Stem cell-derived vasculature: A potent and multidimensional\ntechnology for basic research, disease modeling, and tissue\nengineering
Proper blood vessel networks are necessary for constructing and re-constructing tissues, promoting wound healing, and delivering metabolic necessities throughout the body. Conversely, an understanding of vascular dysfunction has provided insight into the pathogenesis and progression of diseases both common and rare. Recent advances in stem cell-based regenerative medicine \xe2\x80\x93 including advances in stem cell technologies and related progress in bioscaffold design and complex tissue engineering \xe2\x80\x93 have allowed rapid advances in the field of vascular biology, leading in turn to more advanced modeling of vascular pathophysiology and improved engineering of vascularized tissue constructs. In this review we examine recent advances in the field of stem cell-derived vasculature, providing an overview of stem cell technologies as a source for vascular cell types and then focusing on their use in three primary areas: studies of vascular development and angiogenesis, improved disease modeling, and the engineering of vascularized constructs for tissue-level modeling and cell-based therapies.
1. Introduction<!>2. Advances in stem cell-derived vascular models<!>3. Modeling vascular development, pathophysiology, and disease using stem\ncell-based and tissue-engineered models<!>3.1. Modeling vascular development and physiology<!>3.2. Disease modeling using stem-cell derived vascular cells<!>4. Stem cell-derived vasculature as cell therapy and functional tissue\nengineering<!>4.1. Direct cell-based therapy for in situ tissue vascularization<!>4.2. In situ vascularization<!>4.3. Pre-vascularization<!>5. Future directions<!>
<p>Being able to move toward the creation of more complex models of disease
pathophysiology and tissue-engineered therapies has required advances in our
understanding of and ability to replicate blood vessel formation, growth, and
support of surrounding tissues. Primitive vascular structures appear early in human
development, helping to guide the specification and organization of underlying
tissues and defining a wide variety of congenital defects. The process of
neoangiogenesis has long fascinated researchers studying diseases as diverse as
macular degeneration and arthritis. Indeed, vascular biology has been a primary
focus of research into tumor formation, growth and metastasis; inhibitors of
vascular processes have been investigated as anti-cancer therapeutics for decades.
More recently, the presence of a functional blood supply has been identified as
critical to the success of organ and tissue transplantation efforts –
including graft survival and rejection – and in the engineering of functional
replacements.</p><p>With recent advances in regenerative medicine, the field of vascular biology
has seen rapid progress. Pluripotent stem cell biology has garnered significant
attention in the last two decades, headlined by the establishment of human embryonic
stem cell lines (ESCs); the discovery and development of somatic cell
"reprogramming" to created induced pluripotent stem cells (iPSCs); and
the development of protocols to differentiate pluripotent and multipotent stem cells
or directly convert other terminally differentiated cell lineages into vascular
precursors. Advances in tissue engineering and biomaterials have led to new
scaffolds and strategies for creating three-dimensional (3D) vascular networks and
new insights into how the extracellular matrix and surrounding microenvironment
contributes to angiogenesis. The concomitant development of increasingly precise
techniques for genome editing, including Zinc-finger nucleases, TALENs, and
CRISPR-Cas9, opens up the possibility of editing cell lines and animal models to
further study vascular development and disease pathogenesis.</p><p>In this brief review, we survey recent advances in stem cell biology and
tissue engineering as they have been applied to the creation of stem cell-derived
vasculature through two main approaches. After presenting advances in pluripotent
stem cell-derived vascular modeling, we discuss the ways in which this technology
has been: 1) applied to studies of basic developmental biology and vascular
pathophysiology; 2) used to model vascular-related disease pathogenesis; and 3)
incorporated as a three-dimensional foundation for functionally vascularized,
bioengineered tissues. These vascularized tissue approaches will be loosely
categorized into those utilizing in situ induction (recruiting
endogenous vessel formation in tissue engineered grafts) and those which attempt
graft pre-vascularization (engineering vessels directly into grafts).</p><!><p>Creating realistic cell-based vascular models requires sources of each of the
cellular components of the desired vessels: endothelial cells (ECs), pericytes
(perivascular, or support, cells), vascular smooth muscle cells (v-SMCs) appropriate
to the desired vessel type, and other tissue-specific cell types that interact with
the vasculature (astrocytes in the central nervous system, for example). A diverse
array of stem cell technologies have matured as potential sources for vascular
precursors: pluripotent cells [1] such as ESCs
[2] and iPSCs [3]; and various types of multipotent (or "adult")
SCs such as mesenchymal stem cells, umbilical cord blood-derived stromal cells,
amniotic fluid-derived stem cells, adipose-derived stem cells, and hemangioblasts
[4–6]. With these new SC sources, researchers have been able to move beyond
primary cell culture and develop lines with particular characteristics, sourced from
human patients with particular genetic characteristics or mutations [7]. Vascular cell types can now be generated
using stem cell technology through three main pathways: 1) differentiation directly
from stem cells obtained from human sources; 2) reprogramming of terminally
differentiated cells (often fibroblasts or peripheral blood) through a pluripotent
intermediate and then differentiated; or 3) through direct
conversion/transdifferentiation from another cell type.</p><p>Investigators have been working to develop more robust, efficient, defined,
and GMP-compliant (clinically applicable) SC differentiation protocols to generate
the necessary vascular cell types for research and eventual therapy (Fig. 1) [2,3,8–13]. The role of
individual culture components, culture conditions, biomechanical stimuli, and
microenvironmental factors has been elucidated using both standard 2D in
vitro culture techniques as well as more advanced suspension culture
systems, 3D microenvironments, and biomaterials-based approaches [14]. For example, various standard techniques
of SC culture have been modified with stimuli to promote early vascular linear
specification, as diversely illustrated by the use of nitric oxide to inhibit
multipotent vascular stem cell differentiation in two dimensions [15], of TGF-β1 to induce the formation
of tubular structures in ESC embryoid body (pseudo-3D) cultures [16], and biomechanical strain to induce
enhanced ECM production in v-SMCs [17].</p><p>Meanwhile, investigators have developed biomaterial-based platforms such as
micropatterned surfaces [18] and electrospun
nanofibrous scaffolds [19], incorporating
chemical as well as biomechanical stimuli [20] to improve the efficiency of vascular differentiation and to study
cellular responses to these stimuli. Specifics of the exact SC differentiation
protocols is beyond the scope of this review and can be found elsewhere [21].</p><p>Much of the focus has been on the ECs themselves, and extensive work has gone
into studying the molecular pathways governing endothelial specification from
pluripotent cells [22]. However, there is
also an increasing consensus around the importance of including a robust population
of perivascular cells in engineered blood vessel models [23–25]. It has
been shown that pericytes and related cells are highly angiogenic, either when used
as direct cell therapy in vivo or when co-cultured in 3D constructs
with ECs that form vascular networks [26].</p><p>Alongside advances in vascular stem cell biology have been advances in
biomaterials to support stem cell culture, differentiation, and self-organization
into functional tissues. Combining cells with scaffolds allows the formation of
three-dimensional (3D) culture systems and the development of rudimentary networks
of blood vessels. Researchers have applied a wide variety of biodegradable scaffolds
[27], both natural and synthetic. The
advantage of natural scaffolds is that of biocompatibility. However, synthetic
scaffolds are often more durable, have greater mechanical stability, and are tunable
to manipulate cellular behavior and network formation [28–30]. These
scaffolds and hydrogels can often be engineered with modified microenvironmental
features to enhance the proliferation and self-organization of vascular cells
embedded within. For example, locally hypoxic conditions are a particularly
attractive feature that investigators have attempted to precisely modulate [31–34] because of the demonstrated ability of controlled hypoxia to enhance
vessel formation in 3D tissue-engineered structures in vitro [35] and the relevance to the tumor
microenvironment in cancer biology.</p><!><p>As our abilities to derive vascular cell types and combine them with other
technologies continue to grow, so too has our ability to use these components to
realistically model human vessel development [36], the vascular niche [37], and
vascular remodeling [38]. Using these models,
researchers hope to gain insight into processes of angiogenesis and wound healing,
to model blood flow dynamics and vessel mechanics under certain conditions, to
develop more robust vascular grafting techniques, and to probe the microenvironments
necessary for enhanced vessel growth and inform future biomaterial and hydrogel
development.</p><!><p>Vessel development and maturation are frequently modeled in vitro to
study the growth of EC vessel- and tube-like structures. Traditional approaches
have involved studying primary and stem-cell derived vascular cells in standard
2D tissue culture using matrix coatings and co-culture formulations. More
recently, vascular development has been modeled within 3D extracellular
biomaterials. Through these rudimentary vascular models, tube morphogenesis can
be studied in the presence of cytokines, defined factors, matrix components, and
supporting cells in co-culture [39,41]. These models have become more advanced
over the past decade, with more complete control of the 3D topographical cues
that guide microvascular development [42,43] and the introduction
of genetic controls [44]. These
approaches have long been used with primary EC cultures; however, with the
increased use of iPSC-derived EC, models of network formation, barrier
formation, and sprouting have become more robust and easily modifiable [45].</p><p>Proliferation of stem cell technologies has allowed more extensive
investigation of tissue-specific vascular niches. This knowledge, in turn, has
great potential to further inform PSC-derived vascular models especially as they
apply to the study (and eventual engineering of) particular tissue types [46]. For example, even within seemingly
straightforward populations of ECs and v-SMCs lie subtle diversity in
developmental origin yielding unique properties, necessitating the development
of protocols to differentiate these subtly different populations (for example,
coronary vascular smooth muscle cells as opposed to those found in bladder and
aorta [47]). Unique vascular populations
have been found to differentiate alongside cardiomyocytes from a common
PSC-derived cardiovascular progenitor cell (CPC) precursor [48].</p><p>The neurovascular sphere has been studied particularly extensively due
to its unique features, its relevance to disease pathogenesis
(neuroinflammation, cerebrovascular accident, psychiatric disorders, and more),
and its import as a barrier to drug delivery to the CNS. Studies to more fully
define the cerebrovascular niche and to characterize cell types will help
distinguish different phases of differentiation from stem cells and allow more
precise models of vascularized neural tissue [49] and the healthy and diseased bloodebrain barrier. The hope is
that the use of stem cell-derived vasculature as a basis for early-stage CNS
modeling can serve as a paradigm for in vitro developmental process capture. As
stem-cell derived neurovascular cell types are purified at all stages of
differentiation, researchers have used them to recapitulate neural development
in a 3D context [50], and multiple groups
have studied the development of the vascular microenvironment from common neural
stem cell precursors [51]. The pathways
are also now known to go in reverse: in the setting of cerebrovascular
ischemia/hypoxia, resident vascular pericytes have demonstrated multipotent stem
cell activity [52].</p><p>The presence of a bloodebrain barrier and the intimate interaction of
astrocytes and other additional cell types with the cerebral vasculature has
long fascinated investigators interested in vascular biology. Initial protocols
have been developed to generate brain-specific EC from PSCs and to cultivate
these cells in multicellular cultures [53,55]. Models of the
bloodebrain barrier fabricated entirely from stem cell sources have shown
promise over primary cell culture for advanced cerebrovascular modeling [54,55].</p><p>The vascular and hematopoeitic stem cell niches have also been found to
be intimately connected, as demonstrated by the use of vascular cell types to
promote the differentiation, proliferation, and engraftment of PSC-derived
hematopoietic cell types [56]. This
interaction has been investigated with great interest, as efforts to study the
development of and engineer the bone marrow niche will likely involve
concomitant modeling of vascular and hematopoietic niches [57].</p><!><p>It has long been known that endogenous vascular SC populations are
implicated in a variety of vascular diseases through both innate and acquired
dysfunction [58]. Among the most common
of these processes are diabetic vasculopathy, arteriovenous malformation,
atherosclerosis, calcification, and ectopic tissue deposition within blood
vessels [59]. With this knowledge have
come efforts to model these disorders using increasingly robust stem-cell
derived vascular models. For example, because of the relevance of the DNA damage
response to vascular dysfunction in the setting of atherogenesis, vasospasm, and
migraines (among others) [60,61], stem cell-derived vascular cells have
been used to model genotoxic insults in order to detect changes in multicellular
"vascular" responses and functionality [62]. Stem cells with particular genetic characteristics,
including from patients with rare diseases, have enabled a deeper mechanistic
understanding of rare vascular phenotypes [63]. More advanced 3D cultures have been used to model tumor
development and metastasis through intravasation [64,65] and to create
a model of perfusable solid tumor for novel drug screening [66].</p><p>Organ-on-chip technologies have incorporated stem cell-derived vascular
cells and cell types of many other organ systems throughout the body into
microfluidic platforms mimicking human circulatory physiology [67,68]. These devices have been demonstrated to have a wide variety of
possible applications, including high-throughput stem cell screening in response
to chemical and mechanobiological cues, models of drug delivery, and full organ
system modeling, both healthy and diseased [68,71]. Vascularspecific
diseases such as atherosclerosis, deep vein thrombosis, and pulmonary
hypertension have both been successfully modeled using this technique, and these
chips have also been used to study the interplay of vascular dysfunction with
other organ-level dysfunction in complex diseases of the lung and liver [68].</p><!><p>Vascular tissue engineering first consolidated as a field around the goal of
developing vascular graft replacements for individual vessels [72,73]. Researchers used
everything from decellularized cadaveric donor vessels to completely synthetic
materials such as Dacron and Gore-Tex to more complex mixtures of cells and
bio-materials. As the field began attempting smaller diameter grafts, neointimal
hyperplasia and thrombus formation bedeviled the transplants and many grafts failed
either due to insufficient transport properties or biomechanical failure. As these
problems surfaced, so did novel biomaterials and combinations with various stem cell
components attempting to solve it [74,76]. Investigators have likewise been creating
more advanced tissue-engineered vascular grafts utilizing novel bioreactor/culture
systems and stem cell sources, including iPSCs [77,78].</p><p>From an initial focus on individual vascular grafts, the field has matured
toward the creation of complex vascular networks e vascular "beds" e
as support structures on which tissue regeneration can occur in vivo and on which
engineered tissues can be created in vitro for transplantation [79]. Through the application of insights from
early vascular development [36],
investigators have developed increasingly complex and successful strategies for
engineering vascular networks.</p><p>Stem-cell derived vasculature is generally envisioned to have therapeutic
applications through three major general approaches: direct cell therapy, in situ
induction, and tissue-engineered prevascularization [80,81] (the last of which can be
subclassified into biomaterial-directed vs. cell-directed/self-organizing). This
review is not meant to be comprehensive of all possible strategies for vascularizing
tissue-engineered constructs, only to highlight some major themes; a more
comprehensive review can be found elsewhere [82].</p><!><p>There is great excitement surrounding the use of SC derived vascular
cell types as a direct cell therapy, either transplanted (injected) locally or
infused systemically for a variety of diseases, most prominently in the setting
of peripheral vascular disease/ critical limb ischemia [83,85]. As a
paradigmatic example, ECs and mesenchymal precursor cells generated from iPSCs
derived from both healthy and type 1 diabetic human sources were shown to form
functional, competent, and durable vessels in vivo when injected in mouse models
[86]. Ophthalmologic and cardiac
applications are being heavily investigated as well: iPSC-derived vascular
progenitors have demonstrated the ability to regenerate ischemic retinal
vasculature, relevant in multiple disease contexts such as diabetic retinopathy
and macular degeneration [9], while
simultaneous delivery of human pericytes and cardiac stem cells additively
improve healing the setting of myocardial infarction [87].</p><!><p>In situ vessel development can be incorporated into tissue engineered
constructs by taking inspiration from endogenous repair processes: creating
bioactive natural and synthetic scaffold impregnated with growth factors,
chemotactic molecules, nano-particles, and sometimes populations of support
cells.</p><p>The paradigmatic approach has been to engineer bioactive acellular
scaffolds to promote endogenous neovascularization capability. Synthetic or
naturally-derived scaffolds can act as proangiogenic grafts without any embedded
cell sources: through the use of incorporated bioactive molecules (such as
factors in the PDGF, VEGF, and SDF families), often under spatial and/or
temporal control in the scaffold (chemokine gradients, controlled release
nanoparticles, etc.), scaffolds can encourage vascularized neotissue development
[88].</p><p>Beyond simple acellular scaffolds, there exist multiple strategies for
in situ induction. One promising strategy involves the delivery of stromal cells
and other types of support cells (either directly or embedded in a scaffold) to
the site requiring neovascularization. Multiple cell lineages can exert
paracrine effects encouraging vascular, with a biomaterial scaffold and then
implanting the cell-seeded scaffold. This allows vessels to form and
self-organize utilizing both resident and transplanted cells after implantation
[89]. In a way, this approach can be
considered a hybrid of in situ and prevascularization, as stem cell-derived
populations other than the vascular cells themselves are pre-embedded.
Vascularized adipose tissue was generated in this manner using stromal-vascular
fraction-derived adipose cells embedded in precultivated fibrin gels for soft
tissue substitution [90]. A similar
strategy has been applied to evaluate the use of both adipose-derived stem cells
co-cultured with EC [91] and
amniotic-fluid derived stem cells [92] as
sources of vascular and perivascular cells for in situ capillary network
formation.</p><p>Complex platforms have been developed to modulate the paracrine effects
of embedded stem cells through customized, self-assembled matrices [93] and by engineering pro-angiogenic
factor release capability into the embedded cell populations themselves [94]. One demonstration of this is through
the transduction of HIF-1a into MSCs embedded into gelatin sponges for bone
tissue engineering, yielding enhanced transcription of pro-angiogenic gene
programs and dramatically improved blood vessel formation within implanted
tissue engineered bone [95].</p><!><p>Pre-vascularization can be scaffold-directed, with vessel channels
preformed and then seeded with cells of interest, or cell-directed, where stem
cell-derived vascular cells are embedded within the scaffold and allowed to
self-organize into tube-like structures. Once a method is chosen, researchers
have a great deal of flexibility over how cells are applied to the scaffolds
(embedded vs. seeded using various methods [96]) and how growth within these scaffolds can be controlled.</p><p>One of the canonical strategies for prevascularization of engineered
tissues is perfusion decellularization, a technique that involves antegrade
perfusion of complex tissues with detergents to remove all cellular components
and leave behind the endogenous extracellular matrix scaffold e including the
channels formed by blood vessels in the tissue [97]. This technique first demonstrated downstream applicability
through tissue-engineered vessel grafts formed from seeding of PSC-derived
endothelial and/or smooth muscle cells onto a decellularized vessel scaffold in
a bioreactor, creating fully cellularized vessels whose cell sources can be
patient-and application-specific [98].
This strategy has matured beyond individual vessel grafts, however; it has been
applied to the creation of decellularized vascular beds within whole tissues,
allowing pre-seeding of vascular networks within complex scaffolds for
whole-organ tissue engineering [99,101].</p><p>While decellularization has received much attention for the presence of
ready-made, physiologic channels within these constructs, other methods of
creating "scaffold-directed" prevascularized constructs have
gained favor as well, including 3D bioprinting [102,104], the use of
"sandwich" cell-accumulation cultures [105], and micropatterning [106]. A high profile illustration of biomaterial-defined
pre-vascularization using biomaterial-based microchannel networks came with the
demonstration that filamentous carbohydrate networks could be printed and
precisely manipulated to generate cylindrical vessel-like networks [107]. Once seeded with endothelial cells,
these networks allowed perfusion with blood under high-pressure pulsatile flow
and supported hepatocytes in large tissue-engineered constructs, overcoming an
obstacle that has bedeviled solid organ tissue engineering: hypoxia and necrosis
in the core of non-perfused constructs [107]. A similar strategy has been used in hollow channel-modified
porous silk scaffolds [108,109].</p><p>To highlight a more "cell-directed" approach, research
groups have successfully generated functional, 3D vascular networks by embedding
stem cells, vascular progenitors, and stem cell-derived
terminally-differentiated cells in a diverse array of biomaterial-based
hydrogels and scaffolds (Fig. 2) [30,110]. The power of this technique is that these cell populations can
all be sourced from the same stem cell precursor and, given the right
microenvironmental cues and support structures, cell populations can
self-organize according to their own developmental programs into multicellular,
functional microvessel networks. Such networks derived from hPSCs have been
shown to be fully mature, integrate with host vasculature, and establish blood
flow within animal models.</p><p>Engineered tissue pre-vascularization has received considerable
attention in the fields of hepatic and cardiac tissue engineering in particular.
Mixtures of primary and stem cell-derived lines of both vascular and
tissue-specific phenotype (e.g. fetal liver cells mixed with umbilical vein
endothelial cells and mesenchymal stem cells) have been successfully embedded in
3D scaffolds and allowed to form rudimentary vascular networks among maturing
and proliferating hepatic cells, forming engineered pre-vascularized human
hepatic tissue [111]. Investigators have
also created prevascularized cardiac tissue constructs using multiple different
strategies [81,112,115]. Sheets
of entirely iPSC-derived cardiac tissue with both cardiomyocytes and vascular
cells have been created and found suitable for transplantation into rat
infarcted hearts and were found to successfully engraft and induce
neovascularization [116]. Investigators
have also created 3D cardiac tissues using stem cell sources and porous
scaffolds in advanced cell culture and bioreactor systems [117], and have used vessel explants surrounded by
engineered microenvironments to stimulate capillary network growth as a
vascularized scaffold [118,119].</p><!><p>Disease modeling using stem cell-derived 3D models is still in its relative
infancy. However, with the advent of patient-derived iPSCs as well as novel genome
editing techniques, one can expect these models to yield insights about areas as
diverse as connective tissue diseases, autoimmune vasculitis, neurodegenerative
disease, cardiomyopathy, muscular dystrophy, even hematopoietic diseases such as
hemoglobinopathies and leukemias. All of these have a suspected vascular phenotype
and thus a role to play for vascularized models of disease.</p><p>As stem cell-derived vascular models continue to expand, so too will their application to create increasingly complex tissue-engineered constructs. Following
the model of perfusion decellularization leads us to believe that engineered lung,
pancreas, muscle, and skin will soon be layered atop vascularized scaffolds,
improving outcomes in tissue engineering. Extension of vascular tissue engineering
strategies to the lymphatic system to address such problems as lymphedema and
lymph-mediated immunologic dysfunction has also been contemplated [120], and investigators have begun to discuss
the use of vascular stem cells in conjunction with tissue engineered models of
neural tissue and the bone marrow niche.</p><!><p>Transparency document</p><p>Transparency document related to this article can be found online at
http://dx.doi.org/10.1016/j.bbrc.2015.09.127.</p><p>stem cell</p><p>pluripotent stem cell</p><p>induced pluripotent stem cell</p><p>embryonic stem cell</p><p>vascular smooth muscle cell</p><p>endothelial cell</p><p>(A–H) Spontaneous vascular differentiation in embryoid bodies (EBs).
Confocal microscopy of stained 10-15-day-old human EBs. (A)
CD34+ cells forming vascular networks along the EBs
(×200). (B) Projection of CD34+ cells serial
sections within an EB (×600). (C) Occasionally round
cells are stained with CD34+ (×600). (D and
E) CD31+ cell groups organized in specific
channel-like structures (×600). (F) Elongated smooth
muscle actin (SMA)+ cells surrounding voids with no detectable
network formation (×200). (G) SMA+ and
(H) smooth muscle myosin heavy chain (SM-MHC)+
cells surrounding voids, forming flat thick tubes (×600). (I–M)
Sprouting from differentiated human embryonic stem cells. Differentiated cells
were allowed to aggregate for 24 h in medium supplemented with human vascular
endothelial growth factor (VEGF165), after which they were seeded
into type-I collagen or Matrigel in the same medium. (A)
Tube-like formation after 7 days in type-I collagen gel and (B)
aggregate sprouting in Matrigel (low and high magnification).
(C) Two different areas of 3-dimensional sprouting on
Matrigel, documented with Hoffman microscopy. (D) Longitudinal
histological sections showed (i) seeded cells (arrow)
penetrating into the Matrigel (M) and (ii) forming primitive tube-like
structures (arrow) (higher magnification). (E)
Transverse histologic sections of the cell-seeded Matrigel exhibited different
sizes and shapes of tube-like structures formed within the Matrigel (Scale bars:
100 Î¼m.). [Both adapted from Gerecht-Nir et. al. Lab
Invest 2003; 83:1811–1820].</p><p>(A–F) Self-assembly of EVCs to multicellular networks in a 3D matrix.
(A) Network formation from BC1 (iPSC-derived)-EVCs in
collagen (i) and Hyaluronic Acid (HA) hydrogels
(ii). (B) Sorted VEcad+ and
VEcad− cells encapsulated within collagen gels were unable
to form networks. (Insert) Example of a cell with typical
stellate morphology, with phalloidin in green and nuclei in blue (Scale bars:
100 Î¼m.) (C) Vacuole formation was observed after one
day as evidenced by light microscopy (LM) (i) and confocal images
(ii) of vacuole vital stain, FM4-64, in red and nuclei in
blue (Scale bar: 10 Î¼m.) (D) On day 2, network formation
with enlarged lumen (i and ii) and cell
sprouting (iii and iv) were visualized by LM
images (i and iii) and confocal images
(ii and iv) of FM4-64 in red and nuclei in
blue. (Scale bars: 10 Î¼m in i and iii;
20 Î¼m in ii; 50 Î¼m in iv.)
(E) On day 3, complex networks were observed with enlarged
and open lumen, as indicated by confocal z-stacks and orthogonal sections of
FM4-64 in red and nuclei in blue (Scale bar: 20 Î¼m.) (F)
After 3 d, multilayered structures were also detected, as demonstrated by a 3D
projection image of NG2 (green), phalloidin (red), and nuclei (blue) showing
NG2+ pericytes integrated onto hollow structures. Images shown
are typical of the independent experiment (Scale bars: 50 Î¼m.) [Adapted
from 110]. (G–I) Vascular tube
morphogenesis by ECFCs within Hypoxia Inducible (HI) hydrogels.
(G) Light micrographic images of ECFCs encapsulated within
HI hydrogels during 3 days of culture (Scale bar: 50 Î¼m.)
(H) Confocal microscopic images of ECFCs encapsulated
within nonhypoxic gel and hypoxic gel; (I) confocal
z-stacks and orthogonal sections show lumen formation
(indicated by arrows) within the vascular networks (phalloidin in green; nuclei
in blue) (Scale bars: 50 μm.) [Adapted from 32]. (J–M) ECFC vascular network growth and
complexity in HA hydrogels. (J) Left panel: Growth of
comprehensive vascular networks at day 3 are demonstrated using LM images at low
magnification. Right panel: (i and ii) are high magnifications of white boxes
(arrows indicate branched and elongated vascular networks) (Scale bars: 100
Î¼m.) (K) Confocal analysis of vacuole vital stain FM4-64
(cyan; nuclei in blue) demonstrating large lumen within the networks, and
(L) demonstrated using orthogonal view (indicated by
arrowhead) (Scale bars: 50 Î¼m.) (M) TEM high-resolution
images show cross-sections of matured vascular tube networks with enlarged
lumens (Scale bars: 20 Î¼m.) [Adapted from Hanjaya-Putra et. al.
Blood 2011; 118(3):804–15. doi:
10.1182/blood-2010-12-327338.]. (For interpretation of the references to color
in this figure legend, the reader is referred to the web version of this
article.)</p>