Sheng BMC Developmental Biology (2015) 15:44
DOI 10.1186/s12861-015-0094-5

RESEARCH ARTICLE

Open Access

The developmental basis of mesenchymal
stem/stromal cells (MSCs)
Guojun Sheng

Abstract
Background: Mesenchymal Stem/Stromal Cells (MSCs) define a population of progenitor cells capable of giving rises
to at least three mesodermal lineages in vitro, the chondrocytes, osteoblasts and adipocytes. The validity of MSCs
in vivo has been questioned because their existence, either as a homogeneous progenitor cell population or as a stem
cell lineage, has been difficult to prove. The wide use of primary MSCs in regenerative and therapeutic applications
raises ethical and regulatory concerns in many countries. In contrast to hematopoietic stem cells, a parallel concept
which carries an embryological emphasis from its outset, MSCs have attracted little interest among developmental
biologists and the embryological basis for their existence, or lack thereof, has not been carefully evaluated.
Methods: This article provides a brief, embryological overview of these three mesoderm cell lineages and offers aframework of ontological rationales for the potential existence of MSCs in vivo.
Results: Emphasis is given to the common somatic lateral plate mesoderm origin of the majority of body’s adipose
and skeletal tissues and of the major sources used for MSC derivation clinically. Support for the MSC hypothesis alsocomes from a large body of molecular and lineage analysis data in vivo.
Conclusions: It is concluded that despite the lack of a definitive proof, the MSC concept has a firm embryological basis
and that advances in MSC research can be facilitated by achieving a better integration with developmental biology.

BackgroundThe concept of mesenchymal stem/stromal cells(MSCs) [1, 2], also referred to as skeletal stem cells[3] or adipose stem cells [4], was first introduced byAlexander Friedenstein about half a century ago.
Building on the hematopoietic stem cell (HSC) work
pioneered by another Russian scientist Alexander
Maximow, Friedenstein described a population ofbone marrow derived cells which are distinct fromthe HSC population and are osteogenic in vivo andclonogenic in vitro [5–7]. These bone marrow-derived
MSCs were later shown to be able to self-renew, formcolonies and differentiate into a multitude of mesodermal cell types in vitro [8]. MSC populations withsimilar multi-lineage differentiation potentials in vitro
have since been obtained from many non-bone marrow tissues [9], including the adipose tissue [10, 11],amniotic fluid [12, 13], placenta [14], umbilical cord
[15–17] and peripheral blood [18]. Clinical relevance
Correspondence: sheng@kumamoto-u.ac.jp
Sheng Laboratory, International Research Center for Medical Sciences,
Kumamoto University, Kumamoto 860-0811, Japanof MSCs has been highlighted by their capacity forin vivo differentiation and engraftment and by theirefficacy in promoting wound healing, tissue regeneration and immunosuppression [19–25].
Common for a field attracting a wide scope of interest
from researchers, MSC biology has witnessed confusions
and controversies concerning its name, definition, isolation and characterization criteria, in vivo relevance, andinstitutional and ethical regulations of its clinical use. In
an attempt to standardize studies in this field, theInternational Society for Cellular Therapy came up withguidelines in 2006 for MSC characterization [1]. The
name “multipotent mesenchymal stromal cells” was preferred and three minimal criteria were outlined: 1) being
plastic-adherent in culture; 2) exhibiting a set combination of surface antigens (CD73+, CD90+, CD105+,
CD34-, CD45-, CD11b-, CD14-, CD19-, CD79a- and
HLA-DR-); and 3) being able to differentiate in vitro into
osteoblasts, chondrocytes and adipocytes. These standards, however, have not been widely adopted and criteria for MSC isolation and identification continue tovary, making cross-study comparison difficult [3, 26–31].

© 2015 Sheng. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International
License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any
medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative
Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://
creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Sheng BMC Developmental Biology (2015) 15:44

As a consequence, physiological nature of their therapeutic effect and cellular and molecular nature of theirdifferentiation potentials in vivo remain ill-characterized.
This article will take an embryological approach toevaluate the evidence for the possible existence of MSCs
in vivo. From the perspectives of both cell lineage specification and mesoderm germ layer patterning, developmental ontogeny of the three main mesoderm cell types ofconcern to the MSC biology, the adipocytes, osteoblasts
and chondrocytes, will be discussed in detail. The evidence
for multi-potential progenitor cell populations from molecular and lineage analysis studies in vivo will be examined. Conceptual differences between mesenchymal stem
cells and mesenchymal stromal cells and between mesenchymal stem cells and mesodermal stem cells will also becompared in the broader context of stem cell biology.

Results and discussion
Adipogenesis

Except for a small, cephalic neural crest-derived population in the head, all adipocytes in the adult body are of
the mesoderm origin [32, 33]. Based on their morphology and location, adipocytes are categorized as eitherof a white or brown adipose tissue type (WAT and BAT,
respectively) [34–38]. WAT adipocytes function asenergy store and BAT adipocytes as heat dissipater. A
third, minor type (brite or beige adipocytes) exhibits anintermediate feature with their location associated with
WATs and their function resembling BAT adipocytes[39]. It is noteworthy that cellular and molecularfeatures associated with mammalian adipocytes, e.g.,
regulated fusion of cytoplasmic lipid droplets throughperilipins and respiratory uncoupling of lipid breakdown
through mitochondrial uncoupling protein UCPs, areevolutionarily ancient and are present not only in adipocytes, but also in other mesoderm cell lineages and incells derived from other germ layers [40–43]. The WAT
adipocytes, of relevance to the MSC biology, are further
divided into visceral and subcutaneous subtypes [34–38].
The visceral WATs have recently been shown to comefrom the splanchnic/visceral lateral plate mesoderm(LPM) [44]. The subcutaneous WATs, which constitutethe bulk of human body fat and are found mainly in theabdominal and gluteofemoral regions, are primarily
derived from the somatic/parietal LPM (discussed in
more detail below). Understanding WAT ontogeny andadipogenesis in development therefore requires proper
understanding of the LPM.
Chondrogenesis and osteogenesis

These are two separate, but tightly-linked skeletogenic
processes. Neural crest-derived cells make a significant
contribution to the cranial bones and cartilages [45–47].
All other skeletal elements are of mesoderm origin [48, 49].

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With the exception of the clavicle, which is generated
through a mixture of intramembranous and endochondrial ossification [50], all post-cranial bones form throughendochondrial ossification, i.e., secretion of bone-specificmatrix proteins and subsequent mineralization of thismatrix take place in a tissue architecture modeled by thechondrocytes. Therefore, percentage-wise, most cartilaginous tissues in the embryo exist only temporarily. Threemesoderm lineages in development, the axial, paraxial andlateral plate, are capable of generating skeletal elements.
The axial mesoderm gives rise to the embryonic notochord and the adult nucleus pulposus and expresses many
cartilage-specific markers (e.g., type II collagen andaggrecan) [51, 52]. However, cellular morphogenesis in
the notochord and the nucleus pulposus is very different
from what is known for the cartilage and these axial
mesoderm-derived cells are generally not considered to bechondrocytes. The paraxial/somitic mesoderm generates
all axial and associated skeletal elements (the vertebrae,ribs and part of the shoulder girdle), whereas all distal
skeletal elements (bones in the limbs, the pelvic girdle, thesternum and part of the shoulder girdle) are derived fromthe somatic/parietal layer of the LPM [53, 54]. In
endochondrial bones, chondrogenic differentiation of
osteochondrogenic progenitors proceeds first, and osteogenic differentiation takes place later from a pool ofquiescent progenitors located on the cartilage surface(perichondrium) [55]. Recent reports suggested that
osteogenesis can also take place through chondrocyte
de-differentiation or transdifferentiation [56, 57].
Major lineages of the mesoderm germ layer

The above brief overview indicates that the three major
cell types of special interest to the MSC biology share acommon connection to the somatic LPM. Prior to the onset of gastrulation which generates an embryo with threegerm layers, mesoderm precursors are specified molecularly when they are still part of the epiblast [58–61]. These
mesoderm precursor cells ingress from the epiblast to become bona fide mesoderm cells through an epithelial tomesenchymal transition (EMT) process, which takes placein an embryonic structure called primitive streak [62–66].
EMT of mesoderm precursors at the primitive streak proceeds in a temporally and spatially ordered manner(Fig. 1a). Temporally speaking, early-ingressing precursor
cells will migrate early and contribute to rostrally-locatedmesoderm populations. Those ingressing later from theprimitive streak will form progressively more caudal
mesoderm populations. The temporal difference in mesoderm EMT therefore translates into an antero-posteriordifference in their final destination. Spatially speaking,those precursor cells that ingress from the anterior end of
the primitive streak will contribute to the medially-located(embryologically dorsal) mesoderm lineages, e.g., theSheng BMC Developmental Biology (2015) 15:44

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Fig. 1 Schematic diagram of mesoderm formation and patterning during vertebrate early development. a Mesoderm precursors located in theprimitive streak (PS) undergo epithelial-to-mesenchymal transition (EMT) and migrate between the ectoderm and endoderm germ layers to their
final destinations in a spatially and temporally coordinated manner (white stippled lines). b Major mesoderm lineages (axial, paraxial, intermediate,
lateral plate and extraembryonic) are laid out along the medio-lateral axis of the early embryo. IM: intermediate mesoderm; LPM: lateral plate
mesoderm; ExEM: extraembryonic mesoderm; NT: neural tube; NC: neural crest. White stippled line represents the boundary between the LPM
and ExEM

notochord and the medial half of the paraxial mesoderm.
Those ingressing from the posterior end of the primitive
streak will give rise to lateral (embryologically ventral)
mesoderm lineages, e.g., the extraembryonic mesoderm(all mesoderm cells in the yolk sac, allantois, amnion andthe fetal part of the chorio-allantoic placenta). Therefore,
the antero-posterior spatial difference in mesoderm EMTtranslates into a medio-lateral (embryologically dorsoventral) difference in their final destination. In such spatial
coordinates, the LPM lineage lies between the intermediate mesoderm (giving rise to the urogenital tract) and theextraembryonic mesoderm, and is considered as thelateral-most (embryologically ventral-most) intraembryonic mesoderm lineage (Fig. 1b). The laterally-positioned
LPM in a flat-disc shaped embryo is brought to its final
ventral position during the process of body wall closure.

The somatic layer of the LPM gives rise to the dermisand hypodermis in the lateral and ventral body wall, thechondrocytes and osteocytes in all distal skeletal elements as discussed above, the vast majority of subcutaneous adipocytes including those in the abdominal andgluteofemoral regions and to potential resident progenitor and stem cells (MSC-like cells) in adipose and bonemarrow tissues (Fig. 2). Ontologically speaking, therefore, attention concerning the embryonic origin and molecular regulation of MSCs in vivo should be focused on
the epithelial-shaped somatic LPM and its subsequent
EMT, proliferation, morphogenesis and lineage diversification. The splanchnic LPM, albeit vital for the body’s
cardiovascular and hematopoietic functions, does notcontain the full differentiation potential for mesoderm
cell lineages attributed to the MSCs.

The LPM and its somatic and splanchnic layers

In vivo evidence for multipotential progenitors in thesomatic layer of the LPM

Nascent mesoderm cells do not maintain their mesenchymal morphology for long. All mesoderm cells
undergo at least one round MET (mesenchymal toepithelial transition) after their initial EMT, and many
undergo several rounds of subsequent EMT/MET processes before their final differentiation. Nascent LPM
cells polarize after a brief period of cell migration toform two epithelial layers, one located adjacent to theendoderm and called the splanchnic/visceral layer andthe other located adjacent to the ectoderm and called
the somatic/parietal layer (Fig. 1b). The apical side ofboth epithelial layers faces the enclosed internal space,
the coelomic cavity. Cells from the splanchnic layer ofthe LPM contribute to nearly the entire cardiovascularsystem, including the cardiac and smooth muscles,
endothelial cells, pericytes and HSCs, and to the mesothelial lining of visceral organs and visceral adipocytes.

The ontological evidence discussed above suggests thethree cell types of interest to the MSC biology have thesomatic LPM as their common developmental origin. It
is also evident that the majority of the bone and adipose
tissues that are used as clinical sources for MSC enrichment/purification in vitro are derived from the somatic
LPM. Although differentiation of MSCs in vitro may notnecessarily mimic their in vivo behavior, it is worthwhile
first reviewing the in vivo evidence for the existence of
MSC-like progenitor/stem cell populations in thesomatic LPM.
The somatic LPM, after its formation but before its
differentiation, expresses many genes (e.g., αSMA) which
are considered to be MSC markers later on [67–71].
Inducible αSMA-Cre-mediated lineage labeling suggested that αSMA positive cells in the bone marrow andSheng BMC Developmental Biology (2015) 15:44

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Fig. 2 Schematic diagram of somatic LPM morphogenesis. Epithelial-to-mesenchymal transition (EMT) of the epithelial somatic LPM produces ahomogenous mesenchymal cell population located between the ectoderm and the remaining LPM epithelium. These mesenchymal cells differentiate
into many mesoderm lineageslineages, including the adipocytes, chondrocytes and osteoblasts. MSCs are hypothesized to exist both in the naïve
somatic LPM population and in a more differentiated LPM tissue environment

periosteum are positive for MSC markers and these cells
can give rise to chondrocytes and osteoblasts during
fracture repair in vivo and to multiple mesenchymal
lineages in vitro [72]. Furthermore, αSMA also marks anadult adipose stem cell population [73] which resembles
adipose and bone marrow MSCs with multilineage differentiation potential in vitro [70, 71]. TWIST1, anothermarker for early somatic LPM [74], is highly expressed
together with its close homolog TWIST2 in primary
MSCs [75, 76]. TWIST1 and TWIST2 expression
prevents MSC differentiation in vivo [75, 76] andTWIST2-positive cells were shown to give rise to both
osteoblasts and chondrocytes in vivo in a Cre-mediatedlineage analysis [77].
The epithelial-shaped early somatic LPM cells differentiate by undergoing EMT and generate a populationof mesenchymal cells located between the overlying
ectoderm and the remaining epithelial somatic LPM(the future parietal mesothelium). This population ofsomatic LPM-derived mesenchymal cells is present
throughout the antero-posterior axis of the embryo, but ismost prominent in areas where limb buds develop [78].
These mesenchymal cells are initially homogenous and at
the limb levels they are uniformly positive for Prx1, apaired-related homeobox gene [79]. Results from virusmediated single-cell labeling of the limb mesenchyme suggest that these are multi-potential cells capable of giving
rise to two or more of the limb cell types, including thosein the cartilage, perichondrium, tendon, muscle connective and dermis [80]. Sox9, an early molecular marker forchondrogenic condensation, labels in fact a multi-lineage
sub-population of these mesenchymal cells, including
those contributing to the cartilage, bone, tendon andligament [81, 82]. Regulated by a pre-adipocyte marker
Pref-1, Sox9 also inhibits the adipogenic potential of thesemesenchymal cells [83].
In a more differentiated tissue environment derived
from the somatic LPM, multi-potential progenitors havebeen shown to exist for the osteoblasts, adipocytes andperivascular stromal cells by Osterix-Cre mediatedlineage labeling [84], for the osteoblasts, chondrocytes,
endothelial and bone marrow stromal cells by Nestin-Cre
mediated lineage analysis [27, 85], for the osteoblasts,chondrocytes and marrow stromal cells by Gremlin1Cre mediated lineage and Rainbow Actin-Cre mediatedclonal analyses [86, 87], for the chondrocytes and osteoblasts by Col2-Cre mediated lineage labeling [88], andfor the osteoblasts and adipocytes by fate-mapping andtransplantation analyses of leptinR+ cells in the adultbone marrow [89].
Mesenchymal stem cells or mesenchymal stromal cells

Taken together, data from the ontological, molecular andcellular analyses strongly support the hypothesis that
there exists a MSC-like progenitor population both during somatic LPM differentiation and during homeostatic
maintenance of somatic LPM-derived tissues. The multipotential stemness of MSCs in vivo, with the capacity togenerate tissues including the bone, cartilage, tendon,muscle, fat and marrow stroma [8], however, has notbeen satisfactorily demonstrated. This gap can be viewed
from three perspectives in the broader context of stem
cell biology.
First, the nature of MSC multipotency in vivo may require further clarification and more precise definition(Fig. 3). Uni-potential stem-like behavior of MSC-related
lineages has been reported in many studies, and bipotential or multi-potential stemness of closed related
lineages (such as the chondrocytes, osteoblasts andbone marrow stromal cells) has also been documented [4, 27, 86, 87, 90]. However, multi-potential
stemness with differentiation potentials for all MSCrelated cell lineages has not been demonstrated yet. An
evolving concept of the MSCs, similar to that of the HSCs,
is to view them as a heterogeneous mix of subpopulations each harboring a unique set of multipotency

Sheng BMC Developmental Biology (2015) 15:44

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Fig. 3 Comparison of MSC-related phenomena in vivo and in vitro. In vivo, progenitor populations that will give rise to the adipocyte, chondrocyte
and osteoblast lineages pass through developmental phases that progressively restrict their fate choices. This is correlated with progressively limited
differentiation potentials of corresponding stem cell populations cultured in vitro. ICM: inner cell mass; ESCs: embryonic stem cells; Epi-SCs: epiblaststem cells; LPM: latral plate mesoderm; MSCs: mesenchymal stem cells. Corresponding stem cell populations for the mesoderm (pan-mesoderm stem
cells) and LPM (pan-lateral plate mesoderm stem cells) have not yet been reported[3, 4, 86, 87, 91]. Such a concept is in agreement with thefindings that some MSCs may represent a subpopulationof pericytes or fibroblasts in vivo [92–95]. This modified
concept of MSCs, however, does not preclude the potential existence of a bona fide multi-potential mesenchymal
stem cell population either in development or during
adult tissue homeostasis. Secondly, many well accepted
concepts, such as the embryonic stem cells (ESCs) andepiblast stem cells (Epi-SCs), represent in vitro phenomena without embryological counterparts. Although ESCs
have been likened to the inner cell mass (ICM) during
early mammalian development and Epi-SCs to the young
epiblast before gastrulation (Fig. 3), neither the ICM nor
the epiblast in vivo meets the stemness criteria either froma single cell or from a cell population point of view. Thesecells, like the MSCs, are multi-potential progenitor cells inthe so-called “epigenetic landscape” of successive lineage
restrictions during animal development [96]. Nevertheless,the stem cell nature or the in vivo relevance of ESCs andEpi-SCs has seldom been questioned in the scientific literature, and likewise the MSCs should not either. Thirdly,
the ESCs and EpiSCs are derived from embryologicallyspeaking more naïve tissues. As a consequence, cellular
purity and molecular homogeneity of ESCs and Epi-SCs
are much more rigorously defined and regulated than the
MSCs. Better characterization and standardization of
MSC sources in vivo and cellular and molecular featuresin vitro is therefore essential for the future progress of
MSC biology. Promising novel sources for MSC derivation
are the naïve somatic LPM, the undifferentiated population of somatic LPM-derived mesenchyme and thesomatic LPM-like cell populations derived from guided
differentiation of ESCs, Epi-SCs or iPSCs.

Mesenchymal stem cells or mesodermal stem cells

Although most of the cell and tissue types which havebeen associated with MSCs come ontologically from thesomatic LPM, many of them do not. For example, as target differentiation lineages, the cardiac and most of thesmooth muscles come from the splanchnic LPM and theskeletal muscles are from the somitic mesoderm. As tissue sources, the neural crest is of ectoderm origin, andaxial bones and dorsal dermis are of somitic mesoderm
origin. The peripheral blood in the adult and the umbilical cord blood in the fetus are of splanchnic LPM/
splanchnic extraembryonic mesoderm origin, the amniotic mesoderm is of somatic extraembryonic mesoderm
origin and the placental and the umbilical cord mesenchyme is derived from a mixture of somatic andsplanchnic extraembryonic mesoderm cells.
Leaving aside the neural crest-derived cell populations(not discussed here), one may ask whether the term
“mesodermal stem cells” is more suitable to describe theproperties associated with the MSCs. To answer thisquestion one needs to take a fresh look at the epigenetic
landscape proposed by Waddington about 60 years ago[96]. The original concept of epigenetic landscape ofanimal development outlined progressive restriction ofdifferentiation potentials from a totipotent zygote to terminally differentiated functional cell types. Uni-, bi- or
multi-potential stem cells are perceived to exist in eachbranch or at the branching point. Cell lineages that areclosely related ontologically are more likely to be represented by a common progenitor/stem cell populationand those distantly related less likely so. For example,
paths and barriers for the germ cells and the soma andfor the three principal germ layers are set early inSheng BMC Developmental Biology (2015) 15:44

development and fate conversion crossing these barriers
is almost never observed in vivo. One may therefore
argue that the MSCs are a sub-population of, and are
distinct from, a pan-mesodermal stem cell population,
the latter of which can be viewed as the equivalent ofprimitive streak-like mesoderm progenitors in vivo
(Fig. 3). Although other sub-populations of lineagebiased, multi-potential mesodermal stem cells may exist,
cell types not strictly derived with the somatic LPM
should preferably not be associated with the MSCs.
Advances from iPSC-related research, however, havecomplicated such a simplistic paradigm. Reversion from aterminally differentiated fate to a pluripotent fate can now
be achieved relatively easily through molecular or chemical
perturbations of this epigenetic landscape and paths of reprogramming may not necessarily mirror the conventionallandscape of progressive differentiation in vivo. As a consequence, it is unclear whether the knowledge obtained fromin vivo studies should serve as the guiding principle forachieving fate reversion and targeted lineage differentiationin vitro. It will be fascinating to see whether the epigenetic
landscape manifested in vitro can teach us about
unrealized potentials in vivo. MSC studies in vitro therefore should not be confined by MSC behaviors in vivo.

Conclusions
This article has outlined the ontological, molecular and cellular evidence in support of the existence of MSCs in vivo.
The somatic LPM is the most important mesoderm compartment for the cells and tissues commonly associatedwith MSCs. The term “mesenchymal stem cells” is preferred by this author and a clear distinction should be madebetween somatic LPM-derived MSCs and mesenchymalshaped stem-like cells derived from other mesodermalcompartments. Such understanding of MSCs based onin vivo evidence would benefit in vitro endeavors in harnessing the therapeutic powers of MSCs. The ultimate goal of
MSC-related research is to integrate in vitro-reconstituted
cells or tissues into an in vivo environment, which will befacilitated by an awareness of how endogenous MSC populations differentiate and self-organize in vivo.
Methods
Views expressed in this article are based partially on
research carried out in the author's laboratory at RIKEN
Center for Developmental Biology. All animal experiments performed in the author's laboratory were approvedby the institute's ethical committee.
Competing interests
The author declares that he has no competing interests.
Authors’ contributions
GS wrote the manuscript.

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Acknowledgements
I would like to thank RIKEN for financial support and past and current membersof my laboratory for the concepts presented in this article. The author would
also like to thank Dr. Wei Weng for critical reading of the manuscript.
Received: 17 April 2015 Accepted: 13 November 2015

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