-gunn@babraham.ac.uk
http://orcid.org/0000-0002-9601-5949
http://orcid.org/0000-0003-1910-5454
http://orcid.org/0000-0001-6950-8388
http://orcid.org/0000-0001-6950-8388


legal review processes, and supplies samples to registered research
projects. This is an efficient way to oversee and distribute precious
samples that are in limited supply. Support for biobanks requires
stable long-term funding and strong scientific advocacy, both for
existing biobanks and for prioritising new biobanks to meet specific
demand. Centralised banking of week 1-8 embryonic materials
donated for research may overcome the obstacle of sourcing and
coordinating the supply of embryonic materials for research on early
human development.

Likewise, studies of historical human embryo collections have
provided invaluable information about the anatomical characteristics
of developing embryos, and yet there are several major technical
limitations of these resources. For example, the collections have
provided structural image data of the embryo either in toto or from
histological sections, which primarily capture gross morphological
features of the samples. The datasets of these archival materials are
also inherently static. Encouragingly, recent studies have shown that
some archived samples from biobanks are amenable to spatial and

Access to materials Ex vivo culture

In vitro culture

Legislature

Supply

Quality

Clinical
collaboration

Regulatory barriers

Lack of dependable 
resources

Bias against ‘good’ 
samples reserved
for reproduction

Difficulty setting
up networks

Stages

Only samples
older than 4

weeks available

Static info

 No dynamic data

Historical methods

Only morphological 
information available

Benchmarking

Lack of comparison
with an embryonic 

‘ground truth’

Reproducibility

Robust protocols
between experiment

and lab groups

Off-target cells

Cross-species
comparisons

Species-specific 
differences

Longer-term
viability

Manipulations

Optimising genetic
and environmental 

perturbations

Implantation

Developmentally
faithful implantation

?

Production or 
addition of 

abnormal cells

Maintenance in
longer-term culture

Technical 
challenges of
early human
development

research

Collections

Culturing embryos
Cl

in
ica

l s
am

ples

Em
br

yo
-li

ke
m

od
el

s

Fig. 1. Major technical challenges of studying early human development. Some of the challenges facing researchers include the broad categories of
access to research materials, which includes both accessing clinical samples directly (upper left) and extant collections of embryonic and fetal material, such
as the Kyoto and Carnegie collections (lower left), as well as challenges of ex vivo culture of pre-implantation embryos (upper right) and in vitro culture of
embryo-like models (lower right). Each of these categories faces distinct technical challenges, but many also present opportunities to improve with technical
advances, scientific progress, concerted community efforts and improved regulatory frameworks.

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multiomic analysis (Arutyunyan et al., 2023; Zhang et al., 2023),
indicating that the existing collections might be used to generate new
insights by applying modern technologies that were not previously
feasible.
Access to human materials between the peri-implantation and

immediate postimplantation stages, between week 2 to week 4 of
development, is significantly limited because it is often too early in
pregnancy to acquire abortus material and the culture of human
embryos beyond day 14 is forbidden in most jurisdictions (Mathews
and Morali, 2020). Although one 14-17 day [Carnegie stage (CS)7]
gastrula-stage embryo recently became available for embryological
study (Tyser et al., 2021) and, likewise, a study reported the
expression profiling of seven embryos spanning 4-6 weeks (CS12-
CS16) (Xu et al., 2023), the scarcity of embryonic specimens at
weeks 2-4 leaves a gap of knowledge of early development,
precisely at the stage when the embryo is establishing the early body
plan. Obtaining multiple embryos under 8 weeks with consistent
quality and genome integrity is particularly impractical, making the
study of this period of development especially challenging.

Current experimental challenges for research directly on
embryonic materials
When early embryonic materials are obtained, researchers are
confronted with further technical challenges, including limitations
associated with growing embryos ex vivo and in experimental
tractability. In particular, identifying conditions to model the
implantation of human embryos in vitro have been difficult to
achieve. Although protocols exist for culturing human embryos over
the second week of development (Deglincerti et al., 2016; Shahbazi
et al., 2016; Xiang et al., 2020), these embryos lack the appropriate
recapitulation of morphogenesis of embryos implanted in vivo and
the absence of maternal tissues from the extra-embryonic structures
raise concerns that the physiological equivalence of these post-
blastocyst embryos might be compromised. To improve embryo
implantation and development in culture, recent studies have
co-cultured human blastocysts and stem cell-based blastoids with
endometrial cells, which seem to facilitate attachment and could
help to install the signals between trophectoderm and endometrium
(Kagawa et al., 2022; Rawlings et al., 2021; Yu et al., 2022
preprint). Identifying conditions to culture postimplantation
embryos that replicate the morphology of those in vivo is,
therefore, a major goal. This challenge is further impeded by the
current inability to compare in vitro embryo models directly with
matching in vivo postimplantation embryos at the molecular level.
For now, postimplantation human embryo cultures can be
benchmarked against non-human primate embryos as a proxy
(Bergmann et al., 2022; Cui et al., 2022; Nakamura et al., 2016,
2017; Zhai et al., 2022), but evident species-specific differences
may confound such comparisons, and examples of this include
differing transcriptional profiles during early development
(Boroviak et al., 2018; Cui et al., 2022) and differences in the
implantation of embryos into the endometrium (Siriwardena and
Boroviak, 2022).
The difficulty in maintaining later stages (weeks 5-8) of embryo

development in vitro for experimental embryology experiments also
raises a technical challenge that limits the options for dynamic
readouts, interventional experiments and lineage analysis. Although
recent advances have enabled mouse embryos to be cultured for
prolonged periods (Amadei et al., 2022; Tarazi et al., 2022), this has
not yet been achieved for human embryonic samples. Current
efforts to culture embryonic tissue explants are ongoing and can
sustain tissues for a short period in vitro.

Finally, there are challenges regarding the ability to perform targeted
genetic manipulation of human embryonic material, which are crucial
methods for functional genomics studies and, for example, to generate
cell-type and function reporters as means of experimental readout.
Furthermore, the regulations in many jurisdictions that prohibit the
genetic modification of human embryos for research purposes present
an obstacle that impacts the scope of experimentation. A small number
of studies have generated gene knockouts in human embryos (Fogarty
et al., 2017; Stamatiadis et al., 2021, 2022), but these methods are
technically challenging (e.g. often requiring significant protocol
optimisation at each stage of the genetic modification process and
hampered by the limited information about DNA repair processes in
human embryos), and can lead to unintentional off-target genetic
events (Alanis-Lobato et al., 2021; Zuccaro et al., 2020). In addition,
such optimisation experiments that are necessary to improve the
efficiency of genetic manipulations require the use of large numbers of
embryos, which is prohibitive for many research groups. Where
feasible, further investigation of DNA replication and repair pathways
in early human development, combined with new CRISPR-based
technologies, could be the solution to increasing editing efficiencies
and minimising off-target errors.

Opportunities and limitations of stem cell-derived embryo
models
In part to overcome some of these limitations related to the
accessibility and tractability of embryonic material, in vitro stem
cell-based models have been developed that aim to replicate all or
parts of an embryo (Fu et al., 2021; Rossant and Tam, 2021).
Examples include non-integrated stem cell-based systems that
model specific developmental tissues (including amnion and
primordial germ cell formation; Shao et al., 2017), those that
model specific stages (such as gastruloids; Moris et al., 2020), and
those that model particular tissues or selections of tissues [such as
segmentoids, somitoid and axioloids (Miao et al., 2023; Yamanaka
et al., 2023), neural tube-like structures (Karzbrun et al., 2021), and
organoids (Corsini and Knoblich, 2022)]. Other models, termed
integrated models, aim to replicate the development of the entire
conceptus, including the embryo and its extra-embryonic tissues,
such as blastoids and ETX-based models (embryonic-trophoblast-
extra-embryonic endoderm) (Amadei et al., 2022; Heidari Khoei et
al., 2023; Kagawa et al., 2022; Lau et al., 2022; Liu et al., 2021;
Tarazi et al., 2022; Yanagida et al., 2021; Yu et al., 2021).

The availability of integrated and non-integrated embryo models
provides new opportunities for well-controlled interventional
experiments and may provide insights into the biology of human
embryos at this hiatus (Rossant and Tam, 2022). These models are
well suited to investigate the ability of cells to undergo specific
developmental processes, such as morphogenesis, self-organisation
and patterning, without the complexity of the whole conceptus.
Furthermore, their stem cell origins and potential for scalability also
create new possibilities for chemical and genetic screens, physical
manipulation and disease modelling, and are highly tractable for
live-cell and genetic analyses.

However, many of these models are still in their infancy, and
although the range and scope of available systems is wide and ever
increasing, there are significant challenges that limit the questions
they can address and the relevance of knowledge gained from them
(Rossant and Tam, 2021). As a consequence, most models that are
based on a reductionist approach are currently less suited to
examining interactions between multiple tissue or lineage types.
Future endeavours will need to meet these challenges to maximise
their potential for yielding valuable insights into early development.

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Although not exhaustive, we discuss some of these technical
challenges (Fig. 1) and potential solutions below.

Technical limitations of stem cell-based embryo models
A common confounding factor of stem cell-based embryo models is
the formation or inclusion of off-target cell types, i.e. cells that are
not present in an embryo at the developmental stage that is being
modelled. This was a problem that afflicted early blastoid studies,
but now seems to be largely resolved (Kagawa et al., 2022; Zhao
et al., 2021b preprint). However, an ongoing technical challenge
involves the variability in hypoblast induction that is frequently
observed when using current blastoid methods, where the
localisation of hypoblast cells is usually accurate, but for reasons
not fully understood, the number of hypoblast cells in blastoids
varies beyond the range typically detected in blastocysts. Our
limited understanding of how the hypoblast forms in vivo restricts
our ability to optimise blastoid methodologies, although recent
studies have employed innovations such as a two-step lineage
induction system (Yu et al., 2022 preprint) and altering signalling
modulators and engineering parameters (Vrij et al., 2022). Such
examples confirm the need to define sample similarities not only by
transcriptomic methods – including out-group samples to prevent
over-integration (Sozen et al., 2022; Zhao et al., 2021b preprint) –
but also based on additional characterisation, such as spatial
localisation, subsequent cellular and tissue morphogenesis or,
ideally, functional validation. In examples such as this, multi-
lineage reporter cells could be useful for screening for factors that
modulate the induction of different lineage fates in embryo-like
models.
An area for further improvement is that of additional and better-

characterised starting cell types for downstream differentiation and
for assembling into embryo models. For example, efforts could
focus on deriving human hypoblast stem cells directly from
embryos, which has so far not been achieved, but their
availability could lead to improvement of integrated models. This
principle also extends more broadly to other cell types, such as
trophoblast, amnion and mesenchymal cells, to better enable the
formation of ‘assembloids’ involving multiple cell types (Lau et al.,
2022). Concerted efforts to provide well-characterised and highly
controlled culture conditions will also be important for enabling
efficient and reproducible self-organising model generation, where
small deviations can result in significant downstream effects.
Perhaps the most crucial challenge facing embryo-like models is

that the developmental competence of integrated models is unknown,
and there is no applicable technique to assess this. Attempts at the
prolonged culture of human blastoids have so far been few, with
studies demonstrating that blastoids fail to develop for more than a few
days when attached to tissue culture plastic or to monolayers of
endometrial cells (Kagawa et al., 2022; Yanagida et al., 2021; Yu
et al., 2022 preprint). Clearly, improved culture systems that better
mimic the implantation niche are required for maximising the research
opportunities opened up by integrated models.
In parallel, studies in other species could shed light on the

question of developmental competence. Mouse blastoids transferred
into the receptive uteri of mice did not survive for more than 1 or 2
days (Rivron et al., 2018). Bovine blastoids transferred into bovine
uteri triggered a pregnancy-associated signalling reaction in the
recipient animals; however, no assessment of implantation or
blastoid growth was reported (Pinzón-Arteaga et al., 2023). With
further developments in the field, future efforts are likely to focus on
the non-human primate; one recent example using cynomolgus
monkeys has reportedly achieved implantation and pre-gastrulation

development of blastoids after transfer into maternal uteri (Li et al.,
2023). These are exciting advances, but given that we still only have
competent naïve-state pluripotent stem cells from a limited number
of species, and methods to generate and grow blastoids might vary
to some extent between species, there are still substantial hurdles to
be overcome.

Robustness and reproducibility of stem cell-based embryo models
One challengewith any stem cell-based embryomodel is to understand
to what extent they can mimic key features of embryogenesis, and
which features are not replicated in vitro. Additional data from human
embryos themselves will be crucial in enabling accurate interpretation
of observations from embryo models and enable researchers to assess
both the quality and relevance of their models.

Benefits of stem cell-based embryo models include their tractability
and scale, and the improved standardisation that these models should
provide. An imperative is providing quantification when reporting
results, as this information is crucial to understanding the ‘standard’ for
the model. Furthermore, the models must be reproducible both within
and between experiments, to gain statistical power to determine the
differences of outcome after manipulation. In addition, many stem cell-
based systems are subject to cell line differences, media composition
dependence and unknown sources of variability, which reduces the
overall robustness of the protocol. This problem is exacerbated by
reliance on commercial products that may have batch-to-batch
variation, are often proprietary in formulation and can lead to
obstacles outside the control of researchers, such as product
discontinuation or global supply-chain issues. Particularly for the
putatively self-organising systems, where any deviation from an ideal
set-point can strongly affect the experimental outcome, this dependence
can create severe constraints to overcome.

Potential solutions to the reproducibility challenges abound, and
it may help to look to fields outside biology and across disciplines.
For example, studies have used bioengineering approaches, such as
constraining 2D culture to balance the proportions of cell types
(Warmflash et al., 2014), or microfluidic systems to control access to
morphogens (Zheng et al., 2020). Likewise, scaffolds that support
or shape large structures and assembloids that bring several systems
together can devise more-complex and more-complete
representations of embryonic processes (Gupta et al., 2021; Liu
and Warmflash, 2021; Vianello and Lutolf, 2019). In addition,
introducing vascularisation might be able to support the continued
growth and development of structures for longer time periods, and
the addition of supporting cells, such as immune cells, could
improve tissue homeostasis (Zhao et al., 2021a). Similarly,
biochemistry approaches to produce defined and standardised
medium components, and computational advances, including
machine learning to optimise protocols (Anand et al., 2023), have
the potential to enhance our technological capabilities while
improving reproducibility. It is vital that the field promotes and
encourages openness and sharing to enable some of these technical
challenges to be overcome quickly and equitably, and to help refine
the model systems used across research.

Perspectives
Besides the ethical and regulatory hurdles to studying early human
development, there are technical constraints to consider. Some of these
are remarkably challenging and will require a concerted effort to bring
about real change. In our opinion, a key priority is to fully benchmark
embryo-like models to embryos – this is not easy and has still not been
carried out in many cases. Ideally, we need to established not only
whether models are similar in their transcriptional profiles, but also

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whether the molecular pathways, regulatory mechanisms and tempo at
which the models develop are conserved and can replicate the
regulatory logic and dynamics that occur in the natural embryo.
Determining whether or not they do, or whether they follow novel or
atypical pathways through development, will still be of value, but
knowing this status will inform the interpretation of the knowledge
gained from these systems. Likewise, we need to analyse sufficient
embryonic samples to establish the variability in natural embryos in
order to assess the variability observed across the models. Presently,
the burning questions are: what is the range of ‘normal’ values and
parameters, and how can we build systems that are robust and
reproducible, yet allow the cells to display this variability?
To fulfil this goal, we will need solutions to fill the gap in

knowledge at 2-4 weeks of embryo development. One route forward
might lie in being able to culture embryos beyond day 14, which
would shed light on a developmental stage that is all but
inaccessible. Before doing so, scientists will need to tackle the
challenges of keeping high-quality embryonic material embedded in
implantation-like conditions and capable of undergoing sustained
development. Scientists must also engage meaningfully with
regulatory bodies and with society for consideration of the
scientific merit of the investigation, and the legal and ethical
issues of human embryo research (Piotrowska, 2020; Fabbri et al.,
2023). Such groundwork will be necessary for communicating the
scientific merit of this research to policymakers and the public, in
terms of what new knowledge this would bring and the potential
healthcare benefits, which are important for subsequent regulatory
and ethical decision making.
Alongside this, attention should be paid to establishing resources

and frameworks that enable new opportunities for research and
collaboration, both within science and in engaging more widely
with stakeholders. This includes sustainable funding to material
banking and resource collections, setting up interdisciplinary teams
and networks, and new training opportunities. Efforts such as these
would underpin research and lower barriers to teams entering this
field. Such mechanisms facilitate growth of the research discipline
by bringing in new ideas and widening engagement with human
developmental biology.

Acknowledgements
The authors thank members of their groups for helpful discussions, in particular
Dr Irene Zorzan for comments on the manuscript.

Competing interests
The authors declare no competing interests.

Funding
Research in P.J.R.-G.’s group is supported by grants from the Biotechnology and
Biological Sciences Research Council (BBS/E/B/000C0421), the UK Medical
Research Council (MR/T011769/1 and MR/V02969X/1) and the Wellcome Trust
(215116/Z/18/Z and 225839/Z/22/Z). N.M.’s group is supported by the Medical
Research Council-Japan Agency for Medical Research and Development (MR/
V005367/2) and the Francis Crick Institute, which receives its core funding from
Cancer Research UK (CC2186), the UK Medical Research Council (CC2186) and
the Wellcome Trust (CC2186). P.P.L.T. was supported by a Senior Principal
Research Fellowship from the National Health and Medical Research Council of
Australia (1110751) . Open Access funding provided by the University of Cambridge.
Deposited in PMC for immediate release.

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