Reprogramming of T Cells from Human Peripheral Blood

Article (PDF Available)inCell stem cell 7(1):15-9 · July 2010with36 Reads
DOI: 10.1016/j.stem.2010.06.004 · Source: PubMed
Cell Stem Cell
Brief Report
Reprogramming of T Cells
from Human Peripheral Blood
Yuin-Han Loh,
1,2
Odelya Hartung,
1,2
Hu Li,
3,4
Chunguang Guo,
5,6,7
Julie M. Sahalie,
1,2
Philip D. Manos,
1,2
Achia Urbach,
1,2
Garrett C. Heffner,
1,2
Marica Grskovic,
8
Francois Vigneault,
7
M. William Lensch,
1,2,5
In-Hyun Park,
1,2
Suneet Agarwal,
1,2
George M. Church,
7
James J. Collins,
3,4,5
Stefan Irion,
8,
*
and George Q. Daley
1,2,5,9,10,
*
1
Stem Cell Transplantation Program, Division of Pediatric Hematology Oncology, Children’s Hospital Boston and Dana-Farber Cancer
Institute; Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA
2
Harvard Stem Cell Institute, Cambridge, MA 02138, USA
3
Department of Biomedical Engineering and Center for BioDynamics, Boston University, Boston, MA 02215, USA
4
Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA 02115, USA
5
Howard Hughes Medical Institute, Boston, MA 02115, USA
6
Immune Diseases Institute, Children’s Hospital Boston
7
Department of Genetics
Harvard Medical School, Boston, MA 02115, USA
8
iPierian, Inc. South San Francisco, CA 94080, USA
9
Division of Hematology, Brigham and Women’s Hospital, Boston, MA 02115, USA
10
Manton Center for Orphan Disease Research, Boston, MA 02115, USA
*Correspondence: stefan.irion@ipierian.com (S.I.), george.daley@childrens.harvard.edu (G.Q.D.)
DOI 10.1016/j.stem.2010.06.004
Human induced pluripotent stem cells
(iPSCs) derived from somatic cells of
patients hold great promise for modeling
human diseases. Dermal fibroblasts are
frequently used for reprogramming, but
require an invasive skin biopsy and a pro-
longed period of expansion in cell culture
prior to use. Here, we report the derivation
of iPSCs from multiple human blood sour-
ces including peripheral blood mononu-
clear cells (PBMCs) harvested by routine
venipuncture. Peripheral blood-derived
human iPSC lines are comparable to
human embryonic stem cells (ESCs) with
respect to morphology, expression of
surface antigens, activation of endoge-
nous pluripotency genes, DNA methyla-
tion, and differentiation potential. Analysis
of immunoglobulin and T cell receptor
gene rearrangement revealed that some
of the PBMC iPSCs were derived from
T cells, documenting derivation of iPSCs
from terminally differentiated cell types.
Importantly, peripheral blood cells can
be isolated with minimal risk to the donor
and can be obtained in sufficient numbers
to enable reprogramming without the
need for prolonged expansion in culture.
Reprogramming from blood cells thus
represents a fast, safe, and efficient way
of generating patient-specific iPSCs.
Somatic cells can be induced to the
pluripotent state by the enforced expres-
sion of several transcription factors in-
cluding OCT4, SOX2, KLF4, MYC,
NANOG, and LIN28 (Takahashi et al.,
2007; Yu et al., 2007; Park et al., 2008a).
Human iPSCs are commonly generated
from dermal fibroblasts harvested by sur-
gical skin biopsy (Park et al., 2008b).
Exposure of the dermis to ultraviolet light
increases the risk for chromosomal aber-
rations (Ikehata et al., 2003), raising
concerns for whether iPSCs will reflect
the patient’s constitutional genotype. For
routine clinical application, it would be
desirable to reprogram cell types that
are safe and can be collected noninva-
sively in large numbers.
Blood is a cell source that can be easily
obtained from patients. Mouse B and
T cells are amenable to reprogramming
by overexpressing Oct4, Sox2, Klf4, and
Myc with the ectopic expression of Cepba
and p53 knockdown, respectively (Hanna
et al., 2008; Hong et al., 2009). iPSC lines
have also been generated from mouse
bone marrow progenitor cells (Okabe
et al., 2009). We have previously reprog-
rammed cytokine-mobilized human
CD34
+
peripheral blood cells to pluripo-
tency, but such harvests are cumber-
some, expensive, and time consuming
(Loh et al., 2009). Several recent studies
reported the generation of iPSCs from
human bone marrow and cord blood (Ye
et al., 2009; Giorgetti et al., 2009; Haase
et al., 2009), but bone marrow harvesting
is an invasive procedure, and cord blood
is available for only a minority of individ-
uals who have their samples banked at
birth. A recent study with peripheral blood
from donors with myeloproliferative dis-
order (MPD) isolated iPSC colonies that
contain the JAK2-V617F mutation (Ye
et al., 2009), but MPD is characterized
by abnormally high numbers of circulating
CD34
+
cells from the bone marrow. These
previous studies demonstrating success-
ful reprogramming of blood cells into
iPSCs have relied on specialized blood
cell sources with high proliferative poten-
tial.
CD34
+
hematopoietic stem/progenitor
cells mobilized into the donor’s peripheral
blood by pretreatment with granulocyte
colony-stimulating factor (G-CSF) can be
successfully reprogrammed to pluripo-
tency (Loh et al., 2009). To test whether
we can reprogram cells from routine
peripheral blood (PB) sources, we ob-
tained CD34
+
purified blood samples
from a healthy 49-year-old male donor
who had undergone simple apheresis
without cytokine priming. We also iso-
lated mononuclear cells (PBMCs) from
the peripheral blood samples collected
by venipuncture of four healthy donors
(28- to 49-years-old) via Ficoll density
centrifugation.
To induce reprogramming of enriched
CD34
+
blood cells, we infected with lenti-
viruses expressing OCT4, SOX2, KLF4,
and MYC reprogramming factors (Fig-
ure 1A). Colonies with well-defined hESC-
like morphology were first observed 21
days after transduction (Figure 1B). For
reprogramming of fresh peripheral blood
mononuclear cells (PBMCs), we em-
ployed two rounds of lentiviral infection
(day 0 and day 8) and isolated colonies
Cell Stem Cell 7, July 2, 2010 ª2010 Elsevier Inc. 15
Figure 1. Reprogramming of Peripheral Blood Cells to Pluripotent iPSCs
(A) Scheme for reprogramming human peripheral blood (PB) mononuclear cells (PBMCs) and CD34
+
cells (PB CD34
+
). Morphology of the typical peripheral blood
cells and images of hESC-like iPSC colonies are shown.
(B) Images of PB34 iPSC colonies. Bright-field images were acquired with a standard microscope (Nikon, Japan) with a 103 objective. Immunohistochemistry of
PB-derived iPSC colonies expressing markers for OCT4, NANOG, Tra-1-60, and alkaline phosphatase (AP). Hoechst staining indicates the total cell content per
field. Fibroblasts surrounding human iPSC colonies serve as internal negative controls for immunohistochemistry staining. Images were acquired with a standard
microscope (Nikon, Japan) with a 103 objective.
(C) Images of PBMC (Donor GH) iPSC colonies. Bright-field images were acquired with a standard microscope (Nikon, Japan) with a 103 objective.
Immunohistochemistry of PB-derived iPSC colonies expressing markers for OCT4, NANOG, Tra-1-60, and alkaline phosphatase (AP). Hoechst staining indicates
the total cell content per field.
(D) Images of PBMC (donors 34, 50, 76) iPSC colonies. Bright-field images were acquired with a standard microscope (Nikon, Japan) with a 103 objective.
Immunohistochemistry of PB-derived iPSC colonies expressing marker for NANOG. DAPI staining indicates the total cell content per field.
(E) Quantitative reverse transcription-PCR analyses for the expression of ESC-marker genes NANOG, hTERT, GDF3, and REX1 in PB CD34
+
and PBMC-derived
iPSCs and human H1 ESCs (with their respective standard errors). Individual PCR reactions were normalized against b-ACTIN and plotted (Log
10
scale) relative to
the expression level in the H1 ESCs, which was set to 1.
(F) Scatter plots comparing PB34 iPSCs and PBMC iPSCs global gene expression profiles to parental (left) and H1 human ESCs (right). The black lines indicate the
linear equivalent and 2-fold changes in gene expression levels between the paired cell types. Positions of pluripotency genes OCT4, SOX2, NANOG, and LIN28 in
scatter plots are indicated.
(G) Bisulfite genomic sequencing of the NANOG promoters reveals demethylation in the iPSC lines. Each horizontal row of circles represents an individual
sequencing reaction for a given amplicon. Open and filled circles represent unmethylated and methylated CpGs dinucleotides, respectively. Percentage of
methylation is indicated for each cell line.
For further characterization of the peripheral blood-derived iPSC clones, see also Figure S1 and Table S1.
Cell Stem Cell
Brief Report
16 Cell Stem Cell 7, July 2, 2010 ª2010 Elsevier Inc.
with distinct flat and compact mor-
phology with clear-cut round edges remi-
niscent of hESCs after a slightly longer
latency of around 35 days (Figure 1C).
Interestingly, a previous study with a
single round of lentiviral infection of
PBMCs failed to observe iPSC colony
formation (Haase et al., 2009). In a sepa-
rate set of experiments, we tested the
ability of retroviruses encoding the human
reprogramming factors to generate iPSCs
from human PBMCs, and despite low
infection efficiency, we observed iPSC
colonies after 25–35 days (Figure 1D).
With immunohistochemistry and flow
cytometry, we analyzed the iPSC lines
for expression of markers shared with
hESCs. Consistent with their hESC-like
morphology, both PB34 iPSCs and
PBMC iPSCs stained positive for Tra-
1-81, NANOG, OCT4, Tra-1-60, SSEA4,
and alkaline phosphatase (AP) staining
(Figures 1B–1D; Figures S1A–S1C avail-
able online; Chan et al., 2009). We
routinely observed a reprogramming effi-
ciency of 0.002% for PB CD34
+
cells
(Table S1), comparable to prior experi-
ence with primary fibroblasts, mobilized
PBMCs, and cord blood cell reprogram-
ming (Takahashi et al., 2007, Park et al.,
2008a, Loh et al., 2009; Haase et al.,
2009). For PBMCs, we obtained hESC-
like colonies at the lower efficiency of
0.0008%–0.001% (Table S1).
We further characterized the PB34
iPSC and PBMC iPSC lines for properties
specific to hESCs. Efficient transgene
silencing is essential for the derivation of
pluripotent iPSC lines (Brambrink et al.,
2008). qRT-PCR via primers specific for
endogenous and total transcripts of the
reprogramming factors confirmed that
OCT4, SOX2, KLF4, and MYC transgenes
were efficiently silenced in the blood-
derived iPSCs (Figure S1D). Additional
analysis via quantitative PCR revealed
the activation of pluripotency markers
NANOG, hTERT, REX1, and GDF3 to a
level similar to the expression in H1
hESCs (Figure 1E).
We next performed global gene expres-
sion analysis of the peripheral blood-
derived iPSCs comparing it to hESCs,
fibroblast iPSCs, and somatic parental
cells. Clustering analysis revealed a high
degree of similarity among the reprog-
rammed iPSCs (dH1F-iPS, PBMC iPS1,
PB34 iPS1, PB34 iPS2), which clustered
together with the H1 and H9 ESCs and
were distant from the parental somatic
cells, as determined by a Euclidean
distance metric (Figure S1E). Analysis of
scatter plots similarly shows a tighter
correlation among reprogrammed iPSCs
(PB34 iPSCs, PBMC iPSCs) and human
ESCs (H1 ESCs) than between differenti-
ated parental cells and their reprog-
rammed derivatives (Figure 1F). Consis-
tent with the activation of endogenous
pluripotency-associated gene expres-
sion, reprogramming of the blood cells
was accompanied by the demethylation
of CpG dinucleotides at the NANOG
promoters (Figure 1G). Moreover, cytoge-
netic analysis showed normal karyotypes
for the iPSC lines (Figure S1F).
Next, we evaluated the developmental
potential of the iPSC lines by in vitro
embryoid body differentiation, hemato-
poietic colony forming assays, and
in vivo teratoma induction. The iPSCs
readily formed embryoid bodies upon
induction (Figure S2A). qRT-PCR of the
differentiated cells showed strong sup-
pression of the pluripotency genes and
activation of lineage-specific genes rep-
resenting the three germ layers (Figures
S2B and S2C). Hematopoietic differentia-
tion of iPSC lines resulted in erythroid,
myeloid, and granulocytic colony forma-
tion (Figures 2A and 2B). Interestingly, all
PB CD34
+
-derived iPS lines we tested
show greater hematopoietic colony form-
ing activity than PBMC iPSCs (Figure 2A).
The most rigorous test for pluripotency
of human ESCs is the formation of tera-
tomas in immunodeficient mouse hosts
(Lensch et al., 2007). Upon subcutaneous
injection into immunodeficient Rag2
/
gc
/
mice, the iPSC lines generated
well-differentiated cystic teratomas rep-
resenting all three embryonic germ layers
(Figures 2C and 2D). DNA fingerprinting
analysis verified that these cells were
indeed derived from the parental blood
cells and not a result of contamination
from existing hESC or iPSC lines (Table
S2). The iPSC clones have been propa-
gated for at least 20 passages as of this
submission.
Because peripheral blood mononuclear
cells consist of both myeloid and lym-
phoid elements (Figure S2D), we were
interested in determining the lineage of
origin of the reprogrammed cells. We
tested the iPSC clones for the presence
of functionally rearranged immunoglob-
ulin and T cell receptor genes by using
probes specific for IgH, TCR-d, and
TCR-b2. Among 12 independent clones
from 3 separate individuals, we failed to
detect IgH recombination, indicating that
none of our lines arose from B lympho-
cytes (Figure S2E). As reported for the
mouse, reprogramming human B lympho-
cytes may require additional factors like
CEBPa (Hanna et al., 2008). Next, we
analyzed the iPSC lines for TCR-d and
TCR-b2 recombination (Figures 2E and
2F; Figure S2F). No PBMC iPSC lines
demonstrated TCR-b2 recombination,
whereas six of seven PBMC iPSC lines
isolated from a single donor sample
exhibited rearrangement of the TCR-d
locus, indicative of derivation from cells
of the T lineage (Figure 2F). In contrast,
PBMC iPSC lines from donors 34 and 76
lacked rearrangement of IgH, TCR-d,
and TCR-b2, indicating derivation from
nonlymphoid lineages (Figure 2F; Figures
S2E and S2F).
Isolation of iPSCs from T lymphocytes
represents definitive proof that even
terminally differentiated human cells are
susceptible to reprogramming to pluripo-
tency. Distinct protocols of cytokine stim-
ulation and viral infection of the PBMC
cells may predispose to derivation from
lymphoid versus nonlymphoid hemato-
poietic cells from peripheral blood sour-
ces, as can preselection of lymphoid
target cells prior to reprogramming
(Hong et al., 2009). PBMCs from donor
GH were grown in medium containing
IL-3, which is known to stimulate the
growth of subsets of CD4
+
T cells
(Figure S2D; Mueller et al., 1994). In
contrast, PBMCs from donors 34 and
76 were cultured in medium promoting
expansion of dendritic cells and yielded
iPSCs with germline IgH and TCR alleles.
For applications in regenerative medicine,
iPSCs containing antibody or T cell
receptor gene rearrangement may be
undesirable (Serwold et al., 2007).
In conclusion, we have successfully
reprogrammed cells from peripheral
blood sources including samples ob-
tained through routine venipuncture. Our
study provides a strategy for the reliable
generation of induced pluripotent stem
cells from peripheral blood mononuclear
cells. Although the per-cell derivation
efficiency is low, peripheral blood is an
accessible source of a large number
of primary cells (easily 10
5
–10
6
), thus
enabling reliable iPSC isolation from only
Cell Stem Cell 7, July 2, 2010 ª2010 Elsevier Inc. 17
Cell Stem Cell
Brief Report
a few milliliters of whole blood. Future
application of viral and transgene-free
reprogramming or protein transduction
(Kaji et al., 2009; Woltjen et al., 2009; Yu
et al., 2009; Kim et al., 2009; Zhou et al.,
2009) to peripheral blood reprogramming
will greatly facilitate the development of
efficient and safe ways of generating
patient-specific pluripotent stem cells.
Figure 2. Pluripotency and V(D)J Rearrangement of Peripheral Blood-Derived iPSCs
(A) Embryoid bodies derived from PB34 and PBMC iPSCs yield hematopoetic colonies in semisolid methylcellulose media: burst forming unit-erythroid (BFU-E),
colony forming unit-granulocyte (CFU-G), colony forming unit-macrophage (CFU-M), colony forming unit-granulocyte, macrophage (CFU-GM), and colony
forming unit-granulocyte, erythroid, macrophage (CFU-GEMM). Total number of each type of colony was counted.
(B) Representative images of various types of hematopoietic colonies. Images were acquired with a standard microscope (Nikon, Japan) with a 203 objective.
(C and D) Hematoxylin and eosin staining of teratomas derived from immunodeficient mice injected with PB34 iPSCs (C) and PBMC iPSCs (D) show tissues
representing all three embryonic germ layers.
(E) Genomic DNA from peripheral blood-derived iPSC lines grown was digested with NcoI and analyzed for V(D)J rearrangements at the TCR-d (T cell receptor
Delta) locus by Southern blotting with a 3
0
Jd3 probe.
(F) TCR-d V(D)J recombination of blood-derived iPSC clones. Lanes 2–8 and lanes 13–17 are PBMC iPSC lines. B cell lines on lanes 8 and 9 showed no rearrange-
ment. TCR-d rearrangement was observed for some PBMC-derived iPSC lines (lanes 2–6 and 8). Lanes 1, 11, 12, 18, and 19 are H1 hESCs, PB34 iPSCs,
fibroblast cells, fibroblast-derived iPSCs via retrovirus and lentivirus, respectively. The red arrow indicates expected size of the germline band. Orange arrow
indicates rearranged bands.
For further information on the pluripotency, V(D)J rearrangement, and fingerprint analysis performed on the peripheral blood-derived iPSC clones, see also
Figure S2 and Table S2.
18 Cell Stem Cell 7, July 2, 2010 ª2010 Elsevier Inc.
Cell Stem Cell
Brief Report
ACCESSION NUMBERS
The array data have been deposited in the GEO
database under the accession number GSE22167.
SUPPLEMENTAL INFORMATION
Supplemental Information includes Supplemental
Experimental Procedures, two figures, and two
tables and can be found with this article online at
doi:10.1016/j.stem.2010.06.004.
ACKNOWLEDGMENTS
This research was funded by grants from the
National Institutes of Health (NIH) and the Howard
Hughes Medical Institute to G.Q.D. J.J.C. is sup-
ported by SysCODE (Systems-based Consortium
for Organ Design & Engineering), NIH grant
# RL1DE019021. Y.-H.L. is supported by the over-
seas fellowship from Agency of Science, Tech-
nology, and Research (A
*
Star) and the Institute of
Medical Biology, Singapore. We are grateful to
Ann M. Mullally, Anupama Narla, Benjamin L.
Ebert, Lars U.W. Mu
¨
ller, Axel Schambach, and Ste-
lios Andreadis for technical assistances. We
acknowledge Sabine Loewer for helpful discus-
sions and critical comments on the manuscript.
G.Q.D. is a member of the Scientific Advisory
Board of iPierian, Inc. M.G. and S.I. are employed
by iPierian, Inc., a biotechnology company using
iPSCs for drug discovery.
Received: May 11, 2010
Revised: May 31, 2010
Accepted: June 5, 2010
Published: July 1, 2010
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Note Added in Proof
A manuscript has appeared online demonstrating
isolation of iPSCs from peripheral blood, including
a single line that showed evidence for both TCR-b
and TCR-g rearrangement by PCR (Kunisato, A.,
Wakatsuki, M., Shinba, H., Ota, T., Ishida, I., and
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Cell Stem Cell
Brief Report
    • "Blood can be an ideal cell source since extraction is minimally invasive compared to skin fibroblasts [3]. However, the reprogramming of floating blood cells is relatively difficult [4] . Nucleated cells in peripheral blood consist of granulocytes including neutrophils, monocytes, T lymphocytes, B lymphocytes, and progenitor cells. "
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    • "Their use also enables the creation of iPSCs from large number of donor samples stored in biorepositories worldwide. Efforts to develop such methods have yielded iPSCs from human CD34+ blood cells and T-lymphocytes [22][23][24][25]. It was revealed in a particularly key study that hiPSCs can be efficiently derived from T lymphocytes from as little as 1 mL of whole blood [26]. "
    [Show abstract] [Hide abstract] ABSTRACT: The year 2006 will be remembered monumentally in science, particularly in the stem cell biology field, for the first instance of generation of induced pluripotent stem cells (iPSCs) from mouse embryonic/adult fibroblasts being reported by Takahashi and Yamanaka. A year later, human iPSCs (hiPSCs) were generated from adult human skin fibroblasts by using quartet of genes, Oct4, Sox2, Klf4, and c-Myc. This revolutionary technology won Yamanaka Nobel Prize in Physiology and Medicine in 2012. Like human embryonic stem cells (hESCs), iPSCs are pluripotent and have the capability for self-renewal. Moreover, complications of immune rejection for therapeutic applications would be greatly eliminated by generating iPSCs from individual patients. This has enabled their use for drug screening/discovery and disease modelling in vitro; and for immunotherapy and regenerative cellular therapies in vivo, paving paths for new therapeutics. Although this breakthrough technology has a huge potential, generation of these unusual cells is still slow, ineffectual, fraught with pitfalls, and unsafe for human use. In this review, I describe how iPSCs are being triumphantly used to lay foundation for a fully functional discipline of regenerative dentistry and medicine, alongside discussing the challenges of translating therapies into clinics. I also discuss their future implications in regenerative dentistry field.
    Full-text · Article · Jun 2016
    • "The original protocol using retroviral vectors expressing Yamanaka factors (OCT4, SOX2, KLF4, and MYC) has been found to be successful in many cell types, including hematopoietic cells (Aasen et al., 2008; Broxmeyer et al., 2011; Loh et al., 2009; Mali et al., 2008 Mali et al., , 2010 Park et al., 2008). Reprogramming of T cells, a major subpopulation of MNCs, into pluripotency has been achieved by many laboratories using different approaches (Loh et al., 2010; Okita et al., 2013; Staerk et al., 2010 ) and T cell reprogramming has the potential to rejuvenate aged T cells for immunotherapy (Nishimura et al., 2013; Wakao et al., 2013). However, induced pluripotent stem cells (iPSCs) from non-lymphoid cells may be more useful, since mature T cells harbor a single T cell receptor (TCR) after somatic recombination and are unable to regenerate the T cell repertoire with unlimited possibilities. "
    [Show abstract] [Hide abstract] ABSTRACT: We previously reported the generation of integration-free induced pluripotent stem cells from adult peripheral blood (PB) with an improved episomal vector (EV) system, which uses the spleen focus-forming virus U3 promoter and an extra factor BCL-XL (B). Here we show an ∼100-fold increase in efficiency by optimizing the vector combination. The two most critical factors are: (1) equimolar expression of OCT4 (O) and SOX2 (S), by using a 2A linker; (2) a higher and gradual increase in the MYC (M) to KLF4 (K) ratio during the course of reprogramming, by using two individual vectors to express M and K instead of one. The combination of EV plasmids (OS + M + K + B) is comparable with Sendai virus in reprogramming efficiency but at a fraction of the cost. The generated iPSCs are indistinguishable from those from our previous approach in pluripotency and phenotype. This improvement lays the foundation for broad applications of episomal vectors in PB reprogramming.
    Full-text · Article · May 2016
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