Wednesday, October 07, 2020

Rebuilding the Infarcted Heart Testicular germ cells for heart repair

  Rebuilding the Infarcted Heart Volume:

Author(s): Loren J. Field, Kai C. Wollert



germline stem cells may provide

a new source of pluripotent cells for regenerative

medicine. From mouse studies, we know that

germline stem cells can be cultured in vitro for an

indefinite period and are able to differentiate into

cells from all three germ layers including functional

cardiomyocytes. For stem cell-based therapy,

a major challenge will be to generate strategies that

enable the development of multipotent cells from

human adult SSCs and to develop further techniques

for their use in organ regeneration.


Modern reperfusion strategies and advances in

pharmacological management of acute myocardial

infarction (AMI) have resulted in an increasing

proportion of patients surviving the acute event.

Many of these patients eventually develop adverse

left ventricular remodeling and heart failure. None

of our current therapies addresses the underlying

cause of the remodeling process, i.e. the damage to

the cardiomyocytes and the vasculature in the

infarcted area. The alleged transdifferentiation

capacity of adult stem cells and the recent discovery

of endogenous cardiac repair mechanisms have

suggested that cardiac repair (i.e. replacement of

necrotic or scarred tissue with viable myocardium)

might be achieved in the clinical setting.

Stem cells are capable of self-renewal, transformation

into dedicated progenitor cells, and differentiation

into specialized progeny. Traditionally,

adult stem cells were believed to differentiate only

within tissue lineage boundaries (e.g. hematopoietic

stem cells giving rise to mature hematopoietic

cells). The fairly recent concept of adult stem cell

plasticity implies that stem cells can transdifferentiate

into cell types outside their original lineage.2

Along this line, it has been reported that

hematopoietic stem cells when transplanted into

infarcted mouse myocardium transdifferentiate into

cardiomyocytes and vascular cells.3 Another

hypothesis that has created some excitement

recently predicts that limited myocardial regeneration

can occur after tissue injury through the

recruitment of resident and cardiac stem cells.4

Ironically, while these new ideas have already triggered

clinical trials, fusion of transplanted stem

cells with resident cardiomyocytes has been offered

as an alternative explanation for previous claims of

transdifferentiation.5–8 Moreover, it has been proposed

that stem cells secrete cytokines and growth

factors which may promote angiogenesis, suppress

cell death of resident cardiomyocytes, modulate

interstitial matrix composition, and maybe even

recruit cardiac stem cells.9,10 Regardless of the mechanisms,

it appears that stem cell therapy has the

potential to improve perfusion and contractile performance

of the injured heart (Figure 13.1).

Potential Donor Cells

Conceptually, a variety of stem and progenitor cell

populations could be used for cardiac repair after

AMI, and many of these potential donor cells are

discussed in detail in this book. Each cell type has

its own profile of advantages, limitations, and

practicability issues. Studies comparing the regenerative

capacity of distinct cell populations are

scarce. Most investigators have therefore chosen a

pragmatic approach by using unfractionated

nucleated bone marrow cells, which contain different

stem and progenitor cell populations,

including hematopoietic stem cells, endothelial

progenitor cells, and mesenchymal stem cells.

Endothelial progenitor cells

Endothelial progenitor cells (EPCs) have been

defined by their cell surface expression of the

hematopoietic marker proteins CD133 and CD34

and the endothelial marker vascular endothelial

growth factor receptor 2, and their capacity to

incorporate into sites of neovascularization and to

differentiate into endothelial cells in situ. The cell

surface antigen CD133 is expressed on early

hematopoietic stem cells and EPCs, both of which

collaborate to promote vascularization of

ischemic tissues. There is increasing evidence that

culture-expanded EPCs contain a CD14+/CD34−/

CD133− -mononuclear cell population with “EPC

capacity”, which mediates its angiogenic effects

by releasing paracrine factors.11

Mesenchymal stem cells

Mesenchymal stem cells represent a rare population

of CD34−and CD133− cells present in bone

marrow stroma and other mesenchymal tissues.12

Mesenchymal stem cells can differentiate into

osteocytes, chondrocytes, adipocytes, and cardiomyocyte-

like cells under specific culture conditions.

When injected into infarct tissue,

mesenchymal stem cells may enhance regional

wall motion and prevent adverse remodeling. It

has been suggested that these effects may be

related to paracrine effects rather than differentiation

of mesenchymal stem cells into cardiomyocytes.

13,14 Since mesenchymal stem cells can be

expanded in vitro, and reportedly have a low

immunogenicity, they might be used in an allogeneic

setting in the future.12

Skeletal myoblasts

Skeletal myoblasts (satellite cells) are progenitor

cells which normally lie in a quiescent state

under the basal membrane of mature muscular

fibers. Myoblasts can be isolated from skeletal

muscle biopsies and expanded in vitro.

Myoblasts differentiate into myotubes and retain

skeletal muscle properties when transplanted

into an infarct scar. Although grafted myotubes

may contract in response to electrical stimulation,

they do not express intercalated disc proteins,

indicating that the majority are not

electromechanically coupled to their host cardiomyocytes.

Nevertheless, myoblast transplantation

has been shown to augment systolic and

diastolic performance in animal models, possibly

through the release of paracrine factors.15

Resident cardiac stem cells

Recently, several groups have detected stem and

progenitor cells within the heart that are capable

of differentiating into cardiomyocytes and/or vascular

lineages. It has been suggested that these

cells can be clonally expanded and used for cardiac

repair in an autologous setting.4 Clearly, independent

confirmation of these provocative

findings is required. If confirmed, cardiac resident

stem and progenitor cells hold great promise for

clinical applications, although it is conceivable

that the bone marrow contains a pluripotent stem

cell population with similar properties.16,17

Embryonic stem cells

Embryonic stem cells (ESCs) are totipotent

stem cells derived from the inner cell mass of

blastocysts. Under specific culture conditions, ESCs

differentiate into multicellular embryoid bodies

containing differentiated cells from all three

germ layers including cardiomyocytes. Human

ESC-derived cardiomyocytes display structural

and functional properties of early-stage cardiomyocytes

that couple with host cardiomyocytes

when transplanted into normal or

infarcted myocardium.18,19 In theory, infinite

numbers of cardiomyocytes could be obtained

from human ESC clones. However, unresolved

ethical and legal issues, concerns about the

tumorigenicity of residual ESCs in ESC-derived

cardiomyocyte preparations, and the need to use

allogeneic cells for transplantation currently

hamper their use in clinical studies. Eventually,

nuclear transfer techniques may provide a

means of generating an unlimited supply of histocompatible

ESCs for the treatment of cardiac

disease.

Multipotent adult germline stem cells

A recent study has highlighted the pluripotency

and plasticity of murine spermatogonial stem

cells, which are responsible for maintaining spermatogenesis

throughout life in the male. In culture,

adult spermatogonial stem cells acquire ESC

properties. These multipotent adult germline

stem cells spontaneously differentiate into derivatives

of the three embryonic germ layers in

vitro, including cardiomyocytes and vascular

cells.20 Eventually, establishment of human multipotent

adult germline stem cells from testicular

biopsies may allow individual cell-based therapy

without the ethical and immunological problems

associated with human ESCs.20

Modes of Cell Delivery in the

Setting of Acute Myocardial

Infarction

The goal of any cell delivery strategy is to transplant

sufficient numbers of cells into the

myocardial region of interest and to achieve

maximum retention of cells within that area.

Retention may be defined as the fraction of

transplanted cells retained in the myocardium

for a short period of time (hours). The localmilieu is an important determinant of cell retention,

as it will influence short-term cell survival

and, if a transvascular approach is used, cell

adhesion, transmigration through the vascular

wall, and tissue invasion. Transvascular strategies

are especially suited for the treatment of recently

infarcted and reperfused myocardium when

chemoattractants are highly expressed.21 Direct

injection techniques have been used in patients

presenting late in the disease process when an

occluded coronary artery precludes transvascular

cell delivery (e.g. patients with chronic myocardial

ischemia) or when cell homing signals are

expressed at low levels in the heart (scar tissue).

However, cell delivery by direct injection may

not be safe in patients with AMI and friable

necrotic tissue.

Selective intracoronary application delivers a

maximum concentration of cells homogeneously

to the site of injury. Unselected bone

marrow cells, circulating blood-derived progenitor

cells, and mesenchymal stem cells have been

delivered via the intracoronary route in patients

with AMI. In these studies, cells were delivered

through the central lumen of an over-the-wire

balloon catheter during transient balloon inflations

to maximize the contact time of the cells

with the microcirculation of the infarct-related

artery.22 In experimental models, intravenous

delivery of endothelial progenitor cells and mesenchymal

stem cells has been shown to improve

cardiac function after AMI. However, cell homing

to non-cardiac organs limits the applicability

of this approach. Indeed, in a recent clinical

study, homing of unselected bone marrow cells

to the infarct region was observed only after

intracoronary stop-flow delivery but not after

intravenous infusion (Figure 13.2).23 Considering

that the acutely infarcted myocardium recruits

circulating stem and progenitor cells to the site

of injury, mobilization of stem and progenitor

cells by cytokines may offer a non-invasive strategy

for cardiac regeneration. Indeed, it has been

reported that the stem cell mobilizing cytokines

stem cell factor (SCF) and granulocyte colony

stimulating factor (G-CSF) improve cardiac function

after AMI in mice.24,25 Notably, G-CSF can

accelerate infarct healing directly by enhancing

macrophage infiltration and matrix metalloproteinase

activation, and suppressing cardiomyocyteapoptosis, suggesting that stem cell-independent

mechanisms may contribute to the favorable

effects of G-CSF post-AMI.26,27

Current Status of Cell Therapy in

Patients with Acute Myocardial

Infarction

Inspired by experimental data suggesting that

functional recovery after AMI can be augmented

by stem cell transfer, clinical trials were initiated

to assess the therapeutic potential of cell therapy

in patients post-AMI. All clinical studies have

included patients who had undergone primary

angioplasty and stent implantation to reopen the

infarct-related artery and who received optimal

medical treatment during the acute phase and

follow-up, and cells were infused intracoronarily

by using the stop-flow balloon-catheter approach.

Current trials may be categorized into studies

using unselected bone marrow cells or selected

stem cell populations.

Unselected bone marrow cells

Following initial safety and feasibility studies,

four larger randomized trials of bone marrow cell

therapy after AMI have now been completed

(Table 13.1).28–31 The combined experience from

these trials indicates that intracoronary delivery

of unselected bone marrow cells is feasible and

safe in the short and mid-term (up to 18 months).

Bone marrow harvest and intracoronary cell

delivery are not associated with bleeding complications

or ischemic damage to the myocardium.

No increased rates of in-stent restenosis have

been observed. Clinical surveillance, Holter

monitoring, and data from an electrophysiological

study indicate that bone marrow cell transfer

is not associated with an increased propensity to

ventricular arrhythmias; moreover, intramyocardial

calcifications or tumor formation were not

observed after intracoronary bone marrow cell

delivery.28–32

In the BOne marrOw transfer to enhance STelevation

infarct regeneration (BOOST) trial,

intracoronary transfer of nucleated bone marrow

cells resulted in an improvement of global left

ventricular ejection fraction of 6 percentage

points after 6 months as compared to the control

group. This improvement of ejection fraction

was due mostly to improved regional wall

motion in the infarct border zone.28 For comparison,

improvements of 3–4 percentage points are

achieved by primary angioplasty and stent

implantation in AMI, suggesting that the further

improvement of ejection fraction by cell therapy

may be clinically meaningful.

The beneficial effects of intracoronary bone

marrow cell transfer were confirmed in the

recent Reinfusion of Enriched Progenitor cells

And Infarct Remodeling in Acute Myocardial

Infarction (REPAIR-AMI) trial. In this study,

mononucleated bone marrow cell transfer promoted

an increase in left ventricular ejection

fraction of 2.5 percentage points after 4 months

as compared to a control group that also underwent

bone marrow aspiration but received an

intracoronary infusion of a placebo.29

In contrast to BOOST and REPAIR-AMI, two

other randomized studies did not report significant

improvements of left ventricular ejection

fraction after intracoronary cell transfer.30,31

While the exact reasons for the differing results

remain elusive, it is worthwhile to take a closer

look at the design of these studies (Table 13.1). In

one of the negative studies, cells were delivered

already 24 hours after coronary reperfusion.30 In

BOOST and REPAIR-AMI, cells were transplanted

several days later. Subgroup analyses in REPAIRAMI

indicate that the timing of cell delivery may

be important, and that the beneficial effects on

ejection fraction are lost when the cells are delivered

too early.29 The Autologous Stem cell

Transplantation in Acute Myocardial Infarction

(ASTAMI) trial also did not find a significant

effect of bone marrow cell transfer on left ventricular

ejection fraction recovery.31 While the

cells were delivered late after reperfusion in

ASTAMI (after 4–6 days), a particular cell preparation

method that leads to an enrichment of

lymphocytic bone marrow cells was employed. It

is not clear whether this method actually recovers

the bone marrow cell populations that are

required to achieve functional improvements

after AMI. Together, these data remind us thatprocedural issues, such as the cell preparation

method and timing of cell transfer, need to be

further refined.

It should be noted that none of the trials so far

has revealed a significant effect of bone marrow

cell transfer on left ventricular end-diastolic

volumes, an index of left ventricular remodeling.

However, larger studies may be required to settle

this issue. Moreover, few data are available

regarding the long-term effects of bone marrow

cell transfer after AMI. Follow-up data from the

BOOST trial indicate that the improvements of

left ventricular ejection fraction are maintained

18 months after cell transfer;32 however, ejection

fraction also increased somewhat in the control

group during long-term follow-up,32 which

would be expected in AMI patients on chronic

angiotensin converting enzyme inhibitor and

β-blocker therapy. Tissue Doppler echocardiography

analyses in BOOST indicate that bone marrow

cell transfer may prevent the development

of diastolic dysfunction during long-term followup.

Significant effects of cell transfer on E/A ratio

(peak early diastolic/late diastolic velocities) and

tissue Doppler Ea/Aa (annular velocities) ratio,

but not on isovolumic relaxation time, suggest

that cell therapy positively affects left ventricular

stiffness but not active relaxation.33

Selected bone marrow cell populations

The Transplantation Of Progenitor Cells And

Regeneration Enhancement in Acute Myocardial

Infarction (TOPCARE-AMI) trial compared

mononucleated bone marrow cells with circulating

blood-derived progenitor cells (mostly EPCs)

in post-AMI patients a few days after reperfusion.

Both cell types appeared to have similar safety

and efficacy profiles.34 However, since TOPCAREAMI

was not randomized, firm conclusions

regarding the efficacy of endothelial progenitor

cells post-AMI cannot be drawn at the present

time. In the recent randomized Transplantation Of

Progenitor Cells And Regeneration Enhancement

in Chronic Heart Failure (TOPCARE-CHF) trial,

patients received an intracoronary infusion of

unselected mononucleated bone marrow cells or

circulating blood-derived progenitor cells months

or years after an AMI. Notably, a significant

improvement in left ventricular ejection fraction

was observed only in patients receiving mononucleated

bone marrow cells in this setting.35 The

effects of culture-expanded mesenchymal stem

cells after AMI have been investigated in one randomized

clinical trial.36 While no serious sideeffects

were reported, it is not known whether

intracoronary mesenchymal stem cell delivery

promoted ischemic damage to the myocardium,36

a complication that has occurred after intracoronary

mesenchymal stem cell infusions in dogs.37

Six months after cell transfer, regional wall

motion and global left ventricular ejection fraction

were improved and left ventricular enddiastolic

volumes were decreased.36 These striking

effects need to be confirmed by additional studies.

In another trial, CD133+ enriched bone

marrow cells were infused into the infarct-related

artery in post-AMI patients. Higher luminal losses

within the stented segment and the distal,

non-stented segments of the infarct-related artery

were observed, raising the concern that CD133+

cells may promote in-stent restenosis and atherosclerosis

progression.38 As these data were

obtained from retrospective analysis and lacked

randomized controls, future studies need to

define the risk and mechanisms of such adverse

effects on the epicardial coronary circulation

(which were not observed in any of the trials

using unfractionated nucleated bone marrow

cells).38

Stem and progenitor cell mobilization

The Front-Integrated Revascularization and Stem

Cell Liberation in Evolving Acute Myocardial

Infarction by Use of Granulocyte-Colony-

Stimulating Factor (FIRSTLINE-AMI) trial randomized

50 patients with AMI to a control group

or a 6-day open-label course of G-CSF that was

initiated within 1–2 hours after primary angioplasty

and stenting.39 G-CSF therapy after stent

implantation was not associated with an

enhanced rate of in-stent restenosis or other serious

adverse events, and promoted significant

improvements in left ventricular ejection fraction

and metabolic activity in the infarct territory.

39 Critics have pointed out that the

beneficial effects of G-CSF in FIRSTLINE-AMIwere magnified by an unexpected decrease in

ejection fraction in the control group.40 The

favorable safety profile of G-CSF post-AMI was

confirmed in two other recent trials which, however,

did not observe a beneficial effect on left

ventricular ejection fraction.41,42 It remains to

be seen whether differences in study design, e.g.

later start of G-CSF injections in the negative

trials, account for the discrepant results.

The Foreseeable Future of Cell

Therapy in Patients with Acute

Myocardial Infarction

We advocate to no longer perform studies

involving small numbers of patients, but rather

to conduct larger, double-blind, randomized

controlled clinical trials to firmly establish the

effects of cell therapy on surrogate markers, such

as left ventricular ejection fraction and remodeling,

myocardial perfusion, or exercise capacity.43

Most important, upcoming trials should address

procedural issues such as the optimal cell type,

cell dosage, and timing of cell transfer. While

these trials may also look at combined morbidity

and mortality endpoints, they may be too small

to be conclusive in this regard. Some of the

ongoing trials of intracoronary bone marrow cell

transfer after AMI are summarized in Table 13.2.

Ultimately, outcome trials and cost–benefit

analyses will be required.

Notably, the absolute number of transplanted

bone marrow cells did not correlate with subsequent

improvements in ejection fraction in previous

post-AMI studies. This may be because the

cell numbers infused were within a narrow

range, or because differences in the functional

capacity of the cells, such as the ability to home

to and engraft in the infarcted area, to undergo

transdifferentiation, and/or to produce paracrine

factors, may override differences in cell numbers.

Intriguingly, cell labeling studies indicate that

fewer than 5% of unselected nucleated bone

marrow cells are retained in the infarcted area

after intracoronary delivery in patients (Figure

13.2).23 Although this rate of cell retention was

sufficient to improve left ventricular systolic and

diastolic function in the BOOST trial, it is

conceivable that pharmacological strategies

might be used to enhance the homing capacity

or other functional parameters of the cells.

Experimental studies are already pointing in this

direction.44 Post-hoc analyses of the BOOST trial

database suggest that the effects of bone marrow

cell transfer are consistent across several subgroups

defined according to sex, age, infarct territory,

and time from symptom onset to

reperfusion.28 However, patient subgroups that

derive the greatest benefit from cell transfer need

to be identified prospectively in future trials (e.g.

patients presenting late after symptom onset in

whom little myocardial salvage can be expected

from reperfusion therapy). In this regard, data

from REPAIR-AMI indicate that the effects of

bone marrow cell transfer may be more pronounced

in patients with more severely

depressed baseline left ventricular ejection fraction.

29 Cytokines with stem-cell mobilizing

and/or direct cardioprotective properties

should be further evaluated as stand alone therapy

or in combination with cell transfer after

AMI. Eventually, cytokines may emerge as a

non-invasive alternative or as an adjunct to cell

therapy.

Meanwhile, fundamental questions need to be

addressed experimentally. What is the fate of the

injected cells after transplantation? Genetic and

transgenic markers should be employed to determine

lineage commitment of engrafted cells.

Cell labeling and imaging techniques need to be

developed to track stem cell fate in patients and

correlate cell retention and engraftment with

functional outcomes. Can the regenerative

capacity of transplanted stem cells be enhanced

by drugs, cytokines, or gene therapy approaches?

Pharmacological and genetic strategies may help

to enhance stem cell retention, engraftment, differentiation,

and paracrine capability.45,46

For the time being, cardiac repair remains the

holy grail of cell therapy. While unselected bone

marrow cells may have a favorable impact on

systolic function, they probably do not make

new myocardium. This should stimulate further

basic research into the prospects of cell types

with transdifferentiation capacity, such as ESCs,multipotent adult germline stem cells, and,

possibly, multipotent bone marrow-derived stem

cells and resident cardiac stem cells.

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Table13.2 Ongoing randomized cell therapy trials in patients with acute myocardial infarction

Primary Principal

Study Country Design n Groups endpoints investigators

REGENT Poland Randomized, 200 Mononucleated Global LVEF, Michal Tendera

multicenter BMCs vs LV volumes

CD34+/CXCR4+ cells (by MRI)

HEBE The Netherlands Randomized, 200 Mononucleated Regional Alexander Hirsch

multicenter BMCs vs peripheral function in Jan J Piek

mononucleated dysfunctional

cells vs standard segments

therapy (by MRI)

BOOST-2 Germany, Randomized, 200 Placebo vs Global LVEF Kai C Wollert

Norway, multicenter, nucleated BMCs (by MRI) Gerd P Meyer

Bulgaria placebo- (low-dose vs Helmut Drexler

controlled, high-dose,

factorial pretreated vs

design not pretreated)

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