Báo cáo khoa học: Hematopoietic differentiation from human ESCs as a model for developmental studies and future clinical translations Invited review following the FEBS Anniversary Prize received on 5 July 2009 at the 34th FEBS Congress in Prague docx

The study of human hematopoiesis using hESCs and induced pluripotent stem cells hiPSCs has been Keywords blood cells; engraftment; hematopoiesis; hematopoietic differentiation; hematopoi

Hematopoietic differentiation from human ESCs as a

model for developmental studies and future clinical

translations

Invited review following the FEBS Anniversary Prize received on

5 July 2009 at the 34th FEBS Congress in Prague

Inmaculada Moreno-Gimeno1, Maria H Ledran1,2and Majlinda Lako1,2

1 Centro de Investigacio´n Prı´ncipe Felipe, Valencia, Spain

2 Institute of Human Genetics, Newcastle University, International Centre for Life, UK

Why use human embryonic stem cells

or induced pluripotent stem cells for

hematopoiesis studies?

The first paper describing the derivation of human

embryonic stem cells (hESCs) was published in 1998 by

Thomson et al [1] Since then, many studies have been

performed using with hESCs in order to better

under-stand embryonic development and stem cell biology,

with the possibility of clinical application as well as their use as tools for pharmaceutical research and drug discovery being a major impetus for such investigations [2] The study of human hematopoiesis using hESCs and induced pluripotent stem cells (hiPSCs) has been

Keywords

blood cells; engraftment; hematopoiesis;

hematopoietic differentiation; hematopoietic

ontogeny; hematopoietic progenitors; hESC

and iPSC therapeutic applications; human

embryonic stem cells; induced pluripotent

stem cells; SCID repopulating cells

Correspondence

I Moreno-Gimeno, Centro de Investigacio´n

Prı´ncipe Felipe, Valencia 46012, Spain

Fax: 00 34 963289701

Tel: 00 34 963289680

E-mail: imoreno@cipf.es

(Received 22 June 2010, revised 10

September 2010, accepted 18 October 2010)

doi:10.1111/j.1742-4658.2010.07926.x

Human embryonic stem cells (hESCs) and induced pluripotent stem cells are excellent models for the study of embryonic hematopoiesis in vitro, aiding the design of new differentiation models that may be applicable to cell-replacement therapies Adult and fetal hematopoietic stem cells are cur-rently being used in biomedical applications; however, the latest advances

in regenerative medicine and stem cell biology suggest that hESC-derived hematopoietic stem cells are an outstanding tool for enhancing immuno-therapy and treatments for blood disorders and cancer, for example In this review, we compare various methods used for inducing in vitro hematopoi-etic differentiation from hESCs, based on co-culture with stromal cells or formation of embryoid bodies, and analyse their ability to give rise to hematopoietic precursors, with emphasis on their engraftment potential as

a measure of their functionality in vivo

Abbreviations

AGM, aorta–gonad–mesonephros; BFU-E, burst forming unit-erythrocyte; BMP4, Bone morphogenetic protein 4; CFU-E, colony forming unit-erythrocyte; FBS, fetal bovine serum; hESC, human embryonic stem cell; mESC, mouse embryonic stem cell; HSC, hematopoietic stem cells; hiPSC, human induced pluripotent stem cells; NK, natural killer; NOD ⁄ SCID, non-obese diabetic ⁄ severe combined immuno-deficient; VEGF, vascular endothelial growth factor.

one of the most successful fields to date It is known

that hESCs, characterized by their pluripotency and

theoretically unlimited proliferation ability, are capable

of producing all blood cell types when differentiated

under suitable conditions [3–8] Various studies have

shown that hESC differentiation to hematopoietic

lin-eages closely mimics embryonic hematopoiesis [9,10],

making them an incomparable tool for the study of

hematopoiesis during embryonic development in states

of health or disease [11–13] There is hope that the

insights gained by better understanding embryonic

hematopoiesis can meet clinical needs (in the fields of

AIDS, immunotherapy, blood disorders and cancer

treatment, for example) by helping to improve existing

therapies currently based on transfusions or allogeneic

hematopoietic stem cell transplantation [14]

HIV infections are known to affect the

hematopoi-etic system by specifically targeting white blood cells,

substantially weakening the immune system and

even-tually progressing to AIDs, and resulting in death,

usually as a result of secondary opportunistic infection

[15] Therefore, cell-replacement therapy based on

reconstitution of the leukocytic hematopoietic

com-partment using a CD34+hESC-derived starting

popu-lation has been considered as a potential AIDS

therapy, and as a way to alleviate secondary effects

produced by anti-retroviral drugs [16] Various studies

have now shown that functional B cells, natural killer

(NK) cells, dendritic cells and macrophages can be

derived from hESCs and used for immune therapy

[4,7,17–20] Furthermore, by combining cellular and

gene therapy, anti-HIV-1 genes can be transduced into

CD34+ hESC-derived macrophages and T cells,

ren-dering them insensitive to HIV infection, and therefore

giving hope that these types of cells could eventually

restore normal immune system function [21]

Addi-tionally, recent studies have highlighted the importance

of chemokine receptor 5 (CCR5) in HIV-1 infection;

silencing of CCR5 in HSCs (by RNA interference

technology) yields HIV-1-resistant hematopoietic

pro-genitors, potentially capable of restoring a healthy

hematopoietic compartment [22] Moreover, Hu¨tter

et al.[23] showed the viability of this type of treatment

by transplanting CD34+stem cells expressing an

inac-tive form of CCR5 receptor (homozygous to the

CCR5 D32 deletion) into an individual suffering from

AIDS and acute myeloid leukemia Following

trans-plantation and discontinuation of anti-retroviral

ther-apy, no HIV-1 virus was detected in the patient over a

20-month period [22,23] A combination of gene and

cell therapy has also been developed to generate hESC

derived dendritic cells with enhanced in vitro

antigen-presenting function [19] These hESC-derived dendritic

cells could potentially be used in clinical immune ther-apy in order to induce immune responses in an anti-gen-specific manner in patients suffering from cancer

or viral infections [20]

Cancer therapy is a principal goal of the current clinical research on hESCs Recently, regression of metastatic melanoma tumors was achieved by trans-plantation of adaptive T cells specific for tumor anti-gens; however, this technique performed poorly in human trials, as only 10% of the patients retained the genetically engineered cells 1 year after infusion [24,25] A new anti-cancer therapeutic strategy involves targeting of the innate immune system through the cytolytic activity of NK cells A recent study by Kauf-man et al showed that hESC-derived NK cells are competent effector cells that can kill human tumor cells in vivo The authors compared hESC-derived NK cells to those generated from umbilical cord blood, and found that hESC-derived cells form a more mature and homogeneous population with an increased ability

to kill tumor cells, and showed higher expression of effector molecules as well as cytolytic competence, thus representing a great advance in the use of hematopoi-etic stem cells in anti-tumor therapy [26]

The latest studies in stem cell biology have shown how somatic cells can be reprogrammed back to a pluripotent state similar to that in hESCs [27,28] These hiPSCs have opened new avenues for creating

in vitro disease models that can be used to help under-stand the pathology of many genetic diseases and to design new drugs They are also considered a potential source of allogenic cells for cell replacement therapy,

as hiPSCs could be derived specifically for each patient The clinical capacity of hiPSCs has been recently described in mouse models In 2007, Hanna

et al reported the first proof that combined gene and cell therapy could be used in the treatment of blood disorders by transplanting modified cells into a humanized mouse model of sickle cell anemia carrying the human mutant variant of the b-globin gene (bS) that is responsible for the disease Fibroblasts from a diseased animal were reprogrammed into iPSCs, and the sickle cell anemia was corrected by homologous recombination These cells differentiated into hemato-poietic progenitors, and, following irradiation of the donor mouse resulting in the destruction of the hema-topoietic compartment, were transplanted successfully back into the mouse, resulting in recovery and correc-tion of the disease phenotype [29] Using similar meth-odology, another study has shown phenotypic correction of the clotting factor VIII disorder in a hemophilia A mouse model [30] Hemophilia A is a genetic blood disorder characterized by mutations in

the factor VIII gene that provoke impaired clotting

and spontaneous hemorrhages that may result in

death in severe cases Tail-tip fibroblasts from healthy

mice were reprogrammed into pluripotent cells and

subsequently differentiated into endothelial cells

pro-ducing factor VIII, and were transplanted into mice

suffering from hemophilia A The transplanted cells

engrafted and expressed endogenous factor VIII

pro-tein in vivo, resulting in phenotypic correction of the

disease [30] Together, these two studies suggest that

iPSCs are a good cell source for correcting blood

dis-eases (especially monogenic disorders); however, more

work is required to examine engraftment efficiency and

functionality in various animal models of diseases before

clinical medical application can become a reality

Ontogeny of human hematopoiesis

Hematopoietic ontogeny has been the subject of

inten-sive investigation by several groups As early as 1970,

Moore and Metcalf [31] demonstrated that primitive

hematopoiesis starts with the formation of yolk sac

blood islands in mouse embryos, and later studies by

Medvinsky and Dzierzak indicated that the aorta–

gonad–mesonephros (AGM) region of the embryo is a

primary source of definitive hematopoietic progenitors

[32,33] These subsequently enter the circulation and

colonize the fetal liver (and other

hematopoiesis-sup-porting organs), where they mature before migrating

to the bone marrow [34,35] Definitive hematopoiesis

is initiated in the aortic endothelium region of the

embryonic AGM, where blood cells emerge into

the aortic lumen, from CD31+ endothelial cells lining

the lumen, in a process that could be understood as hematopoietic transition Both nascent cells express CD31, but only cells budding into the aortic lumen spe-cifically express CD34, c-kit and CD41, and, at a later stage, CD45 [34] (Fig 1) This process of budding has been described recently in mice and zebrafish [36,37] Because HSCs always emerge in close association with endothelial cells, a common origin for these two cell types has been hypothesized, a parent population known as hemangioblasts [38,39] Using the differenti-ation of mouse ESC as an in vitro model, Keller’s group isolated blast colony-forming cells that were shown to be responsible for the generation of endothe-lial and hematopoietic precursors, and hypothesized to represent the in vitro equivalent of hemangioblasts [40] Similarly, Bhatia’s group identified a population of human cells derived from hESCs that were hypothe-sized to be the in vitro human equivalent of the hemangioblast, characterized by expression of the endothelial markers PECAM-1 (CD31), Flk-1 (KDR) and VE-cadherin (CD144), but not the hematopoietic marker CD45 (termed CD45negPFV cells), and hemo-genic bi-phenotypic differentiation capacity [9]

Although the existence of the hemangioblast in vivo is widely accepted and has been described in Drosophila, zebrafish and mice [41–43], identification of a human he-mangioblast in vivo has not yet been achieved, and is hindered largely by the difficulty of obtaining early human embryonic tissues Nonetheless, insights gained from work on human adult cells do suggest the existence

of a human hemangioblast For example, the BCR⁄ ABL fusion gene has been found not only in bone marrow, but also in the endothelial cells of patients suffering

Fig 1 Schematic representation of hematopoietic development in the embryo The surface markers proving hematopoietic commitment at each stage of the differentiation process are shown Transcription factors required for differentiation are shown above the blue arrows where appropriate Runx1, runt-related transcription factor 1; Scl = Tal1, T-cell acute lymphocytic leukemia 1.

from chronic myelogenous leukemia, suggesting that

translocation of these genes occurred in a progenitor

common to them both [44] Further supporting this

hypothesis, both hematopoietic and endothelial cell

lin-eages can be derived from single CD34+ KDR+ cells

isolated from human bone marrow and cord blood [45]

Single-cell tracing and cell imaging of the aortic lumen

in vitro have shown that hemogenic endothelial cells,

that were initially defined by VE-cadherin and claudin

expression, acetylated low density lipoprotein uptake

and formation of tight junctions, start to co-express

CD41 and c-kit (early markers of definitive

hematopoie-sis) and change morphology over time These cells

con-vert from adherent growth as endothelial colonies

towards a more ‘hematopoietic’ phenotype,

character-ized by loss of their characteristic ‘hemogenic’

expres-sion profile whilst concurrently acquiring expresexpres-sion of

the pan-leukocyte marker CD45, and switching to

growth in suspension Thus these studies firmly prove

the emergence of definitive blood cells from aortic

endo-thelium [35,46,47] These findings have been confirmed

by many studies in mouse and human showing that

definitive human hematopoiesis originates from the

aor-tic embryonic endothelium [36,48]

Molecular studies have identified a number of

tran-scriptional factors that are key regulators of HSC

development For instance, the stem cell leukemia

factor Scl, has been shown to play a pivotal role in

endothelial and hematopoietic differentiation This

transcription factor is usually altered in patients

suffer-ing from T-cell leukemia, and it has been shown that

mESCs lacking Scl are unable to undergo

hematopoie-sis [49] Other studies have shown that Scl is not

required for hemangioblast generation, but is necessary

for subsequent differentiation, as Scl mutants cannot

generate hemogenic endothelium from the

hemangio-blast and consequently no hematopoietic or endothelial

cells can be produced [50] Another important

tran-scription factor required for the generation of blood cells is Runx1 Alterations affecting Runx1 are involved in acute myeloid leukemia and pediatric acute lymphoblastic leukemia In vitro studies using Runx1 null cells have shown that this transcription factor is required for the production of definitive hematopoietic cells, but not primitive hematopoietic or endothelial cells, from hemogenic endothelium [51] Despite this progress, the complete developmental program that facilitates the onset and progression of human embryo-nic hematopoiesis remains to be further investigated Although the AGM region is considered the first site

of definitive hematopoiesis, recent studies have indi-cated that the placenta acts as an additional extrame-dullary hematopoietic organ during embryonic and fetal development [52,53] Hematopoietic precursors found in the human placenta can give rise to erythro-cytic and myeloerythro-cytic lineages, and can also reconstitute the hematopoietic system of myelo-ablated mice These findings suggest the possibility of using placenta as a source of human hematopoietic progenitors for clinical use Following these investigations, Serikov et al [54] developed a protocol for cryopreservation and thawing

of the placenta to optimize the recovery of hematopoi-etic precursors, demonstrating that placenta can yield higher amounts of HSCs than umbilical cord blood, and further suggesting that placenta could be banked and used for clinical transplant in a similar way to umbilical cord blood

Which methods drive differentiation of hESC to hematopoietic lineages?

In vitro differentiation of hESCs into HSCs has been achieved through various methods, some based on cul-turing hESCs in the presence of stromal cells that mimic the embryonic hematopoietic developmental environment, others using methods involving the

for-Table 1 Comparison of hematopoietic differentiation methods reported previously.

Method hESC lines

Differentiation media Stages Cytokines

% CD34 +

in culture References

S17 co-culture H1, H1.1, H9.2 FBS added 1 No 1–2% at day 17 Kaufman et al (2001) [3] OP9 co-culture H1 and H9 FBS added 1 No 20% at day 7 Vodyanik et al (2005) [4] Hematopoietic

embryonic niches

H1, H9, hES-NCL1 FBS added 1 No 16% in AGM

co-culture

Ledran et al (2008) [58]

Embryoid bodies H1, H9 FBS added 1 SCF, Flt-3, IL-3, IL-6,

G-CSF, BMP4

20% at day 15 Chadwick et al (2003) [59]

Embryoid bodies H1, HES2 Serum-free 1 BMP4, bFGF – Kennedy et al (2007) [61]

2 VEGF Spin embryoid

bodies

hES2, hES3, hES4 Serum-free 1 BMP4, VEGF, SCF 23% at day 11 Ng et al (2005, 2008)

[62,63]

2 SCF, VEGF, IL-3, IL-6,

Tpo, Epo

mation of three-dimensional cellular clumps termed

embryoid bodies Various hematopoietic differentiation

methods are summarized in Table 1

Co-culture with stromal cells

In 2001, work by Kaufman et al [3] showed that

co-culture of hESCs with the cells lines S17 (derived from

murine bone marrow) or C166 (from murine yolk sac

endothelium) can induce differentiation towards blood

cells After 17 days of co-culture,  2% of the hESCs

had formed hematopoietic progenitors as evaluated by

expression of CD34, an embryonic and adult

hemato-poietic stem cell marker The kinetics of hematopoiesis

in co-culture with the S17 cell line showed increased

CD34 expression in two distinct stages of the

differen-tiation process The first peak appeared in conjunction

with CD31 expression, and is proposed to mark a

hemogenic endothelium state, and the second started

at day 14, together with expression of the definitive

hematopoietic marker CD45, and was coincident with

observation of the first colonies in hematopoietic

col-ony assays [55] The hematopoietic transcription

fac-tors SCL and GATA-2 were also expressed in these

cells between days 7 and 21 of differentiation

More-over, when hESCs co-cultured with the S17 cell line or

CD34+cells derived from these co-cultures were

trans-ferred to semi-solid medium, they produced erythroid,

myeloid and megakaryocyte colonies, arising between

days 14 and 17, demonstrating the functionality of

hESC-derived CD34+precursors

Using a similar approach, hESCs have also been

co-cultured with the mouse bone marrow stromal cell

line OP9, which is deficient in macrophage

colony-stimulating factor [56] This method generates a higher

level of hematopoietic differentiation than co-culture

with the S17 cell line: almost 20% of initial hESCs

dif-ferentiated into CD34+progenitors after 7 days of

co-culture [4] The kinetics of OP9 differentiation show

expression of CD34 from days 3 to 7, and the CD34+

population showed enhanced CD43 and CD41

expres-sion at approximately day 5, followed by CD45

expression at day 8 The expression of CD43 has been

found to mark the earliest hematopoietic cells arising

from hemato-endothelial commitment [57] Using this

method, expression of hematopoietic transcription

fac-tors GATA-1 and GATA-2 was observed from days 2

and 3 of differentiation, coincident with the onset of

CD34 and SCL expression on days 3 and 4,

respec-tively Additionally, colony-forming cells also arose

from CD34+ cells from day 4, and, after subsequent

co-culture with MS-5 cells, showed production of both

lymphoid and myeloid lineages [4]

In our group, we used stromal cells derived from embryonic hematopoietic niches to direct the differen-tiation of hESCs towards hematopoietic progenitors with greater efficiency With this in mind, we com-pared the ability of primary stromal cells from the AGM and fetal liver regions, as well as three established cell lines derived from the AGM region (AM20.1B4), urogenital ridge (UG26.1B6) and fetal liver (EL08.1D2),

to induce hematopoietic differentiation of hESCs Expression of CD34 was found in all cases from day 6 and peaked at day 18, matching the peak of activity of colony-forming cells Co-culture of hESCs with pri-mary AGM stromal cells produced the highest level of CD34+and CD45+cells at day 18 Most importantly,

we found that some of these hESC-derived hemato-poietic cells engrafted both primary and secondary immunocompromised mice (treated with a sub lethal dose of ionising radiation) at substantially higher levels than described previously [58]

Formation of embryoid bodies Differentiation of hESCs in the form of embryoid bodies (EBs) is an easily modifiable alternative method that is often used to obtain differentiated cell types in serum-free and defined media, depending on the cytokines and⁄ or growth factors added Many methods of EB-mediated in vitro hematopoiesis have been described Chadwick et al described the use of hematopoietic cyto-kines in combination with BMP4 to induce hematopoie-sis Using this method, the proportion of CD34+cells at day 15 of differentiation increased from 10% to 20% when BMP4 and cytokines were added to the medium, and 90% of the CD34+ cells co-expressed CD45 During this differentiation process, expression of topoietic transcription factors was detected, and hema-topoietic colonies including macrophages, granulocytes, megakaryocytes and erythrocytes emerged [59,60]

In 2007, Keller’s group developed a new two-step approach for EB-mediated hematopoietic differentia-tion, the first driven by BMP4 and basic fibroblast growth factor, and the second by addition of VEGF165 The kinetics of EB-derived hematopoiesis showed expression of SCL, GATA-1 and RUNX1 from day 3, consistent with up-regulation of CD34 expression, detectable from day 4 of differentiation The first hematopoietic colony-forming cells colonies arose at day 5, and consisted predominantly of primitive ery-throid progenitors, followed by eryery-throid colonies and macrophages that emerged at approximately day 8 of differentiation It is noteworthy that development of hematopoietic progenitors from EBs in these conditions was dependent on the presence of BMP4 [61]

A further improvement in EB-mediated

hematopoi-etic differentiation was described in 2005 when spin

EBs were first described [62] Spin EBs are embryoid

bodies produced from a defined number of cells that

are centrifuged to produce tight cell compaction, thus

improving experimental reproducibility compared to

standard EB derivation methods The number of cells

used for EB formation and various augmenting factors

were found to be critical; optimum hematopoietic

dif-ferentiation was achieved when spin EBs were formed

from at least 1000 hESCs, and different cytokines were

required at each of two distinct differentiation stages

for efficient differentiation Onset of CD34 expression

using this protocol was detected at day 6, concurrent

with expression of the RUNX1 transcription factor

CD34 expression peaked at approximately day 10 and

was maintained at this level until day 26, when

approximately 30% of cells expressed CD45 More

primitive blast forming units erythroid colonies

(BFU-E) emerged on day 6, whereas more mature

col-ony forming unit (CFU-E) erythropoietic colonies and

myeloid cells arose later, probably from CD34+

hema-topoietic progenitors [62,63]

Successes and failures of

hematopoietic engraftment

The only definitive way to evaluate the full

functional-ity of hESC-derived hematopoietic cells is

transplanta-tion into immunocompromised animals in order to test

their ability to engraft and provide long-term

multi-lineage hematopoietic reconstitution Various mouse

models have been used to study human hematopoiesis

in vivo; the most commonly used are genetically

modi-fied strains of the non-obese diabetic⁄ severe combined

immuno-deficient (NOD⁄ SCID) mouse

To date, only a few studies have described the

engraft-ment potential of hESC-derived hematopoietic cells In

2005, Bhatia’s group tested EB-differentiated cells by

transplanting these cells intravenously into the tail vein

of sub-lethally irradiated NOD⁄ SCID mice However,

more than 60% of the transplanted mice died after the

transplantation due to aggregation of human

ESC-derived hematopoietic cells in the presence of rodent

serum, resulting in the formation of pulmonary emboli

To solve this problem, the authors performed

intra-fem-oral injections to deliver the cells directly into the bone

marrow These animals showed hematopoietic

reconsti-tution 8 weeks after injection; however, the level of

human engraftment was low at 1% [64]

A second study used hematopoietic progenitors

derived from hESC co-culture with S17 cells for

intra-venous and intra-femoral transplantation into

sub-leth-ally irradiated NOD⁄ SCID mice (treated with an antibody against NK cells in order to avoid immune rejection of the hESC-derived cells) Long-term engraftment was achieved using both methods as shown by expression of human CD45 3–6 months post-transplantation Additionally, secondary engraft-ment was achieved following intravenous transplanta-tion of bone marrow from primary recipients, but once again the level of human engraftment was very low (< 0.5% independently of intravenous or intra-femo-ral injection) [65]

Our group has successfully used the NOD⁄ LtSz-SCID IL2rcnull (NOG) mouse model [58] to test the hematopoietic potential of hESCs differentiated in var-ious embryonic hematopoietic environments by intra-femoral injection We found that hESC-derived hema-topoietic cells engrafted primary and secondary recipi-ents with multiple hematopoietic lineages for up to

12 weeks following transplantation Most importantly, engraftment levels were higher compared to previous studies, reaching 16% when hESCs were co-cultured with AM20.1B4, an established cell line derived from the AGM embryonic region [58]

Future therapeutic applications for hESC-derived hematopoietic cells will require a significant improve-ment in the engraftimprove-ment levels achieved to date Low engraftment may result from multiple factors, includ-ing possible rejection by the host immune system, the quantity administered, the developmental stage of the cells derived from hESC differentiation, and the qual-ity and⁄ or viability of the transplanted cells Moreover, the variables affecting the study of human hematopoie-sis in ‘humanized’ mice have been studied, and demon-strate the importance of selecting the appropriate mouse strain when designing experimental work For instance, truncation of IL2 receptor gamma chain (IL2rc as in NSG mice) in the genetic background of NOD⁄ SCID mice has been shown to increase the engraftment ability of NOD⁄ SCID re-populating cells, and consequently significantly improve human hemato-poietic cell engraftment [66–68] Host age is also important, as it has been shown that newborn mice support higher levels of engraftment than adults because HSCs can better mature and develop in an age-matched micro-environment [69] This hypothesis

is in agreement with the results of a study of the long-term engraftment of primary and secondary recipients (22 months after transplantation) by Narayan et al., who studied HSC engraftment in fetal sheep by inject-ing HSCs in utero into the fetal peritoneal cavity [70] The site of transplantation (and therefore the differen-tiation cues provided by the niche) is also very impor-tant, as a recent study has shown than hESC-derived

CD34+ cells differentiate preferentially to endothelial

cells upon transplantation into the liver of

immuno-compromised recipients, suggesting that the

environ-ment surrounding the transplanted cells could

determine their final lineages [71]

Advantages and disadvantages of using

hESCs and hiPSCs for therapeutic

applications

The successful use of stem cells for clinical purposes is

one of the most appealing prospects in regenerative

medicine today HSCs can be isolated from peripheral

blood, bone marrow and umbilical cord blood for

exam-ple, but problems arising from immunogenic matching,

resulting in rejection or graft-versus-host disease, as well

as the overall lack of donors, impair use of these cell

types for therapy For these reasons, in vitro production

of hESC-derived HSCs represents a remarkable

oppor-tunity for regenerative medicine, as they represent not

only a theoretically unlimited source of more closely

tis-sue-matched donor cells, but can also reduce immune

responses following transplant of other cell types

derived from the same hESCs if hematopoietic

chimer-ism is first induced [60,72] However, there are many

advantages and disadvantages regarding the use of

hESCs in medical practice, including some ethical

objec-tions regarding the use of human embryos for research,

even when discarded embryos are used

Use of hiPSCs bypasses these ethical issues [27,28],

and several tissue-specific cells have been successfully

obtained using hiPSC differentiation, including

hema-topoietic and endothelial cells [73] In theory, hiPSC

technology could allow the production of specific cell

types for the treatment of various patients or diseases

[74], but the suitability of these cells for therapeutic

use is still open to debate [75,76] Experiments by Choi

et al [73] suggest that HSCs derived from various

hiPSC lines differentiate following the same profile

than those derived from hESCs However, recent work

by Lanza’s group showed that endothelial and

hemato-poietic cells obtained from hiPSC-derived

hemangio-blasts show decreased proliferation potential, an

apoptotic phenotype and early senescence, features

that were not observed in hESC-derived counterparts

The same features were observed in hiPSC-derived

reti-nal pigment epithelial cells and cardiomyocytes [77,78],

evidence against a differentiated cell type-specific

phe-nomenon The mechanism that results in such

abnor-malities is still unknown, but may be related to the

integration of viral vectors carrying the transcriptional

factors required for hiPSC reprogramming, or the

result of a perturbed epigenome

A new method to reprogram hiPSCs based on epi-somal vectors, which avoid the expression of transg-enes in the iPSC lines generated, was described recently [79] However, it has been shown that the vec-tors are not responsible for the differences in effective-ness of hiPSC versus hESC differentiation, as neural cells obtained from lentiviral, retroviral or episomal vector iPSCs exhibit the same variability compared to their hESC counterparts [80] In order to clarify this, comparative studies at the level of genomic DNA, cod-ing RNA, microRNA and epigenetic events have recently been performed [81–83] These findings dem-onstrated that, although very similar, hiPSCs and hESCs are not identical in either their genomic DNA, gene expresion pattern, epigenetic profile, teratoma formation efficiency or latency [84] In agreement with this, two recently published studies using mouse mod-els have shown that iPSCs retain a temporary tran-scriptional and epigenetic memory of their original somatic lineages, leading to preferential differentiation

of these cells towards their original tissue type [85,86] This phenomenon was only observed when pluripotent stem cells were obtained by exogenous expression of reprogramming transcription factors, but not by somatic cell nuclear transfer or embryonic stem cells derived from fertilized embryos [85] These findings suggest that certain sets of somatic cells may be selected prior to reprogramming with the aim of enhancing the iPSC differentiation capacity using their tissue-specific epigenetic memory However, in many cases, full pluripotency may be advisable or required prior to re-differentiation, and therefore erasing the epigenetic⁄ transcriptional memory from iPSCs is an important goal Work towards achieving this by serial reprogramming or by treating iPSCs with chromatin-modifying chemicals has been described [85] An alter-native method for avoiding problems associated with partially retained cellular epigenetic memory, by repro-gramming human cord blood un-restricted somatic stem cells, has recently been suggested [87] It was found that the iPSCs obtained from these cells are very similar to hESCs in terms of morphology, molecular signature and differentiation potential; nonetheless, further studies on epigenetics and comparative analysis using iPSCs obtained from other cells are required [87]

Finally, some recent studies have suggested that hiP-SCs might potentially be oncogenic It is known that some transcriptional factors used for hiPSC repro-gramming, such as Lin28 and c-Myc, are tumorigenic, and although their expression should be abolished in hiPSCs, it is not known whether they can be activated again at any time during differentiation [77,88]

Another recent study showed that the p53 pathway

acts not only as a tumor suppressor, but also

sup-presses iPSC generation as determined using small

interfering RNA and p53 mutation [89] These findings

indicate the need to improve hiPSC technology as well

as for a complete evaluation of the possible

conse-quences of use of hiPSCs for drug screening, as disease

models and for medical purposes

Conclusions

The use of stem cells for the treatment of human

diseases is one of the principal goals in regenerative

medicine today hESCs and hiPSCs, capable of

direc-ted differentiation into specific cell types with

theoreti-cally no limits on cell availability, are a promising

source of cells, but detailed investigation is required to

enable their application in clinical situations In vitro

differentiation of hESCs to hematopoietic progenitors

has been successfully achieved by various methods and

groups, but the cells obtained using these approaches

are not completely functional in vivo, as demonstrated

by the low proportion of cells able to reconstitute the

hematopoietic system of immunodeficient animals It is

worth highlighting that co-culture of hESCs with the

stromal cell line AM20.1B4, derived from the

embry-onic AGM region, yields the highest engraftment level

in NOD⁄ LtSz-SCID ⁄ IL2rcnull (NOG) mice, increasing

this proportion from < 1% to 16% [58]; however, this

is still not sufficient for any useful clinical application

Human iPSCs are a promising alternative to the use

of hESCs for hematopoietic studies and in drug

devel-opment, but reasonable doubts regarding the

suitabil-ity of these cells for clinical therapies are starting to

emerge The main objection to the potential clinical

use of hiPSC-derived cells is the use of viral vectors

required for initial reprogramming, which could confer

tumorigenic potential to these cells and their

deriva-tives However, this is not the only problem: low

dif-ferentiation efficiencies and early senescence have also

been detected in hiPSC-derived cells

For these reasons, future studies should strive to

improve differentiation protocols, in order to attain

higher engraftment levels Additionally, the

develop-ment of safer and more efficient reprogramming

meth-ods for obtaining pluripotent stem cells may guarantee

the future safe application of hiPSC technologies for

clinical therapy

Acknowledgements

The authors are grateful to financial support received

by from Leukemia and Lymphoma UK, Fanconi

Hope UK and the Fanconi Anemia Research Fund USA, and funds for research in the field of regenera-tive medicine through the collaboration agreement between the Conselleria de Sanidad (Generalitat Valen-ciana) and the Instituto de Salud Carlos III (Ministry

of Science and Innovation)

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