Accepted Manuscript Human embryonic stem cells versus human induced Pluripotent Stem cells for cardiac repair Lili Barad, MSc Revital Schick, MSc Naama Zeevi-Levin, PhD Joseph Itskovitz-Eldor, MD, DSc Ofer Binah, PhD PII:
S0828-282X(14)00429-2
DOI:
10.1016/j.cjca.2014.06.023
Reference:
CJCA 1258
To appear in:
Canadian Journal of Cardiology
Received Date: 16 April 2014 Revised Date:
26 June 2014
Accepted Date: 29 June 2014
Please cite this article as: Barad L, Schick R, Zeevi-Levin N, Itskovitz-Eldor J, Binah O, Human embryonic stem cells versus human induced Pluripotent Stem cells for cardiac repair, Canadian Journal of Cardiology (2014), doi: 10.1016/j.cjca.2014.06.023. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Human embryonic stem cells versus human induced Pluripotent Stem cells for cardiac repair
Barad: Stem cells for cardiac regeneration Lili Barad, MSc, a, b, cRevital Schick, MSc, c, dNaama Zeevi-Levin, PhD,
b, c, d
a, b, c
Ofer Binah, PhD
SC
Joseph Itskovitz-Eldor , MD, DSc,
RI PT
a, b, c
a
Department of Physiology, bThe Rappaport Family Institute, cRuth & Bruce
Cell Center, Technion, Haifa, Israel
TE D
Corresponding author
M AN U
Rappaport Faculty of Medicine and dThe Sohnis and Forman Families Stem
AC C
EP
Ofer Binah, Ph.D. Department of Physiology Rappaport Faculty of Medicine POB 9649, Haifa, 31096 Israel Email: [email protected]; Tel: +972-4-8295262; Fax: +972-4-8513919
Word count: 7245
1
ACCEPTED MANUSCRIPT Brief summary Human embryonic stem cells (hESC) and human induced Pluripotent Stem Cells (hiPSC), with their ability to differentiate into functional
RI PT
cardiomyoctes, offer a potential cell-source for cardiac repair applications intended to rehabilitate myocardial function. In this review we discuss the advantages and limitations of these two candidates for cell-based therapy, and provide an overview of the main studies performed in myocardial
SC
infarction models. These studies demonstrate the challenges and future
AC C
EP
TE D
M AN U
perspectives of these cells for cardiac regeneration.
2
ACCEPTED MANUSCRIPT Abstract Human embryonic stem cells (hESC) and human induced pluripotent stem cells (hiPSC) have the capacity to differentiate into any specialized cell
RI PT
type including cardiomyocytes. Therefore, hESC- and hiPSC-derived cardiomyocytes (hESC-CM and hiPSC-CM) offer great potential for cardiac regenerative medicine. Unlike some organs, the heart has limited ability to regenerate, and dysfunction due to a significant cardiomyocyte loss under
SC
pathophysiological conditions such as myocardial infraction, can lead to heart
M AN U
failure. Unfortunately, for patients with end-stage heart failure, heart transplantation remains the main alternative which is insufficient mainly due to limited availability of donor organs. Although left ventricular assist devices are progressively entering clinical practice as a bridge to transplantation and even as an optional therapy, cell replacement therapy presents a plausible
TE D
alternative to donor organ transplantation. During the past decade, multiple candidate cells were proposed for cardiac regeneration and their mechanisms
EP
of action in the myocardium have been explored. The purpose of this article is to critically review the comprehensive research involving the use of hESC and
AC C
hiPSC in myocardial infraction models, and to discuss current controversies, unresolved issues, challenges and future directions.
3
ACCEPTED MANUSCRIPT Introduction Cardiovascular diseases which are the leading cause of morbidity and mortality worldwide, account for ~30% of all deaths, with nearly half resulting
RI PT
from myocardial infraction (MI) (1). Because the regenerative capacity of the adult heart is limited and insufficient to compensate for cell death during MI, cardiac repair techniques to rehabilitate myocardial function hold great potential in overcoming the loss of working myocardium. Since the main goal
SC
of cardiac repair is to create new myocardium that is electrically and
M AN U
mechanically integrated into the recipient heart, many studies have searched for the ultimate cell source capable of fulfilling this immense task. To this end, human embryonic stem cells (hESC) and human induced Pluripotent Stem Cells (hiPSC) are among the key cell types with in vitro cardiogenic differentiation capacity (2, 3) that are considered for cardiac regeneration. In
TE D
this review we discuss the advantages and limitations of hESC and hiPSC as candidates for cell-based therapy (see Table 1), and provide an overview of
AC C
(see Figure 1).
EP
the main in vivo studies using these pluripotent stem cells as therapies for MI
hESC or hiPSC: which cell is a better candidate for cardiac regeneration? hESC obtained from the inner cell mass of blastocysts are pluripotent
cells that possess the ability to differentiate into cells derived from the three germ layers: ectoderm, endoderm and mesoderm, and hold unlimited selfrenewal capacity. Indeed, under specific culture conditions, the differentiation of hESC into cardiomyocytes is already well established (2). hESC-derived 4
ACCEPTED MANUSCRIPT cardiomyocytes (hESC-CM) consist of a mixed population of the three major action potential phenotypes (nodal-, atrial-, and ventricular-like), as found in the human adult heart (4). These properties provide the basis for considering hESC-CM as a likely source of cardiomyocytes for regenerative medicine.
RI PT
The second source is hiPSC originally generated by the expression of the transcription factors Oct4, Sox2, Klf4 and c-Myc in somatic cells (5, 6). The inherent pluripotency of hiPSC, their capacity to differentiate into all three
SC
cardiomyocyte phenotypes, along with their genetic identity to the specific patient, holds tremendous promise for clinical applications and regenerative
M AN U
medicine. While recent studies have shown that hiPSC are similar to hESC (7-9), as discussed herein each cell type has advantages and disadvantages
TE D
for its application in cardiac regenerative applications.
Ethical issues
Because hESC are derived from the inner cell mass of preimplantation-
EP
stage blastocysts (10), the need for using surplus embryos during their
AC C
derivation contributes to the ethical and legislative debate surrounding their use, and also restricts their availability. In contrast, hiPSC which are generated from adult cells, do not create the same ethical hurdles as hESC. Further, hiPSC derivation is not necessarily associated with invasive procedures, since they can be reprogrammed from cells such as hair keratinocytes or blood cells (11, 12).
5
ACCEPTED MANUSCRIPT Genetics and immunogenicity The use of autologous stem cells for cardiovascular medicine has a major advantage over hESC-CM (which can trigger an immune response)
RI PT
since the transplanted cells will not be identified as a foreign and thus an immunosuppressive therapy will not be required. Hence, immune rejection against allogeneic hESC-derived grafts constitutes another major barrier for hESC-based cell therapies. Therefore, hiPSC which carry a genome matching
SC
the genome of the patient from whom it was derived have an important clinical
M AN U
advantage over hESC. This advantage may be preserved even in cases where a patient’s disease is genetic, because gene targeting techniques are extensively being developed to repair known genetic mutations (13, 14). Surprisingly, important studies by Zhao et al demonstrated that isogenic iPSC may still provoke an immune response (15, 16). By using the teratoma
TE D
formation model, they found that some but not all cells derived from murine fibroblasts can be immunogenic, and the immune rejection response was T
EP
cell dependent. This response was attributed to the reprogramming procedure which can induce both genetic and epigenetic defects in iPSC (17, 18).
AC C
Most of the current reprograming techniques utilize viral delivery of
particular factors, involving the risk of altering the original genomics of the target cell. Since these viral factors will likely trigger antiviral immunity or antiDNA antibody production (19, 20), recent studies developed non-viral hiPSC rerprogramming techniques using non-viral vectors (21), RNAs (22, 23) and small molecules accompanied by chemical treatment (24, 25). Thus, based on immunological considerations, non-viral and non-integrating reprogramming methods offer a better option. However, it is still possible that mild 6
ACCEPTED MANUSCRIPT immunogenicity will be developed due to the long-term culture duration, differentiation conditions and epigenetic modulations. Therefore, it is critical to test hiPSC for mutations before therapeutic use, as well as to improve the reprograming process. In any event, it appears that presently immune
RI PT
intervention in the recipient hosts may still be required for effective hiPSC
SC
transplantation.
Cell availability
M AN U
Despite the advantages (compared to hESC cell therapy) of autologous hiPSC-derived tissue transplantation, the logistics of achieving patient-specific cells on a large scale are challenging due to a relatively inefficient reprogramming techniques and high costs. Specifically, the major obstacle
TE D
here is the prolonged period required for hiPSC derivation, and thus the iPSC technology is (currently) irrelevant under 'urgent' circumstances such as acute MI. To overcome this hurdle, a hiPSC banking to provide rapidly accessible
EP
source for cell therapy may be useful. A hiPSC bank comprising human
AC C
leucocyte antigens (HLA)-typed hiPSC is a strategy that will also overcome the immunological barrier by providing HLA-matched (histocompatible) tissue for the target population. However, establishing such a bank (for either hiPSC or hESC) is associated with practical, financial, political and ethical (especially with hESC) issues that need to be overcome before application (26). Another current limitation for cardiac cell therapy is the large number of functional cardiomyocytes required for transplantation. Under spontaneous differentiation conditions, only a small fraction of hESC or hiPSC 7
ACCEPTED MANUSCRIPT spontaneously differentiates into functional cardiomyocytes (27), and therefore for clinical use where a billion myocytes are lost post-MI, the current spontaneous
low-yield
differentiation
methods
are
impractical
(28).
Consequently, new protocols were developed to enable well controlled and
RI PT
highly efficient direct differentiation of hESC or hiPSC into a large supply of
SC
high-purity functional cardiomyocytes (29-33).
Teratoma-formation propensity
M AN U
Another concern related to the clinical application of pluripotent stem cells is their tendency to form tumors or teratomas (34). Undifferentiated cells included
within
the
hESC/hiPSC-CM
population
due
to
incomplete
differentiation efficiency or inefficient isolation techniques, can cause
TE D
undesirable tumors or teratoma formation after transplantation. To obtain a homogenous cardiomyocytes population, it is extremely important to efficiently purify and remove all residual hESC or iPSC. Accordingly, several groups
EP
developed protocols for generating highly purified cardiomyocytes from
AC C
pluripotent stem cells (35, 36). Yet, these methods have a serious disadvantage, since they require genetic modifications of the cells. Recently Hattori et al (37) developed a widely applicable non-genetic cardiomyocyteenrichment and purification method (>99% purity), based on the fact that differentiated cardiomyocytes are extremely enriched in mitochondria. Cardiomyocytes separated using fluorescent-activated cell sorting (FACS) with mitochondria-specific fluorescent dyes did not induced teratoma formation following
transplantation
into NOD/SCID
8
mice. While
this
ACCEPTED MANUSCRIPT mitochondria-based method might be useful for a safe hESC or hiPSC cell therapy, its successful application in additional stem cell lines and further technological improvements are required to enable large scale and high speed flow cytometry for clinical use. Later on, Tohyama et al reported
RI PT
another non-genetic method for mass-producing cardiomyocytes from mouse and human pluripotent stem cells (38). This method is based on the remarkable biochemical differences in glucose and lactate metabolism
SC
between cardiomyocytes and non-cardiomyocytes, including undifferentiated cells. Using this approach the authors obtained cardiomyocytes of up to 99%
M AN U
purity, which importantly, did not form tumors after transplantation. The lactate method compared to the mitochondrial method for purifying cardiomyocytes has a relative advantage due to its simplicity and ease of application (38). Another purification method was published by Dubois et al, using a surface
TE D
protein, signal-regulatory protein alpha (SIRPA), as a cardiac-specific marker in hESC and hiPSC (39). Cell sorting with an antibody against SIRPA allowed for the enrichment of cardiomyocytes from differentiated cultures, yielding up
EP
to 98% purity. However none of these methods are ideal for the therapeutic
AC C
application of hESC-CM or hiPSC-CM because of insufficient purity (
S0828-282X(14)00429-2
DOI:
10.1016/j.cjca.2014.06.023
Reference:
CJCA 1258
To appear in:
Canadian Journal of Cardiology
Received Date: 16 April 2014 Revised Date:
26 June 2014
Accepted Date: 29 June 2014
Please cite this article as: Barad L, Schick R, Zeevi-Levin N, Itskovitz-Eldor J, Binah O, Human embryonic stem cells versus human induced Pluripotent Stem cells for cardiac repair, Canadian Journal of Cardiology (2014), doi: 10.1016/j.cjca.2014.06.023. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Human embryonic stem cells versus human induced Pluripotent Stem cells for cardiac repair
Barad: Stem cells for cardiac regeneration Lili Barad, MSc, a, b, cRevital Schick, MSc, c, dNaama Zeevi-Levin, PhD,
b, c, d
a, b, c
Ofer Binah, PhD
SC
Joseph Itskovitz-Eldor , MD, DSc,
RI PT
a, b, c
a
Department of Physiology, bThe Rappaport Family Institute, cRuth & Bruce
Cell Center, Technion, Haifa, Israel
TE D
Corresponding author
M AN U
Rappaport Faculty of Medicine and dThe Sohnis and Forman Families Stem
AC C
EP
Ofer Binah, Ph.D. Department of Physiology Rappaport Faculty of Medicine POB 9649, Haifa, 31096 Israel Email: [email protected]; Tel: +972-4-8295262; Fax: +972-4-8513919
Word count: 7245
1
ACCEPTED MANUSCRIPT Brief summary Human embryonic stem cells (hESC) and human induced Pluripotent Stem Cells (hiPSC), with their ability to differentiate into functional
RI PT
cardiomyoctes, offer a potential cell-source for cardiac repair applications intended to rehabilitate myocardial function. In this review we discuss the advantages and limitations of these two candidates for cell-based therapy, and provide an overview of the main studies performed in myocardial
SC
infarction models. These studies demonstrate the challenges and future
AC C
EP
TE D
M AN U
perspectives of these cells for cardiac regeneration.
2
ACCEPTED MANUSCRIPT Abstract Human embryonic stem cells (hESC) and human induced pluripotent stem cells (hiPSC) have the capacity to differentiate into any specialized cell
RI PT
type including cardiomyocytes. Therefore, hESC- and hiPSC-derived cardiomyocytes (hESC-CM and hiPSC-CM) offer great potential for cardiac regenerative medicine. Unlike some organs, the heart has limited ability to regenerate, and dysfunction due to a significant cardiomyocyte loss under
SC
pathophysiological conditions such as myocardial infraction, can lead to heart
M AN U
failure. Unfortunately, for patients with end-stage heart failure, heart transplantation remains the main alternative which is insufficient mainly due to limited availability of donor organs. Although left ventricular assist devices are progressively entering clinical practice as a bridge to transplantation and even as an optional therapy, cell replacement therapy presents a plausible
TE D
alternative to donor organ transplantation. During the past decade, multiple candidate cells were proposed for cardiac regeneration and their mechanisms
EP
of action in the myocardium have been explored. The purpose of this article is to critically review the comprehensive research involving the use of hESC and
AC C
hiPSC in myocardial infraction models, and to discuss current controversies, unresolved issues, challenges and future directions.
3
ACCEPTED MANUSCRIPT Introduction Cardiovascular diseases which are the leading cause of morbidity and mortality worldwide, account for ~30% of all deaths, with nearly half resulting
RI PT
from myocardial infraction (MI) (1). Because the regenerative capacity of the adult heart is limited and insufficient to compensate for cell death during MI, cardiac repair techniques to rehabilitate myocardial function hold great potential in overcoming the loss of working myocardium. Since the main goal
SC
of cardiac repair is to create new myocardium that is electrically and
M AN U
mechanically integrated into the recipient heart, many studies have searched for the ultimate cell source capable of fulfilling this immense task. To this end, human embryonic stem cells (hESC) and human induced Pluripotent Stem Cells (hiPSC) are among the key cell types with in vitro cardiogenic differentiation capacity (2, 3) that are considered for cardiac regeneration. In
TE D
this review we discuss the advantages and limitations of hESC and hiPSC as candidates for cell-based therapy (see Table 1), and provide an overview of
AC C
(see Figure 1).
EP
the main in vivo studies using these pluripotent stem cells as therapies for MI
hESC or hiPSC: which cell is a better candidate for cardiac regeneration? hESC obtained from the inner cell mass of blastocysts are pluripotent
cells that possess the ability to differentiate into cells derived from the three germ layers: ectoderm, endoderm and mesoderm, and hold unlimited selfrenewal capacity. Indeed, under specific culture conditions, the differentiation of hESC into cardiomyocytes is already well established (2). hESC-derived 4
ACCEPTED MANUSCRIPT cardiomyocytes (hESC-CM) consist of a mixed population of the three major action potential phenotypes (nodal-, atrial-, and ventricular-like), as found in the human adult heart (4). These properties provide the basis for considering hESC-CM as a likely source of cardiomyocytes for regenerative medicine.
RI PT
The second source is hiPSC originally generated by the expression of the transcription factors Oct4, Sox2, Klf4 and c-Myc in somatic cells (5, 6). The inherent pluripotency of hiPSC, their capacity to differentiate into all three
SC
cardiomyocyte phenotypes, along with their genetic identity to the specific patient, holds tremendous promise for clinical applications and regenerative
M AN U
medicine. While recent studies have shown that hiPSC are similar to hESC (7-9), as discussed herein each cell type has advantages and disadvantages
TE D
for its application in cardiac regenerative applications.
Ethical issues
Because hESC are derived from the inner cell mass of preimplantation-
EP
stage blastocysts (10), the need for using surplus embryos during their
AC C
derivation contributes to the ethical and legislative debate surrounding their use, and also restricts their availability. In contrast, hiPSC which are generated from adult cells, do not create the same ethical hurdles as hESC. Further, hiPSC derivation is not necessarily associated with invasive procedures, since they can be reprogrammed from cells such as hair keratinocytes or blood cells (11, 12).
5
ACCEPTED MANUSCRIPT Genetics and immunogenicity The use of autologous stem cells for cardiovascular medicine has a major advantage over hESC-CM (which can trigger an immune response)
RI PT
since the transplanted cells will not be identified as a foreign and thus an immunosuppressive therapy will not be required. Hence, immune rejection against allogeneic hESC-derived grafts constitutes another major barrier for hESC-based cell therapies. Therefore, hiPSC which carry a genome matching
SC
the genome of the patient from whom it was derived have an important clinical
M AN U
advantage over hESC. This advantage may be preserved even in cases where a patient’s disease is genetic, because gene targeting techniques are extensively being developed to repair known genetic mutations (13, 14). Surprisingly, important studies by Zhao et al demonstrated that isogenic iPSC may still provoke an immune response (15, 16). By using the teratoma
TE D
formation model, they found that some but not all cells derived from murine fibroblasts can be immunogenic, and the immune rejection response was T
EP
cell dependent. This response was attributed to the reprogramming procedure which can induce both genetic and epigenetic defects in iPSC (17, 18).
AC C
Most of the current reprograming techniques utilize viral delivery of
particular factors, involving the risk of altering the original genomics of the target cell. Since these viral factors will likely trigger antiviral immunity or antiDNA antibody production (19, 20), recent studies developed non-viral hiPSC rerprogramming techniques using non-viral vectors (21), RNAs (22, 23) and small molecules accompanied by chemical treatment (24, 25). Thus, based on immunological considerations, non-viral and non-integrating reprogramming methods offer a better option. However, it is still possible that mild 6
ACCEPTED MANUSCRIPT immunogenicity will be developed due to the long-term culture duration, differentiation conditions and epigenetic modulations. Therefore, it is critical to test hiPSC for mutations before therapeutic use, as well as to improve the reprograming process. In any event, it appears that presently immune
RI PT
intervention in the recipient hosts may still be required for effective hiPSC
SC
transplantation.
Cell availability
M AN U
Despite the advantages (compared to hESC cell therapy) of autologous hiPSC-derived tissue transplantation, the logistics of achieving patient-specific cells on a large scale are challenging due to a relatively inefficient reprogramming techniques and high costs. Specifically, the major obstacle
TE D
here is the prolonged period required for hiPSC derivation, and thus the iPSC technology is (currently) irrelevant under 'urgent' circumstances such as acute MI. To overcome this hurdle, a hiPSC banking to provide rapidly accessible
EP
source for cell therapy may be useful. A hiPSC bank comprising human
AC C
leucocyte antigens (HLA)-typed hiPSC is a strategy that will also overcome the immunological barrier by providing HLA-matched (histocompatible) tissue for the target population. However, establishing such a bank (for either hiPSC or hESC) is associated with practical, financial, political and ethical (especially with hESC) issues that need to be overcome before application (26). Another current limitation for cardiac cell therapy is the large number of functional cardiomyocytes required for transplantation. Under spontaneous differentiation conditions, only a small fraction of hESC or hiPSC 7
ACCEPTED MANUSCRIPT spontaneously differentiates into functional cardiomyocytes (27), and therefore for clinical use where a billion myocytes are lost post-MI, the current spontaneous
low-yield
differentiation
methods
are
impractical
(28).
Consequently, new protocols were developed to enable well controlled and
RI PT
highly efficient direct differentiation of hESC or hiPSC into a large supply of
SC
high-purity functional cardiomyocytes (29-33).
Teratoma-formation propensity
M AN U
Another concern related to the clinical application of pluripotent stem cells is their tendency to form tumors or teratomas (34). Undifferentiated cells included
within
the
hESC/hiPSC-CM
population
due
to
incomplete
differentiation efficiency or inefficient isolation techniques, can cause
TE D
undesirable tumors or teratoma formation after transplantation. To obtain a homogenous cardiomyocytes population, it is extremely important to efficiently purify and remove all residual hESC or iPSC. Accordingly, several groups
EP
developed protocols for generating highly purified cardiomyocytes from
AC C
pluripotent stem cells (35, 36). Yet, these methods have a serious disadvantage, since they require genetic modifications of the cells. Recently Hattori et al (37) developed a widely applicable non-genetic cardiomyocyteenrichment and purification method (>99% purity), based on the fact that differentiated cardiomyocytes are extremely enriched in mitochondria. Cardiomyocytes separated using fluorescent-activated cell sorting (FACS) with mitochondria-specific fluorescent dyes did not induced teratoma formation following
transplantation
into NOD/SCID
8
mice. While
this
ACCEPTED MANUSCRIPT mitochondria-based method might be useful for a safe hESC or hiPSC cell therapy, its successful application in additional stem cell lines and further technological improvements are required to enable large scale and high speed flow cytometry for clinical use. Later on, Tohyama et al reported
RI PT
another non-genetic method for mass-producing cardiomyocytes from mouse and human pluripotent stem cells (38). This method is based on the remarkable biochemical differences in glucose and lactate metabolism
SC
between cardiomyocytes and non-cardiomyocytes, including undifferentiated cells. Using this approach the authors obtained cardiomyocytes of up to 99%
M AN U
purity, which importantly, did not form tumors after transplantation. The lactate method compared to the mitochondrial method for purifying cardiomyocytes has a relative advantage due to its simplicity and ease of application (38). Another purification method was published by Dubois et al, using a surface
TE D
protein, signal-regulatory protein alpha (SIRPA), as a cardiac-specific marker in hESC and hiPSC (39). Cell sorting with an antibody against SIRPA allowed for the enrichment of cardiomyocytes from differentiated cultures, yielding up
EP
to 98% purity. However none of these methods are ideal for the therapeutic
AC C
application of hESC-CM or hiPSC-CM because of insufficient purity (
