Efficient commitment to functional CD34+ progenitor cells from human bone marrow mesenchymal stem-cell-derived induced pluripotent stem cells.

Abstract

The efficient commitment of a specialized cell type from induced pluripotent stem cells (iPSCs) without contamination from unknown substances is crucial to their use in clinical applications. Here, we propose that CD34+ progenitor cells, which retain hematopoietic and endothelial cell potential, could be efficiently obtained from iPSCs derived from human bone marrow mesenchymal stem cells (hBMMSC-iPSCs) with defined factors. By treatment with a cocktail containing mesodermal, hematopoietic, and endothelial inducers (BMP4, SCF, and VEGF, respectively) for 5 days, hBMMSC-iPSCs expressed the mesodermal transcription factors Brachyury and GATA-2 at higher levels than untreated groups (P<0.05). After culturing with another hematopoietic and endothelial inducer cocktail, including SCF, Flt3L, VEGF and IL-3, for an additional 7-9 days, CD34+ progenitor cells, which were undetectable in the initial iPSC cultures, reached nearly 20% of the total culture. This was greater than the relative number of progenitor cells produced from human-skin-fibroblast-derived iPSCs (hFib-iPSCs) or from the spontaneous differentiation groups (P<0.05), as assessed by flow cytometry analysis. These induced cells expressed hematopoietic transcription factors TAL-1 and SCL. They developed into various hematopoietic colonies when exposed to semisolid media with hematopoietic cytokines such as EPO and G-CSF. Hematopoietic cell lineages were identified by phenotype analysis with Wright-Giemsa staining. The endothelial potential of the cells was also verified by the confirmation of the formation of vascular tube-like structures and the expression of endothelial-specific markers CD31 and VE-CADHERIN. Efficient induction of CD34+ progenitor cells, which retain hematopoietic and endothelial cell potential with defined factors, provides an opportunity to obtain patient-specific cells for iPSC therapy and a useful model for the study of the mechanisms of hematopoiesis and drug screening.

Full text

Page 1

Efficient Commitment to Functional CD34+ + Progenitor
Cells from Human Bone Marrow Mesenchymal Stem-Cell-
Derived Induced Pluripotent Stem Cells
Yulin Xu1, Lizhen Liu1, Lifei Zhang1, Shan Fu1, Yongxian Hu1, Yingjia Wang1, Huarui Fu1, Kangni Wu1,
Haowen Xiao1, Senquan Liu1, Xiaohong Yu1, Weiyan Zheng1, Bo Feng2, He Huang1*
1Bone Marrow Transplantation Center, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang Province, China, 2School of Biomedical
Sciences, The Chinese University of Hong Kong, Hong Kong, China
Abstract
The efficient commitment of a specialized cell type from induced pluripotent stem cells (iPSCs) without contamination from
unknown substances is crucial to their use in clinical applications. Here, we propose that CD34+ progenitor cells, which
retain hematopoietic and endothelial cell potential, could be efficiently obtained from iPSCs derived from human bone
marrow mesenchymal stem cells (hBMMSC-iPSCs) with defined factors. By treatment with a cocktail containing mesodermal,
hematopoietic, and endothelial inducers (BMP4, SCF, and VEGF, respectively) for 5 days, hBMMSC-iPSCs expressed the
mesodermal transcription factors Brachyury and GATA-2 at higher levels than untreated groups (P,0.05). After culturing
with another hematopoietic and endothelial inducer cocktail, including SCF, Flt3L, VEGF and IL-3, for an additional 7–9 days,
CD34+ progenitor cells, which were undetectable in the initial iPSC cultures, reached nearly 20% of the total culture. This
was greater than the relative number of progenitor cells produced from human-skin-fibroblast-derived iPSCs (hFib-iPSCs) or
from the spontaneous differentiation groups (P,0.05), as assessed by flow cytometry analysis. These induced cells
expressed hematopoietic transcription factors TAL-1 and SCL. They developed into various hematopoietic colonies when
exposed to semisolid media with hematopoietic cytokines such as EPO and G-CSF. Hematopoietic cell lineages were
identified by phenotype analysis with Wright-Giemsa staining. The endothelial potential of the cells was also verified by the
confirmation of the formation of vascular tube-like structures and the expression of endothelial-specific markers CD31 and
VE-CADHERIN. Efficient induction of CD34+ progenitor cells, which retain hematopoietic and endothelial cell potential with
defined factors, provides an opportunity to obtain patient-specific cells for iPSC therapy and a useful model for the study of
the mechanisms of hematopoiesis and drug screening.
Citation: Xu Y, Liu L, Zhang L, Fu S, Hu Y, et al. (2012) Efficient Commitment to Functional CD34+ Progenitor Cells from Human Bone Marrow Mesenchymal Stem-
Cell-Derived Induced Pluripotent Stem Cells. PLoS ONE 7(4): e34321. doi:10.1371/journal.pone.0034321
Editor: Pranela Rameshwar, University of Medicine and Dentistry of New Jersey, United States of America
Received November 26, 2011; Accepted February 28, 2012; Published April 9, 2012
Copyright: ? 2012 Xu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was supported by research funding from National Post Doctor Science Fund of China to YX [20100471736], from Major Program of
Technology Research and International co-operative research of Zhejiang Province to WZ (2009C14011) and from Zhejiang Provincial Key Medical Discipline
(Medical Tissue Engineering) to HH. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: hehuang.zju@gmail.com
Introduction
CD34+ progenitor cells are used to treat many disorders, such
as incurable hematologic and lymphoid malignancies. However,
immune incompatibility, or gene disparity between recipients and
donors, causes graft-versus-host disease (GVHD) and other
transplant-related complications. These can cause graft failure or
even death [1]. Additionally, sources of transplantable CD34+
progenitor cells have been limited to bone marrow, umbilical cord
blood, and mobilized peripheral blood tissues. This makes it
difficult to obtain CD34+ progenitor cells in numbers sufficient to
reconstitute a normal blood system for an adult [2,3]. The high
risk of fatal complications and the quantity requirements for
CD34+ progenitor cells have prevented cell therapy from gaining
wide application. The study and establishment of efficient CD34+
progenitor cell induction systems are thus necessary [4,5].
Human embryonic stem cells (hESCs) are derived from the
inner cell mass of preimplantation embryos. These cells are
totipotent, capable of giving rise to all types of human cells and of
proliferating indefinitely in vitro under specific culture conditions.
Since the establishment of the first hESC line, people have been
immensely interested in using embryonic stem cells as an
alternative source of CD34+ progenitor cells for cell transplanta-
tion [6–13]. However, approaches using embryoid body (EB)
formation or co-culture with a feeder layer of mouse cells, such as
the OP9 stromal cell line or the S17 bone marrow cell line, are not
suitable for clinical applications. This is not only because of the
low differentiation efficiency, poorly defined induction compo-
nents, and contamination from animal sources, but also because of
the immune incompatibility and the ethical and legal restrictions
surrounding embryonic stem cell research [7].
The emerging of induced pluripotent stem cells (iPSCs) was a
breakthrough. Potentially patient-specific cells can be obtained
without the ethical concerns or immune rejection. They represent
a potentially unlimited source of functionally specific cell lineages
[14–17]. iPSCs have been demonstrated to be a viable alternative
to ESCs for generation of CD34+ progenitor cells, which is of
PLoS ONE | www.plosone.org1April 2012 | Volume 7 | Issue 4 | e34321

Page 2

immense importance to clinical treatment, drug discovery, and the
study of disease mechanisms [10,18–21]. Studies performed by
Choi and his colleagues showed similar patterns of differentiation
potential between human iPSC lines and hESC lines, as
determined using an OP9 differentiation system [18]. Woods et
al. developed an optimized differentiation protocol using mouse
stromal cells with cytokines for the generation of hematopoietic
lineages from iPSCs [19]. However, the cells obtained from the
studies given above were contaminated with unidentified factors
from the mouse-derived cells. This is not in line with the crucial
step for cell-based therapy. Although several defined culture
conditions have been identified for directing human iPSCs
differentiation toward CD34+ progenitor cells, the overall efficacy
of these protocols remains low [20,21]. In one study, hiPSC-
derived CD34+ cells cannot develop into hematopoietic cells [21].
Better approaches for more efficient induction of CD34+
progenitor cells merit investigation. Hematopoietic potential is
strictly dependent on cellular signaling, as demonstrated by
Kennedy et al., and existing protocols for the efficient induction
of functional CD34+ progenitor cells appear to require treating
the differentiating cells with complex cocktails of factors that affect
the mesoderm, hematological, and endothelial cells [9,22–25].
Adult human bone marrow-derived mesenchymal stem cells
(hBMMSCs)aremultipotent,meaningthattheycandifferentiateinto
several lineages, such as osteoblasts, adipocytes, and chondrocytes
[26]. They are closely related to hematopoiesis. hBMMSCs have
immunosuppressive properties that make them interesting candidates
for cell-based therapy in organ transplantation. They are currently
used in regenerative medicine for tissue regeneration [27]. Although
hBMMSCs can be easily expanded in vitro, they have limited lifespan
and differentiation potentials in culture. This impedes further clinical
application of these cells. However, hBMMSC-derived iPSCs
(hBMMSC-iPSCs) may have greater potential in clinical applications
with indefinite passages. They are alsocapable of forming three germ
layers and are more immunologically compatible with patients than
hBMMSCs are. They also have other advantages over other types of
cell-derived iPSCs with respect to disease treatment [28]. Regarding
their epigenetic state in hematopoiesis, we propose that hBMMSCs
would be an excellent source of cells for the efficient generation of
functional CD34+ progenitor cells.
In this study, based on the reprogramming of hBMMSCs to
iPSCs by ectopic expression of four transcription factors OCT4,
SOX2, c-MYC, and KLF4, we demonstrated that functional
CD34+ progenitor cells retaining hematopoietic and endothelial
cell potential could be obtained more efficiently from hBMMSC-
iPSCs than from human-skin-fibroblast-derived iPSCs (hFib-
iPSCs) by sequential exposure to two sets of defined chemicals
and growth factors, which exert specific function on mesodermal,
hematological and endothelial cell signaling pathways, including
BMP4, SCF, Flt3L, VEGF, IL-3, and EPO. This induction system
for CD34+ progenitor cells is not contaminated by unidentified
substances because it does not involve mouse feeder layers or other
poorly defined induction components, providing an opportunity to
generate an unlimited number of human-leukocyte-antigen
matched (HLA-matched) transplantable cells and constitutes an
ideal cell model for the study of the mechanisms of hematopoiesis
and related diseases and drug screening.
Results
Generation and characterization of hBMMSC-iPSCs
We cultured hBMMSCs to passage 2–4 before reprogramming
them into iPSC. cDNAs encoding four transcription factors were
retrovirally transduced to hBMMSCs for iPSC generation. ESC-
like colonies were examined with standard characterization
procedures. AP staining showed that some ESC-like colonies
(Fig. 1A) exhibited comparatively strong AP activity (Fig. 1B),
whereas hBMMSCs had weak AP activity. No ESC-like colonies
were observed in hBMMSC cultures. Immunofluorescence assay
revealed that the colonies expressed pluripotent markers such as
OCT4 (Fig. 1C), NANOG (Fig. 1D), SSEA3 (Fig. 1E), and TRA-1-
81 (Fig. 1F). RT-PCR analysis verified the expression of pluripotent
genes such as Nanog, Oct4, Sox2, and Rex1 (Fig. 1G). The
differentiation potential of the colonies was determined with
spontaneous differentiation in vitro (Figs. 1H-I) and teratoma
formation in vivo (Figs. 1J–L). Taken together, the colonies had
features typical of ESC colonies in morphology, expression of
specific markers of pluripotency, and differentiation potential,
indicating that iPSCs had been generated from hBMMSCs.
iPSCs derived from human skin fibroblasts (hFib-iPSCs) were used
for parallel analysis. Because of the highly similar results, data for the
generation and characterization of hFib-iPSCs are not shown here.
Efficient commitment of hBMMSC-iPSCs into CD34+
progenitor cells by stepwise treatment with defined
factors
In this study, we followed a defined culture condition protocol
for CD34+ progenitor cell differentiation from hBMMSC-iPSCs.
We did so to evaluate the potential of iPSCs for therapeutic
applications. The culture scheme is given in Figure 2A. Factors
representing essential lineage-inducing factors for mesodermal,
hematopoietic, and endothelial cells, such as BMP4, PD98059,
Flt3L, SCF, and VEGF, were selected and divided into groups by
function, as shown in Figure 2B. In the initial induction step, the
culture for hBMMSC-iPSCs were depleted of the feeder layer and
bFGF, with the presence of BMP4, PD98059, Flt3L, SCF, and
VEGF for 5 days to induce cell aggregate formation. The results of
RT-PCR and immunofluorescence assays showed that Brachyury
and GATA-2 were up-regulated at this time. Higher expression
levels were observed in the groups treated with the inducer cocktail
than in the spontaneous differentiation groups (Figs. 2C-D). This
indicates that the factors employed here increased the potential of
hBMMSC-iPSC commitment to mesoderm cells. Immunofluores-
cence assays confirmed that the transcription factor GATA-2 was
up-regulated and that efficiency was more pronounced in
hBMMSC-iPSC groups treated with the cocktail than in the
spontaneous differentiation groups. However, the expression of the
pluripotent marker OCT4 was similar in both groups (Fig. 2E).
After treatment for another 7–9 days with the cocktail containing
SCF, Flt3L, VEGF, bFGF, IL-3, and IL-6, the mixed population
displayedaseriesofchanges.CD34+progenitorcells,undetectablein
undifferentiated hBMMSC-iPSCs, increased in number, as assessed
by flow cytometry analysis. The proportion reached 19.5864.37%
(mean 6 SD, n=6, Fig. 3A), higher than that observed in parallel
hFib-iPSCs (13.2063.14%, mean 6 SD, n=6, Fig. 3B), about 10
fold that observed in spontaneously differentiated hBMMSC-iPSCs
(2.1061.47%, mean 6 SD, n=4, Fig. 3C), and about 20 fold that of
spontaneously differentiated hFib-iPSCs (1.3062.56%, mean 6 SD,
n=4, Fig. 3D) (P,0.05). CD34+ progenitor cells were then isolated
and enriched from the induced population by magnetic procedure
and the purity reached more than 90% (Figs. 3E-F). The different
replicates were derived from independent reprogramming cells.
Differentiation of hBMMSC-iPSCs was confirmed by the down
expression of pluripotency markers, Oct4 and Sox2 by flow
cytometry, as shown in Figures 3G-J.
Additionally, we separated the chemically induced cells into
CD34+ and CD34- subsets and demonstrated that the former only
had detectable CD34 transcripts (Fig. 3K).
Efficient Induction CD34+ Progenitors from iPSCs
PLoS ONE | www.plosone.org2 April 2012 | Volume 7 | Issue 4 | e34321

Page 3

Figure 1. Generation and characterization of iPSCs from hBMMSCs. Representative hBMMSC-iPSCs colonies (A) and AP staining (B). (C-F)
Immunoassay of hBMMSC-iPSC colonies expressing ESC specific markers as OCT4 (C), NANOG (D), TRA-1-81 (E) and SSEA-3 (F). 4, 6-Diamidino-2-
phenylindole (DAPI) staining was used to reveal the nuclei. Scale bar, 100 mm. (G) RT-PCR analysis of endogenous pluripotent gene mRNA expression
in hBMMSC-iPSCs. Total RNA was isolated from hBMMSC-iPSCs, ESCs and hBMMSCs. (H-L) Pluripotency assay of hBMMSC-iPSCs. EBs was formed (H)
and spontaneously differentiated into many types of cells during the in vitro potential assay (I). (J-L) Hematoxylin and Eosin staining of teratoma
sections from hBMMSC-iPSCs. 16107of hBMMSC-iPSCs were injected subcutaneously into limbs of NOD/SCID mice. Teratomas were obtained after
8–10 weeks. Three germ layer as ectoderm, neural-like tissue (J); mesoderm, adipose tissue (K) and endoderm, intestinal-like epithelium (L) were
observed.
doi:10.1371/journal.pone.0034321.g001
Figure 2. Exploration the potential of hBMMSC-iPSCs into CD34+ + progenitor cells with the defined factors. (A) Schematic of CD34+
progenitor cell differentiation protocol for the study. (B) Protocol for the chosen defined factors, divided into groups according to their function, to
direct hBMMSC-iPSCs to CD34+ progenitor cells, and then to hematopoietic and endothelial cells. (C-D) Enhanced expression of mesoderm
transcription factor Brachyury and GATA-2 was detected in factor-treated hBMMSC-iPSCs, compared to the spontaneous differentiation groups by RT-
PCR (C) and fluorescence intensity (D) assay after the differentiation for 5 days. The values were the mean 6 SD of 3 independent experiments. (E)
Immunoassay revealed that the hematopoietic transcription factor GATA-2 was up-regulated after factor treatment for 5 days and it was more
efficient in hBMMSC-iPSC groups treated with the factors than in the spontaneous groups (control). The expression of the plutipotent marker Oct4
was similar between two groups. Scale bar, 100 mm.
doi:10.1371/journal.pone.0034321.g002
Efficient Induction CD34+ Progenitors from iPSCs
PLoS ONE | www.plosone.org3April 2012 | Volume 7 | Issue 4 | e34321

Page 4

Dynamic analysis during hematopoietic cell
differentiation of hBMMSC-iPSCs
Flow cytometry and RT-PCR assays were used to detect the
dynamic course of hematopoietic differentiation of hBMMSC-
iPSCs via treatment with the cocktails. The results of flow
cytometry analysis were shown in figure 4A-D. Undifferentiated
hBMMSC-iPSCs expressed no CD34 or CD45 (Fig. 4A), and a
very low percentage of CD34+ cells, but no CD45+ cells were
detected after induction for 5 days (Fig. 4B). After treatment with
the following cocktail for another 7–9 days, the number of CD34+
increased significantly relative to the total number of cells.
However, few CD45+ cells were obtained at this stage (Fig. 4C).
As the hematopoietic induction continued, samples obtained from
the CFU assay developed a subpopulation of CD34+CD45+ cells,
and the percentage increased until about 24 days of culture
Figure 3. Efficient commitment of hBMMSC-iPSCs into CD34+ + progenitor cells by treatment with the defined factors. CD34+
progenitor cells were identified by flow cytometry analysis in the groups. Differentiated hBMMSC-iPSCs treated with the factors produced about 20%
proportion of CD34+ progenitor cells (A), higher than that of nearly 13% from hFib-iPSCs by parallel performance (B). However, the efficiency for the
spontaneous differentiation groups was low. The proportion in hBMMSC-iPSCs treated with no factor was nearly 2% (C) and it was much lower in
hFib-iPSCs of nearly 1% (D). The data demonstrated that the defined factors employed here efficiently improved the efficient differentiation of CD34+
progenitor cells from iPSCs, and iPSCs derived from hBMMSCs was more efficient to produce CD34+ progenitor cells than those derived from human
fibroblasts. The enriched CD34+ progenitor cells were analyzed from hBMMSC-iPSC versus hFib-iPSC differentiation systems by flow cytometry (E-F).
(G-J) Flow cytometry analysis demonstrated that the expression of Oct4 (G and I) and Sox2 (H and J) was down regulated at day 14 after the
differentiation. (K) The chemically induced cells were separated into CD34+ and CD34- subsets and the former only had detectable CD34 transcripts.
doi:10.1371/journal.pone.0034321.g003
Efficient Induction CD34+ Progenitors from iPSCs
PLoS ONE | www.plosone.org4April 2012 | Volume 7 | Issue 4 | e34321

Page 5

(Fig. 4D). This marked increase in CD45+ cells reflects the
development of more mature hematopoietic cells. The result of the
hematologic differentiation of hBMMSC-iPSCs shown here
suggests that the hematopoiesis was a dynamic process. RT-
PCR and Image J analysis demonstrated that the pluripotent
marker Oct4 continued to be down-regulated and the hemato-
poietic special genes TAL-1 and SCL were sustainably up-
regulated during the process (Figs. 4E-F). At day 14 after the
differentiation, the expression of TAL-1 and SCL reached a
considerable level while the expression of Oct4 was not detected
(Figs. 4E-F). The kinetics of CD34+ cells from hBMMSC-iPSCs
versus hFib-iPSCs treated with the cocktails was also detected as
shown in figure 4G, indicating that the efficiency of hematopoietic
differentiation from hBMMSC-iPSCs was more efficient than that
from hFib-iPSCs.
Differentiation of hBMMSC-iPSC-derived CD34+
progenitor cells into hematopoietic cell lineages
CD34+ progenitor cells were isolated to determine their CFU
potential in methylcellulose and in hematopoietic cytokines
including SCF, VEGF, TPO, EPO, IL-3, IL-6, and GM-CSF.
CFUs were enumerated 14 days later and the number of colonies
in the hBMMSC-iPSC groups is higher than that in the hFib-iPSC
groups (Fig. 5A).
Various CFUs were identified in the culture after 10–14 days.
These CFUs included burst-forming units of erythroid lineages
(BFU-E) (Fig. 5B), small colonies of erythroid cells (CFU-E)
(Fig. 5C), CFU-granulocytes and macrophages (CFU-GM)
(Fig. 5D), CFU-granulocytes, erythroid cells, macrophages, and
megakaryocytes (CFU-GEMM) (Fig. 5E), and CFU-granulocytes
(CFU-G) (Fig. 5F). Hematopoietic cell lineages including erythroid
cells, granulocytes, megakaryocytes, and macrophages were
characterized using Wright-Giemsa staining (Figs. 5G–I). Addi-
tionally, representative colony morphologies including BFU-E
(Fig. 6A), CFU-E (Fig. 6B), CFU-GM (Fig. 6C), CFU-GEMM
(Fig. 6D), CFU-G (Fig. 6E), and CFU-M (Fig. 6F) from hFib-
iPSC-derived CD34+ cells were shown as control.
Differentiation of hBMMSC-iPSC-derived CD34+
progenitor cells into endothelial cells
In this study, hBMMSC-iPSC-derived CD34+ progenitor cells
were differentiated into endothelial cells in endothelial cell growth
medium-2 (EGM-2). Immunoassay results showed that some
hBMMSC-iPSC-derived CD34+ progenitor cells could differenti-
ate into endothelial cells, as determined by the expression of
endothelial cell-specific proteins, including CD31 and VE-
CADHERIN (Figs. 5J and 5K). These cells formed vascular-like
structures on Matrigel when the cultures were supplemented with
VEGF-A (Fig. 5L).
Figure 4. Kinetic expression of the hematopoietic and pluripotent genes during hBMMSC-iPSC commitment to CD34+ + progenitor
cells, and then to hematopoietic cells. (A-D) Kinetic expression of CD34+ and CD45+ during the hematopoietic cell differentiation of hBMMSC-
iPSCs with flow cytometry analysis. Undifferentiated hBMMSC-iPSCs expressed no CD34 or CD45 (A). After treatment with the cocktail containing
mesodermal, hematopoietic, and endothelial inducers for 5 days, hBMMSC-iPSCs expressed about 5% percentage of CD34+ but few CD45+ cells (B).
After culturing with the following hematopoietic and endothelial inducer cocktail for additional 7–9 days, the proportion of CD34+ population
increased to nearly 20% and a few CD45+ cells were obtained at this stage (C). In hematopoietic potential assay of CD34+ progenitor cells, about 5%
population of CD34+CD45+ cells were developed from the CFU assay, and the percentage of CD45+ cells increased to about 25% during the
differentiation culture (D). (E-F) Dynamically relative expression of the pluripotent marker Oct4 and the hematopoietic cell markers TAL-1, SCL during
hBMMSC-iPSC commitment to CD34+ progenitor cells, and then to hematopoietic cells by RT-PCR (E) and fluorescence intensity (F) assay in the
differentiation culture. The values were the mean 6 SD of 3 independent experiments. (G) Kinetics of CD34+ cells during hematopoietic
differentiation of hBMMSC-iPSCs versus hFib-iPSCs treated with the inducers.
doi:10.1371/journal.pone.0034321.g004
Efficient Induction CD34+ Progenitors from iPSCs
PLoS ONE | www.plosone.org5 April 2012 | Volume 7 | Issue 4 | e34321