consume the highest amount of oxygen per gram of tissue in
the body, and thus require a continuous source of oxygenated
blood. The human ophthalmic artery supplies 20% of its blood
to the retina and ≈80% to the choroid and uveal tract. If either
the retinal or choroidal vasculature becomes compromised,
Clinical Perspective on p 372
he human retina’s high metabolism makes it uniquely reli-
ant on an intact, functional vasculature. Photoreceptors
neurons in affected ischemic areas rapidly die. Following
branch vein occlusion 1,2 and during the course of diabetic reti-
nopathy,3–5 ischemia results in retinal pericyte and endothe-
lial cell (EC) death. This leads to acellular vascular segments,
rapid death of retinal neurons, secondary inflammation,6–8 and
further retinal damage from subsequent compensatory neo-
vascularization.9–13 In both branch vein occlusion and diabetic
retinopathy, the regeneration of retinal capillaries with cellular
therapies by using vascular progenitors (VPs) could reverse
Background—The generation of vascular progenitors (VPs) from human induced pluripotent stem cells (hiPSCs) has great
potential for treating vascular disorders such as ischemic retinopathies. However, long-term in vivo engraftment of hiPSC-
derived VPs into the retina has not yet been reported. This goal may be limited by the low differentiation yield, greater
senescence, and poor proliferation of hiPSC-derived vascular cells. To evaluate the potential of hiPSCs for treating ischemic
retinopathies, we generated VPs from a repertoire of viral-integrated and nonintegrated fibroblast and cord blood (CB)–
derived hiPSC lines and tested their capacity for homing and engrafting into murine retina in an ischemia-reperfusion model.
Methods and Results—VPs from human embryonic stem cells and hiPSCs were generated with an optimized vascular
differentiation system. Fluorescence-activated cell sorting purification of human embryoid body cells differentially expressing
endothelial/pericytic markers identified a CD31+CD146+ VP population with high vascular potency. Episomal CB-induced
pluripotent stem cells (iPSCs) generated these VPs with higher efficiencies than fibroblast-iPSC. Moreover, in contrast to
fibroblast-iPSC-VPs, CB-iPSC-VPs maintained expression signatures more comparable to human embryonic stem cell VPs,
expressed higher levels of immature vascular markers, demonstrated less culture senescence and sensitivity to DNA damage,
and possessed fewer transmitted reprogramming errors. Luciferase transgene-marked VPs from human embryonic stem cells,
CB-iPSCs, and fibroblast-iPSCs were injected systemically or directly into the vitreous of retinal ischemia-reperfusion–injured
adult nonobese diabetic-severe combined immunodeficient mice. Only human embryonic stem cell– and CB-iPSC–derived
VPs reliably homed and engrafted into injured retinal capillaries, with incorporation into damaged vessels for up to 45 days.
Conclusions—VPs generated from CB-iPSCs possessed augmented capacity to home, integrate into, and repair damaged
retinal vasculature. (Circulation. 2014;129:359-372.)
Key Words: blood supply ◼ diabetic retinopathy ◼ embryonic stem cells ◼ induced pluripotent stem cells
◼ reperfusion injury ◼ stem cells ◼ transplantation
© 2013 American Heart Association, Inc.
Circulation is available at http://circ.ahajournals.org DOI: 10.1161/CIRCULATIONAHA.113.003000
Received April 18, 2013; accepted October 15, 2013.
From the Institute for Cell Engineering, and Kimmel Comprehensive Cancer Center, Johns Hopkins School of Medicine, Baltimore, MD (T.S.P.,
L.Z., J.S.H., J.A., E.T.Z.); Wilmer Ophthalmological Institute, Johns Hopkins University School of Medicine, Baltimore, MD (I.B., R.G., C.M., G.L.);
Department of Radiation Oncology (P.N., F.R.) and Department of Microbiology/Immunology (D.M., R.A.F.), University of Maryland School of Medicine,
Baltimore, MD; Department of Cardiovascular Medicine (A.J.R., J.C.) and Institute for Stem Cell Biology and Regenerative Medicine (A.J.R., R.R.-P.,
J.C.), Stanford University, Palo Alto, CA; and Institute for Basic Biomedical Science at Johns Hopkins School of Medicine, Baltimore, MD (C.T.).
*Drs Park and Bhutto contributed equally to this work.
†Drs Lutty and Zambidis are co-senior authors.
The online-only Data Supplement is available with this article at http://circ.ahajournals.org/lookup/suppl/doi:10.1161/CIRCULATIONAHA.
Correspondence to Elias T. Zambidis MD, PhD, Institute for Cell Engineering and Kimmel Comprehensive Cancer Center, The Johns Hopkins University
School of Medicine, 733 N Broadway, MRB 755, Baltimore, MD 21205. E-mail firstname.lastname@example.org
Vascular Progenitors From Cord Blood–Derived Induced
Pluripotent Stem Cells Possess Augmented Capacity for
Regenerating Ischemic Retinal Vasculature
Tea Soon Park, PhD*; Imran Bhutto, MD, PhD*; Ludovic Zimmerlin, PhD;
Jeffrey S. Huo, MD, PhD; Pratik Nagaria, PhD; Diana Miller, BS; Abdul Jalil Rufaihah, PhD;
Connie Talbot, BS; Jack Aguilar, BS; Rhonda Grebe, BS; Carol Merges, MA;
Renee Reijo-Pera, PhD; Ricardo A. Feldman, PhD; Feyruz Rassool, PhD; John Cooke, PhD;
Gerard Lutty, PhD†; Elias T. Zambidis, MD, PhD†
360 Circulation January 21, 2014
the ischemic death of retinal neurons and associated second-
ary pathological neovascularization, as well, thus potentially
ameliorating or preventing end-stage blindness.
Several groups have demonstrated the feasibility of regen-
erating ischemic retinal vasculature with cellular therapies.
For example, transplanted adult hematoendothelial11,12,15 or
embryonic hemangioblastic14 progenitors were shown to
home to degenerated ocular vascular sites created by severe
ischemia or long-term diabetes mellitus. However, adult VPs
are rare in circulating peripheral blood and bone marrow, and
restricted in expansion capacities. Furthermore, adult VPs
from diabetic patients are limited in their regenerative poten-
tial because they are functionally defective because of chronic
hyperglycemia.16,17 The regeneration of highly proliferative
embryonic VPs from patient-specific or human leukocyte anti-
gen–defined human induced pluripotent stem cells (hiPSCs)
would circumvent these caveats and provide unlimited sources
of pristine, nondiseased progenitors for cellular therapies.
The ability of hiPSCs to differentiate into vascular-endo-
thelial progenitors with in vivo engraftment potential has pre-
viously been demonstrated.18–23 Additionally, both adult and
human embryonic stem cell (hESC)-derived hemangioblasts
possessing endothelial capacities could transiently populate
injured tissue, including the retina.14,24–26 However, long-term
and functional in vivo engraftment of hiPSC-derived vascular
cells has not yet been reported in the retina. Additionally, the
use of viral vectors for expressing reprogramming factors in
somatic cells poses a major obstacle limiting clinically useful
hiPSC-based vascular therapies. Despite overall silencing of
integrated retroviral and lentivector promoters during hiPSC
generation, low levels of viral transgenes or reactivated vector
promoters can result in incompletely reprogrammed states that
can promote insertional mutagenesis or malignant transforma-
tion.27,28 Additionally, many standard hiPSC lines have differen-
tiated to the vascular lineage with poor efficiency, more rapid
senescence, and reduced proliferation rates in comparison with
hESC.29 It is currently unknown whether hiPSCs made with
nonintegrated methodologies30–32 will have similar or fewer lim-
itations for generating therapeutically useful vascular lineages.
We recently described an efficient method for generat-
ing nonintegrated hiPSCs from human myeloid progenitors,
including from cord blood (CB).31,32 These high-fidelity (HF)
CB–induced pluripotent stem cells (iPSC) lines possessed
global and pluripotency-associated transcriptional signatures
that were indistinguishable from hESCs.32 We also recently
described methods for generating hematovascular progenitors
from an optimized differentiation system.33,34 Here, we demon-
strate that VPs differentiated from nonintegrated HF CB-iPSCs
possess an enhanced potential for repairing damaged retinal
blood vessels in a preclinical ischemic retinopathy model.
Detailed Expanded Methods are available in the online-only Data
The hESC lines H1 (WA01), H7 (WA07), H9 (WA09), and ES03 (ES03)
used in these studies were obtained from the Wisconsin International
Stem Cell Bank. The use of all Wisconsin International Stem Cell
Bank–donated hESC lines in these studies was approved by the Johns
Hopkins Institutional Stem Cell Research Oversight and Institutional
Review Board Committees. All animal surgical procedures were per-
formed in accordance with protocols approved by the Johns Hopkins
School of Medicine Institute of Animal Care and Use Committee and
the Association for Research of Vision and Ophthalmology statement
for the Use of Animals in Ophthalmic and Visual Research.
Isolation of Human Embryoid Body–Derived VP
Populations Expressing CD31 and CD146
Our vascular differentiation system was previously described,24,33,35 and
is summarized in Figure 1A. Recipes for all differentiation reagents,
antibodies, and polymerase chain reaction primers are provided in
Tables I through VII in the online-only Data Supplement. In brief, day
8 human embryoid body cells were disaggregated by using collagenase
type IV (1 mg/mL, Sigma-Aldrich, St Louis, MO), and plated onto
fibronectin (10 µg/mL, Life Technologies, Grand Island, NY)–coated
plates in endothelial growth medium-2 (EGM2, Lonza, Walkersville,
MD) supplemented with 25 ng/mL vascular endothelial growth factor
(VEGF)165 (Peprotech, Rocky Hill, NJ). Four to 6 days later, adherent
human embryoid body–derived cells were treated with 0.05% trypsin-
ethylenediaminetetraacetic acid (Life Technologies) for 5 minutes at
37°C, and washed in mouse embryonic fibroblast medium (Table II in
the online-only Data Supplement). Cell clumps were filtered by using
a 40-µm cell strainer (Fisher Scientific, Pittsburgh, PA), centrifuged at
200g for 5 minutes, and resuspended at a concentration of 1×107 cells/
mL in EGM2/phosphate-buffered saline (1:1) solution. Fluorescence-
activated cell sorting (FACS) was conducted at the Johns Hopkins
FACS Core Facility with a FACS Aria III instrument (BD Biosciences,
San Jose, CA). Cell suspensions were incubated with mouse anti-
human CD31-APC (eBioscience, San Diego, CA) and CD146-PE (BD
Biosciences) antibodies for 30 minutes on ice, FACS-purified into 4
fractions based on high CD31 and CD146 expression (Figure 1B),
plated onto fibronectin-coated plates in EGM2, and expanded to 80%
to 90% confluence for 7 to 9 days before in vivo injections.
Ocular Ischemic Reperfusion Injury and Human
Four- to 6-week old male nonobese diabetic-severe combined immu-
nodeficient (NOD-SCID) mice (Jackson Laboratory, Bar Harbor, ME)
were subjected to high intraocular pressure to induce retinal ischemia-
reperfusion (I/R) injury. Mice were deeply anesthetized by intraperi-
toneal injection of ketamine/xylazine (50 mg/kg ketamine+10 mg/kg
xylazine in 0.9% NaCl). The pupils were dilated with 2.5% phenyleph-
rine hydrochloride ophthalmic solution (AK-DILATE, Akorn, Buffalo
Glove, IL) followed by 0.5% tetracaine hydrochloride ophthalmic
topical anesthetic solution (Phoenix Pharmaceutical, St. Joseph, MO).
The anterior chamber of the eye was cannulated under microscopic
guidance (OPMI VISU 200 surgical microscope; Zeiss, Gottingen,
Germany) with a 30-gauge needle connected to a silicone infusion
line providing balanced salt solution (Alcon Laboratories, Fort Worth,
TX), avoiding injury to the corneal endothelium, iris, and lens. Retinal
ischemia was induced by raising intraocular pressure of cannulated
eyes to 120 mm Hg for 90 minutes by elevating the saline reservoir.
Ischemia was confirmed by iris whitening and loss of the retinal red
reflex. Anesthesia was maintained with 2 doses of 50 µL intramus-
cular ketamine (20 mg/mL) during the 90 minutes. The needle was
subsequently withdrawn, the intraocular pressure was normalized, and
reperfusion of the retinal vessels was confirmed by the reappearance
of the red reflex. The contralateral eye of each animal served as a non-
ischemic control. Antibiotic ointment (Bacitracin zinc and Polymyxin
B sulfate, AK-Poly-Bac, Akron) was applied topically. Two days later,
FACS-purified human VPs were injected into either the vitreous body
(50 000 cells in 1 µL/eye), the orbital sinus (100 000 cells in 2 µL/
eye) by using a microinjector (PLI-100, Harvard Apparatus, Holliston,
MA), or into the tail vein (300 000 cells/100 µL per mouse).
To evaluate statistical significance, 2-tailed t tests (between indi-
vidual groups), or 1-way analysis of variance (eg, analysis of
Park et al hiPSC Therapy of Ocular Ischemia 361
Figure 1. Efficient generation of embryonic VP populations from hPSCs. A, Schema for vascular differentiation (diff) and expansion of
VP. B, Flow cytometry plot of day 8 hEB cells from H9-hESC after expansion in EGM2 for 4 days. The average percentage of 4 indicated
quadrants±SEM is shown (n=13 experiments). C, Percentage Dil-Ac-LDL uptake (mean±SEM) of FACS-purified and EGM2-expanded
populations differentiated from 2 hESC lines (gold), 3 CB-iPSC lines (red), and 6 fibroblast-iPSC lines (green). Each data point represents an
independent, replicate experiment. D, In vitro Matrigel assays of purified and expanded populations. Scale bar, 500 μm. E, Representative
Matrigel plugs consisted of vascular structures formed by indicated CB-iPSC-6.2 hEB–derived populations, and immunostained with
anti-CD31 (brown). Scale bars, 100 μm. F, Measurements in Matrigel plug sections: Left, percentages of blood vessels >30 μm diameter
per microscopic field (mean±SEM); Right, total number of blood vessels per microscopic field (mean±SEM) (2-tailed t tests; *P<0.05;
**P<0.01). AM indicates adaptation medium; CB, cord blood; Dil-Ac-LDL, Dil-acetylated low-density lipoprotein; EGM2, endothelial growth
medium-2; FACS, fluorescence-activated cell sorting; hEB, human embryoid body; hESC, human embryonic stem cell; hPSC, human
pluripotent stem cell; iPSC, induced pluripotent stem cell; LDM, liquid differentiation medium; MM, methylcellulose medium; SEM, standard
error of the mean; VEGF, vascular endothelial growth factor; and VP, vascular progenitor.
362 Circulation January 21, 2014
variance-Eisenhart method with Bonferroni correction for ≥3 groups)
were performed. For smaller, non-Gaussian–distributed sample sizes
(n<10), nonparametric (Mann-Whitney) tests were performed. P val-
ues of <0.05 or <0.01, respectively, were considered significant.
The National Institutes of Health Gene Expression Omnibus has
issued the accession number GSE44926.
Enrichment of a VP Population Differentiated
From hPSCs With Enhanced In Vitro and In Vivo
Vascular Pericytic Endothelial Potential
We previously described a differentiation system that generated
mesodermal-hematoendothelial and CD143/ACE+ hemangio-
blast progenitors from human pluripotent stem cells (hPSCs).24
We recently optimized this system to generate hematovascu-
lar progenitors (Figure 1A).33,34 To identify a putative embry-
onic VP population, we expanded day 8 human embryoid
body cells for 4 to 6 days in EGM2 cultures supplemented
with VEGF165 (Figure I in the online-only Data Supplement),
and subdivided adherent CD31+ cells by their coexpression
of the endothelial-perivascular-mesenchymal stem/progeni-
tor marker CD14622,36,37 (Figure 1B, Figures II and III in the
online-only Data Supplement). Four populations were FACS-
purified from adherent cells (CD31+CD146−, CD31+CD146+,
CD31−CD146−, and CD31−CD146+), further expanded in
EGM2 medium, and analyzed for surface coexpression of
established hematoendothelial, mesenchymal, pericytic, and
smooth muscle cell markers (Figures III through V in the
online-only Data Supplement). Fractionating CD31+ vascu-
lar-endothelial populations for high coexpression of CD146+
enriched a putative VP population that coexpressed mesenchy-
mal stem cell markers (CD44, CD90, CD105; Figure III in the
online-only Data Supplement), the hematoendothelial progeni-
tor marker CD117 (C-KIT; Figure V in the online-only Data
Supplement), but lacked hematopoietic-myeloid (Figure VI in
the online-only Data Supplement) or smooth muscle (α-SMA-
positive; Figure IV in the online-only Data Supplement) poten-
tial. Further EGM2 expansion of purified CD31+CD146+ VP
generated 2 discrete cellular phenotypes: an endothelial pro-
genitor cell–like (CD105hiCD144+CD140b−) population, and a
pericytic-like (CD31−CD105dimCD144−CD140b+) population
(Figure VII in the online-only Data Supplement).
To evaluate the endothelial functionality of VP popula-
tions, we differentiated and tested the 4 purified subsets with
Matrigel tube-forming and Dil-acetylated low-density lipo-
protein (Dil-Ac-LDL) uptake assays. As a group, EGM2-
expanded CD31+CD146+ populations from hESC, CB-iPSC,
and fibroblast-iPSC demonstrated significantly higher percent-
ages of Dil-Ac-LDL+ uptake (Figure 1C). Although expanded
CD31+CD146- and CD31+CD146+ populations both formed
organized microtubes in Matrigel (and with capillary-like
lumens in collagen gels), CD31+CD146+ VPs formed larger-
diameter and more extensively branching vascular tubes
(Figure 1D, Figure VIII in the online-only Data Supplement).
To confirm the vascular potential of putative VP popula-
tions in vivo, expanded cells from each FACS-purified group
were resuspended in Matrigel and injected subcutaneously into
NOD-SCID mice. Capacity for 3-dimensional vessel formation
was quantified 2 weeks later in Matrigel plugs. CD31+CD146+
VP populations expanded for 7 to 10 days in EGM2 proved
most optimal for forming significantly greater numbers of
large-diameter chimerized human-mouse blood vessels (>30
µm) in these Matrigel plugs (Figure 1E and 1F, Figures VII and
IX in the online-only Data Supplement). Chimerized vessels
carried circulating murine blood within them, thus demonstrat-
ing successful anastomosis and developmental maturity.
CB-iPSCs Generated CD31+CD146+ VPs With
Higher Efficiency Than Fibroblast-iPSC
To determine the differentiation efficiency of generating this
novel VP population from hPSC, we differentiated 18 hPSC
lines derived via various methods; this included 4 hESC lines
(H1, H7, H9, ES03), 4 viral fibroblast-iPSC lines (IMR90-1,
IMR90-4, HUF3, HUF5), 4 7F-episomal (7F-E) fibroblast-
iPSC lines (7ta, WT2, WT4, SF-iPSC 6.1), and 6 episomal
CB-iPSC lines (7F-E: 6.2, 6.13, 19.11; 4F-episomal (4F-E):
E5C3, E12C5, E17C6). Although most hiPSC lines differ-
entiated to the vascular lineage with efficiencies relatively
comparable to hESC lines, CB-iPSC as a group produced
significantly (P<0.01) higher frequencies of CD31+CD146+
VP cells than fibroblast-iPSC (Figure 2A). These hiPSC-
derived VP populations displayed morphologies similar
to neonatal EC (eg, human umbilical vein endothelial cells
[HUVECs]), and readily took up Dil-Ac-LDL and endothe-
lial-specific Ulex europaeus agglutinin38 (UEA-1) (Figure 2B
through 2D). There were no apparent quantitative differences
in capacity to generate CD31+CD146+ VP between viral and
episomal-derived fibroblast-iPSC, or as a function of passage
number (data not shown). However, HF CB-iPSC VPs pos-
sessed significantly higher capacities for Dil-Ac-LDL uptake
than did fibroblast-iPSC VP, and with minimal hiPSC inter-
line variability (Figure 2E). Purified CB-iPSC-VPs expanded
for several weeks also maintained higher expressions of VP
markers (eg, CD31, CD146, KDR [VEGFR2], CD90, and
CD144 [VE-Cadherin]), and stably maintained their capac-
ity for generating branching, sprouting endothelial-pericytic
microvascular tubes with capillary-like lumens in collagen
gels (Figure 2F and 2G).
CB-iPSC–Derived VP Possessed Transcriptional
Signatures With Closer Resemblance to VP
Generated From hESC
To evaluate the molecular resemblance of embryonic hPSC-
derived VP to adult vascular EC (eg, HUVEC and human
microvascular ECs), we compared whole genome transcrip-
tional signatures of adult EC with FACS-purified embryonic
VPs with Illumina expression microarrays. We computed
unsupervised hierarchical clustering and principal compo-
nent analysis of global expression variance (34 680 genes) of
FACS-purified and expanded CD31+CD146+ VPs from hESC,
CB-iPSC, fibroblast-iPSC, versus adult HUVEC, human
microvascular ECs, donor CB cells, donor fibroblasts, and
undifferentiated hESCs (Figure 3A, Figure X in the online-
only Data Supplement). This global expression analysis did
not sharply distinguish the VPs generated from each hPSC
class, but it did demonstrate that embryonic VPs from all
Park et al hiPSC Therapy of Ocular Ischemia 363
Figure 2. Characterization of VPs generated from hPSCs. A, Efficiency of hEB differentiation (%mean±SEM) of hPSCs to CD31+CD146+
VPs. Data are from 4 hESC lines (H1, H7, H9, ES03; n=13 experiments [gold]), 8 fibroblast-iPSC lines (IMR90-1, IMR90-4, HUF3, HUF5,
7ta, WT2, WT4, fF6.1); n=11 experiments [green]), and 6 CB-iPSC lines (6.2, 6.13, 19.11, E5C3, E12C5, E17C6; n=17 experiments [red]).
Two-tailed t tests: **P<0.01. B, Phase-contrast image of FACS-purified, expanded CD31+CD146+ VP cells differentiated from CB-iPSC-6.2,
with Ulex europaeus agglutinin (UEA) and DAPI costaining (C), and with Dil-Ac-LDL uptake staining (D). E, Percentage Dil-Ac-LDL uptake
(mean±SEM) of expanded CD31+CD146+ VPs from individual differentiations of hESC (gold), CB-iPSC (red), and fibroblast-iPSC (green).
Mann-Whitney tests: *P<0.05. F, TEM image CB-iPSC-derived VPs forming vascular tubes in collagen gel via cooperating endothelial
and pericytic-like cells; all border on lumens and are potential ECs in apparent bifurcation. G, Representative surface marker analyses of
FACS-purified/expanded hPSC-derived CD31+CD146+ VPs and HUVECs. CB indicates cord blood; Dil-Ac-LDL, Dil-acetylated low-density
lipoprotein; FACS, fluorescence-activated cell sorting; fibro, fibroblast; hEB, human embryoid body; hESC, human embryonic stem cell;
hPSC, human pluripotent stem cell; HUVEC, human umbilical vein endothelial cell; iPSC, induced pluripotent stem cell; L, lumen; n, nuclei;
SEM, standard error of the mean; TEM, transmission electron microscopy; and VP, vascular progenitor.
364 Circulation January 21, 2014
Figure 3. Expression signatures of hPSC-derived VPs. A, Principal component analysis of genome-wide expression signatures for
indicated samples of embryonic CD31+CD146+ VPs generated from hESC, CB-iPSC, and fibroblast-iPSC; adult endothelial cells (HUVEC,
HMEC); hiPSC donor cells; and hESC lines. Data were generated from samples in Figure X in the online-only Data Supplement. Pearson
coefficient R2 for hESC-VP vs CB-iPSC-VP=0.974; hESC-VP vs fibroblast-iPSC-VP=0.958. B, Heatmap-dendrogram of Illumina expression
array data of the vascular lineage-specific genes indicated. Individual RNA samples from independent differentiations were obtained from
HUVEC (n=3), HMVEC (n=3), hESC-VP (n=4), CB-iPSC-VP (n=4), fibroblast-iPSC-VP (n=4), donor fibroblast (Fibro, n=3), hESC (n=3), and
donor CB (n=3). C, Quantitation of expression of vascular lineage-specific genes in B. Mean value of the gene signal intensity is shown
(*P<0.05; mean expression among hESC-VP, CB-iPSC-VP, and Fib-iPSC-VP differed significantly (1-way ANOVA, P=0.001). Fib-iPSC-
VPs also significantly differed by individual comparison from hESC-VP (P=0.0002, Bonferroni correction threshold P<0.016), whereas
CB-iPSC-VP vs hESC-VP did not (P=0.09). For list of genes analyzed, see Table IVVV in the online-only Data Supplement. D, Q-RT-PCR
analysis of CD31+CD146+ VPs from hESC-H9, CB-iPSC-6.2, and HUVEC. Relative expression of CD31, CD34, vWF, FLT1, TIE1, and TIE2
of replicate samples was normalized to expression in undifferentiated hESC-H9 (n=2 experiments). ANOVA indicates analysis of variance;
CB, cord blood; Dil-Ac-LDL, Dil-acetylated low-density lipoprotein; Fib, fibroblast; H, hematopoietic-specific genes; hEB, human embryoid
body; hESC, human embryonic stem cell; HMVEC, human microvascular endothelial cell; hPSC, human pluripotent stem cell; HUVEC,
human umbilical vein endothelial cells; iPSC, induced pluripotent stem cell; P, pluripotency-specific genes; Q-RT-PCR, quantitative real-
time polymerase chain reaction; VASCULAR, vascular lineage-specific genes; VP, vascular progenitor; and vWF, von Willebrand factor.
Park et al hiPSC Therapy of Ocular Ischemia 365
hPSC sources were transcriptionally distinct from adult dif-
ferentiated HUVEC/human microvascular ECs.
A focused expression analysis of vascular lineage-spe-
cific genes by microarray revealed that VPs generated from
CB-iPSC shared more congruence in their vascular expression
signatures39 (Table VIII in the online-only Data Supplement)
with hESC-VPs than did VPs from fibroblast-iPSC (Figure 3B
and 3C). Interestingly, CB-iPSC-VP populations expressed
higher transcript levels of endothelial-specific, perivascular/
pericytic–specific genes (eg, PDGFRb [CD140b]), and adhe-
sion/migration–specific genes (eg, integrin a5) than other
hPSC classes (Figure 3B, Table VIII in the online-only Data
Supplement). Quantitative real-time polymerase chain reac-
tion expression analysis of key hematovascular genes (eg,
CD31, CD34, von Willebrand factor, FLT1, TIE1, and TIE2)
further confirmed the notion that embryonic hPSC-derived
VP populations were more primitive than adult HUVEC
(Figure 3D). For example, VPs differentiated from hESC
and CB-iPSC expressed higher transcript levels of immature
endothelial progenitor markers such as TIE1 and TIE2, and
lower expressions of mature endothelial transcripts (eg, von
Willebrand factor) than adult ECs.
To determine differential expression of genes in VP derived
from either CB-iPSC or fibroblast-iPSC that distinguished
them from VPs derived from hESC, we conducted compara-
tive bioinformatics analyses (Figure XI in the online-only Data
Supplement). These studies demonstrated that CB-iPSC-VPs
possessed significantly fewer (344) differentially expressed
genes with hESC-derived VPs than did VPs generated from
fibroblast-iPSC (628). A gene ontology analysis of the genes that
were differentially expressed between fibroblast-derived VP and
hESC-VP revealed systematic differences in fibroblast-iPSC-VP
Figure 4. Senescence and DNA damage sensitivity of expanded VPs. A, Representative β-galactosidase senescence staining in hPSC
classes; legend symbols are as before. Scale bars, 100 µm. B, Percentage of senescent cells (mean±SEM of n=12 microscopic fields per
each line of each hPSC class; n=3 per class). C, Western blots of p53 expression before and 24 hours after 2-Gy irradiation of VPs from
(1) H9 (p46), (2) ES03 (p88), (3) 6.2 (P20), (4) 6.2 (P23), (5) 19.11 (P19), (6) E17C6 (P29), (7) HUF3 (P44), (8) IMR90-1 (P71), and (9) IMR90-1
(p72). D, Fold percentage change in p53 protein expression levels by Western blot densitometry; >1.0 (increase) or <1.0 (decrease)
above baseline). Two-tailed t tests: *P<0.05. CB indicates cord blood; fibro, fibroblast; hESC, human embryonic stem cell; iPSC, induced
pluripotent stem cell; SEM, standard error of the mean; and VP, vascular progenitor.
366 Circulation January 21, 2014
Figure 5. In vivo migration, homing, and engraftment of luciferase-transgenic VP cells into I/R-damaged mouse retina. A, Experimental
design for quantifying human VP engraftment into NOD-SCID mouse retinas. Left, Anatomy of mouse eye indicating I/R location at
anterior chamber and site of human cell injections into vitreous body. Right, Timeline of in vivo engraftment analysis. B, Representative
immunofluorescent retinal sections of I/R-damaged eyes injected with hESC-luciferase-transgenic (green) CD31+CD146+ VP (left) and
CD31+CD146− (right) cells at 3 days postinjection. CD31+CD146+ cells readily migrated into deep retinal layers, whereas CD31+CD146−
cells remained primarily in vitreous. C, Quantification of (a) cell migration into retina and (b) number of engrafted human cells visualized
per retinal cross-sections following injection of hESC-derived CD31+CD146+ VP and CD31+CD146− cells (n=15 and 9 sections evaluated,
Park et al hiPSC Therapy of Ocular Ischemia 367
genes that regulate processes of developmental fate, signal
transduction, cell adhesion, and cell growth/proliferation (Table
IX in the online-only Data Supplement). Taken together, these
genomewide transcriptomic studies suggested that embryonic
VPs generated from CB-iPSC had fewer aberrant transcriptional
patterns, and a significantly greater transcriptional resemblance
to embryonic VPs differentiated from hESCs.
CB-iPSC-VP Demonstrated Reduced Senescence
and Sensitivity to DNA Damage
Previous studies with standard viral fibroblast-iPSC lines
reported significantly diminished and highly variable directed
differentiation to the vascular-endothelial lineage.29 For exam-
ple, differentiations of fibroblast-iPSC were characterized
by poor growth and expansion of vascular-endothelial cells,
with high rates of apoptosis and early senescence. To deter-
mine the functional quality of VPs generated with our system,
purified VP differentiated from hESC, fibroblast-iPSC, and
CB-iPSC were expanded in EGM2 for up to 30 days (≈4 pas-
sages) and senescent cells were quantified via β-galactosidase
activity (Figure 4A). These studies revealed that CB-iPSC-VP
maintained more enhanced proliferation and significantly
less senescence following expansion for several passages in
comparison with fibroblast-iPSC–derived VP (Figure 4B).
To further evaluate the nature of this relative resistance of
CB-iPSC-VP to senescence, we probed the capacity of VPs
generated from the 3 classes of hPSCs to maintain genomic
integrity by assaying for sensitivity to double-stranded DNA
damage following irradiation. Expression of p53 protein,
which is normally activated briefly following DNA damage,
was compared before and after 24 hours of 2-Gy irradiation
(Figure 4C and 4D). These studies revealed that p53 levels
were relatively lower in expanded CB-iPSC-VPs, suggest-
ing a reduced sensitivity to irradiation-induced DNA dam-
age in comparison with fibroblast-iPSC-VPs. Interestingly,
CB-iPSC-derived VP protein levels of RAD51, which also
plays an important role in mediating repair from double-
stranded DNA damage following irradiation, was also more
comparable to hESC-derived VPs (data not shown).
CB-iPSC–Derived VPs Demonstrated Augmented
Capacity for Homing and Engraftment Into Retinal
Vasculature of I/R-Damaged NOD-SCID Mice
To test the potential for in vivo ocular vessel engraftment of
hiPSC-VPs, we generated luciferase transgene–expressing hESC
(H9), fibroblast-iPSC (IMR90-1), and CB-iPSC (6.2) lines repre-
senting the 3 major classes of hPSC being evaluated in this study
(Figure XII in the online-only Data Supplement). These lucifer-
ase-marked hPSC lines were differentiated to VPs as described
in Figure 1A. Fifty thousand luciferase+ human VP cells were
injected directly into the vitreous body of NOD-SCID recipient
eyes 2 days following I/R injury, and human cell engraftment in
murine retina was evaluated at 3, 7, 14, 21, and 45 days later with
antiluciferase immunofluorescent staining (Figure 5A).
Interestingly, hPSC-derived CD31+CD146− cells failed
to migrate efficiently into I/R-damaged retina and home to
blood vessels, and instead remained in the vitreous body, or
adherent to the superficial layer of retina adjacent to vitreous
(Figure 5B, right). In contrast, CD31+CD146+ VPs migrated
efficiently through the deep retinal layers, and homed and
incorporated readily into blood vessels (Figure 5B, left;
Figure 5D, left). The migration distance and cell numbers of
these CD31+CD146+ and CD31+CD146− populations were
quantitatively compared in serial retinal sections (Figure 5C).
CD31+CD146+ cells not only migrated longer distances
into the deeper retinal layers (≈5-fold; P<0.05), but also
higher numbers of cells were detected in comparison with
CD31+CD146− cells (≈5-fold; P<0.05).
To evaluate mechanisms involved in enhanced retinal vessel
homing of VPs, we measured surface expression of CXCR4
(CD184), a critical chemokine that regulates cell migration.40
CD31+CD146+ VP cells expressed higher levels of CXCR4
than CD31+CD146− cells before vitreal injections (Figure XIII
in the online-only Data Supplement). An analysis of migration
of CXCR4+ CD31+CD146+ VP cells into I/R-damaged eyes
versus normal noninjured control eyes revealed that homing to
retinal vessels depended on vascular damage: CD31+CD146+
VP stayed adjacent to the internal limiting membrane in eyes
without injury signals (Figure 5D, right; Tables 1 and 2).
Following I/R injury, robust homing of CD31+CD146+ VPs
from both hESC and CB-iPSC into the retinal vasculatures
was observed as early as 3 days following vitreal injection
(Figure 5E, Tables 1 and 2). In comparison, CD31+CD146+
VPs from fibroblast-iPSC poorly homed and engrafted into
retinal vessels in I/R-damaged eyes in comparison with
CB-iPSC (Tables 1 and 2).
CB-iPSC-VPs Efficiently Engrafted Into Damaged
Retinal Blood Vessels After Local and Systemic
Injection for at Least 45 Days
In our I/R injury model, retinal vessel damage increases
over time following I/R injury. To detect the sequential loss
of murine host ECs, the retinal vasculature was stained with
an antibody specific to mouse anti-CD31, and the vascular
basement membrane was labeled with a murine anticolla-
gen type IV antibody. This method demonstrated that blood
vessel branches lost viable ECs as early as 7 days post-I/R
with the formation of acellular collagen tubelike capillary
structures4 (Figure XIV in the online-only Data Supplement).
This damage was more severe in capillaries and veins presum-
ably owing to their higher collapsible or compressible nature
under high intraocular pressure in comparison with arteries.
Interestingly, VPs injected into vitreous body initially assumed
abluminal (pericytic) positions at early postinjection days 3
Figure 5. Continued. respectively; 2-tailed t tests: *P<0.005; **P<0.001). D, CD31+CD146+ hESC-VP injected to I/R-damaged (injury,
left) and normal eye (no injury, right) demonstrating that cells do not migrate into retinal layers without injury signals. E, CD31+CD146+
hESC-VP (left) and CD31+CD146+ CB-iPSC-VP (right) engrafted into both venules and microcapillaries (arrows) in this flat mount retina
at postinjection day 3. Scale bars, 50 µm. hESC indicates human embryonic stem cell; ILM, internal limiting membrane; iPSC, induced
pluripotent stem cell; I/R, ischemia-reperfusion; NOD-SCID, nonobese diabetic-severe combined immunodeficient; ONH, optic nerve
head; PR: photoreceptor; VB, vitreous body; and VP, vascular progenitor.
368 Circulation January 21, 2014
and 7 (Figure 5E, arrows; Figure 6A through 6C, arrows).
Cryopreservation and sectioning of these retinas demonstrated
stable enwrapping of the retinal blood vessels by human VP
cells (Figure 6D and 6F, arrows).
To avoid the confusion of circulating cells appearing to be
located in the lumen of blood vessels, mice were systemi-
cally perfused before the collection of retinas. At postinjec-
tion day 14, CB-iPSC-VPs were clearly detected engrafting
into both luminal endothelial and abluminal pericytic loca-
tions (Figure 6G, Figure XVA and XVB and Movie I in the
online-only Data Supplement). Although hESC-VPs could
be found sporadically and nonspecifically throughout neural
retina, CB-iPSC-VPs consistently demonstrated more specific
engraftment into blood vessels (Figure XVC and XVD in the
online-only Data Supplement, Tables 1 and 2). At lower mag-
nification of retinal flat mounts, CB-iPSC-VPs appeared to
favor venous engraftment (blood vessels with larger diameter)
than arteries (blood vessels with smaller diameter and more
rigid walls), suggesting again that these cells preferentially
migrated in response to injury signals (Figure XVI and Movie
II in the online-only Data Supplement).
At postsystemic injection day 21, human cells were
observed primarily in luminal locations in murine host retinal
capillaries (Figure 6H, Figure XVII and Movie I in the online-
only Data Supplement). Chimeric capillaries on both linear
(Figure XVIIA through XVIIC, XVIIE in the online-only Data
Supplement), and branch point (Figure XVIIC and XVIID in
the online-only Data Supplement) vascular segments, as well,
were detected, suggesting an injury-induced vasculogenesis,
but that had no short-term impact on I/R-degenerated neuronal
viability (Figure XVIII in the online-only Data Supplement).
Finally, an alternative injection of CB-iPSC-VP via orbital
sinus or tail vein injection resulted in robust engraftment in
endothelial or luminal cell positions that could be detected
for at least 45 days (Figures 6I and 7, Tables 1 and 2).
Intravenously injected human cells were detected and quanti-
fied in both the superficial capillary layer and the deeper reti-
nal vascular networks, as well (Figure 7). CB-iPSC-VPs still
homed to damaged retinal vessels in greater numbers (≈2.4-
fold for orbital sinus and ≈1.6 fold for tail vein) in damaged
eyes in comparison with uninjured normal eyes. This long-
term engraftment is, to our knowledge, the most durable yet
reported for injected hiPSC-derived vascular cells.
We have identified a functional hPSC-derived embryonic VP
population that can integrate long-term into ischemia-dam-
aged mouse retinal vasculature. This study provides a pre-
clinical model for evaluating the potential of patient-specific
hiPSC-VP therapies for vascular degenerative disorders such
as diabetic retinopathy and branch vein occlusion. Both ocular
disorders progress to end-stage death of retinal neurons and
subsequent pathological neovascularization. If VPs could be
used to repopulate acellular retinal capillaries and regenerate
viable blood vessels, areas of ischemia could be reperfused,
potentially avoiding the end-stage blinding complications.
Such novel vascular therapies will require the efficient nonvi-
ral reprogramming of accessible somatic donor cells (eg, from
skin or blood) that can generate hiPSCs with superior vascu-
lar differentiation potential. Nonintegrated patient-specific
hiPSCs could be used to simultaneously generate both retinal
neurons and VPs for treating a variety of blinding disorders.
Our previous studies demonstrated that the barriers for effi-
cient nonintegrated, nonviral pluripotency induction can be
overcome by targeting highly accessible myeloid progenitors
that are readily available from patient bone marrow, peripheral
blood, or human leukocyte antigen–matched CB.31,32,41 We have
proposed that CB progenitors are especially attractive for plu-
ripotency induction, because they carry few somatic mutations
and could be used to create an human leukocyte antigen–defined
stem cell bank for ocular regenerative medicine via worldwide
networks of existing blood bank repositories.31,32 Computational
models predict that a small number of human leukocyte
Table 2. Summary of In Vivo I/R–Damaged Retinal Vessel
Engraftment of VPs Generated From hESCs, CB-iPSCs, and
Fibroblast-iPSCs via Systemic Intravenous Tail Vein Injections
Tail vein injection of CD31+CD146+ cells. CB indicates cord blood; hESC, human
embryonic stem cell; iPSC, induced pluripotent stem cell; I/R, ischemia-reperfusion;
n, number of independent experiments; ND, not done; R, cells engrafted in retinal
blood vessels; VP, vascular progenitor; +, 1–2 cells detected engrafted per
microscopic field; and ++, >2 cells detected engrafted per microscopic field.
Table 1. Summary of In Vivo I/R–Damaged Retinal Vessel Engraftment of VPs Generated From hESCs, CB-iPSCs, and Fibroblast-
iPSCs via Local Vitreous Body Injections
No I/RI/RNo I/RI/RNo I/RI/R
Intravitreal injection of CD31+CD146+ cells. CB indicates cord blood; F, fragmented, nonviable cells detected; hESC, human embryonic stem cell; iPSC, induced pluripotent
stem cell; I/R, ischemia-reperfusion; n, number of independent experiments; ND, not done; R, cells engrafted in retinal blood vessels; V, cells detected in vitreous body; VP,
vascular progenitor; –, zero cells detected; +, 1–2 cells detected engrafted per microscopic field; and ++, >2 cells detected engrafted per microscopic field.
Park et al hiPSC Therapy of Ocular Ischemia 369
antigen–defined hPSCs derived from existing cord and marrow
banks could generate matches to serve the transplantation needs
for the majority of the US population.42 Coculturing human
myeloid progenitors on patient-derived mesenchymal stromal
layers, which our reprogramming system uses, is a routine
method already used in clinical trials in highly reproducible,
good manufacturing practices-standardized conditions.43
Although hESCs and hiPSCs share high molecular simi-
larity, hiPSCs generally possess more variable directed
differentiation potencies than hESCs.27–29,31 Incomplete repro-
gramming and retention of donor-specific epigenetic memory
have been proposed as etiologies for poor hiPSC-differentia-
tion potencies, including to vascular-endothelial lineages. In
these studies, we found that HF CB-iPSCs derived at very
high efficiencies31,32 possessed significantly augmented vascu-
lar potency in comparison with fibroblast-hiPSCs derived via
standard methods. These CB-iPSCs generated CD31+CD146+
VPs that were more akin molecularly to those generated from
Figure 6. In vivo engraftment of CB-iPSC-VP into retinal vasculature. Luciferase-transgenic human VPs were injected directly into the
eye (intravitreal; A through H), or systemically intravenously (orbital sinus; I). G, Transgenic CB-iPSC-VP (green) homed to both damaged
capillaries (arrows) and larger blood vessels (retinal flat mounts). C, Damaged host vessels lacked murine ECs (lack of blue signal from
anti-mouse CD31), but their Coll IV+ basement membranes remained intact (red). D through F, Human cells (green) were often observed
in retinal cross-sections at pericytic positions surrounding host murine ECs (blue) in capillaries. Engrafted human CB-iPSC-VPs detected
in murine retina at 14 days (G), 21 days (H), and 45 days (I) postinjection. The degree of damage was more severe and the density
of functional blood vessels was reduced with time following I/R. Scale bars, 20 µm (A, G–I) and 10 µm (D). Shown are representative
experiments from Tables 1 and 2. CB indicates cord blood; Coll IV, collagenase type IV; EC, endothelial cell; iPSC, induced pluripotent
stem cell; I/R, ischemia-reperfusion; and VP, vascular progenitor.
370 Circulation January 21, 2014
hESCs, and with significantly fewer aberrant hiPSC-specific
genes expressed that are likely attributable to transmitted repro-
gramming errors. Although previous studies demonstrated
high senescence in vascular lineage cells generated from fibro-
blast-derived hiPSCs,29 CB-iPSC-VPs expanded more robustly
in culture, possessed lower rates of culture senescence, and
demonstrated more resistance to DNA damage than fibro-
blast-iPSCs. Moreover, our in vivo engraftment results sug-
gested that CB-iPSC-VPs may possess advantages in survival,
migration, and homing to damaged tissues in comparison with
fibroblast-iPSC-VPs. Derivation methods with more effective
reprogramming capacities may greatly improve the final func-
tional pluripotency of hiPSCs, including to the vascular lineage.
Further studies that explore the role of epigenetic memory will
ultimately confirm if HF CB-iPSCs will have greater clinical
utility for generating multiple transplantable lineages (eg, neu-
ral, vascular, retinal pigmented epithelium) for comprehensive
regenerative therapy of blinding ocular diseases.
Figure 7. Quantification of
human VP engraftment into
murine retinal vasculature.
A, Representative experiments
(from Tables 1 and 2)
(green) engrafting into
murine vessels following
orbital sinus (OS) or tail-vein
(TV) injections. Scale bars,
20 µm. B, Representative
experiments showing detection
of CB-iPSC-VP injected via
orbital sinus (left) or tail vein
(right). Retinas were harvested
at postinjection day 7, and
whole flat-mount retinas were
scanned and human cells
were quantified in superficial
(near vitreous body, red) and
deep retinal vasculatures (blue)
layers. C, Representative
quantification of a retinal
demonstrated that systemic
injections attracted higher
numbers of homing
CD31+CD146+ VPs into
damaged deep retinal blood
vessels (OS>TV). CB indicates
cord blood; Coll IV, collagenase
type IV; iPSC, induced
pluripotent stem cell; and
VP, vascular progenitor.
Park et al hiPSC Therapy of Ocular Ischemia 371
One important aspect of CB-iPSC–derived CD31+CD146+ VPs
was their efficient capacity to home to injured vessels. Despite
damage to ischemic acellular capillaries, the basement mem-
brane shared by EC and pericytes in ischemic retinal capillaries
is normally spared. Although many growth factors regulate hom-
ing to sites of injury, the most likely stimulus is stromal-derived
factor-1 (SDF-1)/CXCR4–mediated migration.40 We have previ-
ously demonstrated that the human retinal vasculature develops
by specialized vasculogenesis, differentiation, and coalescence
of angioblasts to form blood vessels at ≈14 weeks gestation.44,45
Retinal angioblasts during normal development, and the hiPSC-
derived VPs we have described, as well, expressed robust levels
of CXCR4 (the receptor for hypoxia-inducible SDF-1), thus pre-
sumably providing efficient homing of transplanted progenitors
to hypoxic, damaged retinal vessels. As angioblasts migrate to the
inner retina, they continue to express CXCR4 until they differen-
tiate into the ECs that line patent retinal vessel lumens. SDF-1 is
prominently localized to the innermost portion of retina during
retinal vascular development and displays a gradient toward the
outer retina.44 Retinal angioblasts also expressed VEGFR-2/KDR
and C-KIT similar to these embryonic VPs. Thus, hPSC-derived
CD31+CD146+ VP migration, homing, and engraftment in our
I/R injury model likely recapitulate the hypoxic events during
retinal development, when SDF/CXCR4 regulation is prominent.
Another important aspect of this study was the compara-
tive demonstrations of transplanting hPSC-VPs via intravitreal,
orbital sinus, and intravenous administrations. When directly
injected into vitreous, VPs homed to injured blood vessels and
engrafted primarily in pericytic positions on the outside of host
collagenous vascular tubes. In contrast, VPs delivered intrave-
nously engrafted primarily in endothelial positions. This behavior
is reminiscent of early studies in developing retinal vasculature
that suggested position in reference to the basement membrane
determines the developmental fate of VPs.45 The future aim of
selectively delivering hiPSC-derived ECs or pericytes (or both)
by targeting different routes to the damaged retina could have
value in regenerating vascular segments in the diabetic retina
where pericytes die before ECs before yielding acellular capil-
laries. Thus, this model establishes an important tool for evaluat-
ing the further development of clinically relevant hiPSC-based
regenerative therapies for the treatment of ischemic retinopathies.
We thank Dr Takayuki Baba, Scott McLeod, Murilo Rodiriguez, Dr
Malia Edwards, Dr Wayne Yu, and Karan Verma for technical sup-
port, and Dr Jingxia Wang for statistical consultation. We are grateful
to Ada Tam and Lee Blosser for their expertise in FACS. We are grate-
ful to Alan Friedman for reading and editing the manuscript.
Sources of Funding
This work was supported by grants from the National Institutes of
Health (NIH)/National Heart, Lung, and Blood Institute (NHLBI)
U01HL099775 (to Dr Zambidis) and U01HL100397 (to Drs Cooke and
Reijo-Pera), NIH/NEI R01-EY09357(to Dr Lutty), Research to Prevent
Blindness unrestricted funds and EY01761-Wilmer Eye Institute (to
Dr Lutty), NIH/NCI CA60441 (to Dr Huo) and the Maryland Stem
Cell Research Fund 2011-MSCRF-II-0008-00 (to Dr Zambidis);
2007-MSCRF-II-0379-00 (to Dr Zambidis); 2011-MSCRF-E-0023-00
(to Dr Lutty); 2009-MSCRF-III-106570
2007-MSCRFE-0110-00 (to Dr Feldman); 2009-MSCRFII-0082-00
(to Dr Feldman); 2011-MSCRFII-0023-00 (to Dr Rassool).
(to Dr Park);
Under a licensing agreement between Life Technologies and the
Johns Hopkins University, Dr Zambidis is entitled to a share of roy-
alty received by the University for licensing of stem cells. The terms
of this arrangement are managed by Johns Hopkins University in
accordance with its Conflict of Interest policies. This does not alter
the authors’ adherence to journal policies on sharing data and materi-
als. The other authors report no conflicts.
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The regeneration of retinal capillaries with cellular therapies could reverse the ischemic death of retinal neurons, and potentially
ameliorate or prevent end-stage blindness in disorders such as diabetic retinopathy and branched vein occlusion. For example,
unlimited supplies of transplantable vascular progenitors (VPs) along with retinal photoreceptors could be differentiated syn-
chronously from patient-specific human induced pluripotent stem cells for comprehensive regeneration of the damaged retina.
Additionally, cord blood cells offer an especially attractive universal donor source for generating human induced pluripotent stem
cells, because they carry few somatic mutations, and they can more efficiently generate nonviral, clinically relevant pluripotent
stem cell lines that could theoretically be assembled to create an human leukocyte antigen–defined stem cell bank via worldwide
networks of existing repositories. In these studies, we identify an embryonic VP population differentiated from human induced
pluripotent stem cells that can functionally integrate long-term into ischemia-damaged mouse retinal vasculature. We demonstrate
for the first time that, in contrast to VPs differentiated from human induced pluripotent stem cells derived via standard methods,
embryonic VPs from high-fidelity reprogrammed nonviral cord blood-induced pluripotent stem cells demonstrated lower culture
senescence, expanded more robustly in culture, demonstrated more resistance to DNA damage, and were more akin molecularly
to those generated from human embryonic stem cells. More importantly, VPs generated from cord blood-induced pluripotent stem
cell lines possessed an inherent advantage for long-term in vivo survival, migration, homing, and specific engraftment to ischemia/
reperfusion–injured retinal tissues. This humanized vascular regenerative model establishes an important tool for evaluating the
further development of clinically relevant pluripotent stem cell–based therapies for treating blinding ischemic retinopathies.