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Posts Tagged ‘Adult stem cell’


Optical Neurons

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Umbilical Cells Help Eye’s Neurons Connect

Factor released by cells helps connections, not longevity

“By learning more about how these cells work, we are one step closer to understanding the disease states in which these cells should be studied,” said Cagla Eroglu, an assistant professor of cell biology and neurobiology at the Duke University Medical Center, who led the research.
Umbilical cord tissue-derived cells (hUTC) differ from umbilical cord blood cells in that they are isolated from cord tissue itself, rather than the blood. The Duke
team used an established cell culture system to determine whether and how the hUTCs might affect the growth of neurons isolated from the retinas of rat eyes.
In an experimental setup that allowed the two types of cells to bathe in the same fluid without coming into physical contact, retinal neurons in a bath with hUTCs formed new connections between neurons called synapses, and they sprouted new ‘neurites’ — tiny branches that lead to additional connections.
These cells also survived longer than rat neurons placed in a bath lacking the umbilical cord tissue-derived cells.
Something present in the fluid surrounding the neurons in the bath with the hUTCs was apparently affecting the neurons. Through a series of experiments, the researchers determined that relatively large molecules, thrombospondin 1, 2 and 4, were primarily responsible for the effect.
Blocking thrombospondins was found to reduce new connections among neurons. By genetically inhibiting the individual members of the thrombospondin family, the researchers found that TSP1, TSP2, and TSP4 in particular were required to create both neurites and new connections.
“It’s exciting that thrombospondins had a really strong effect on neurite outgrowth,” said Eroglu, who is also a member of the Duke Institute for Brain Sciences (DIBS). She added that making neurites and forming new connections between them are crucial for helping neurons grow when faced with injury and neurodegenerative diseases.
However, blocking TSP1, 2 and 4 did not affect neuron survival, suggesting that there is some other factor in the UTC cells that promotes cell longevity. Her group is now searching for those molecules.
Eroglu’s earlier work has shown that thrombospondins are released by brain cells called astrocytes and boost new synapse formation between neurons in the brain.
Eroglu said there may be deficiencies in thrombospondin signaling in neurodegenerative disease, and the group is actively pursuing this hypothesis in animal studies.
Postdoctoral fellow Sehwon Koh is the lead author of this study and a member of the Eroglu lab. Other authors include Namsoo Kim and Henry H. Yin from Duke’s department of psychology and neuroscience. This research was supported by a research agreement with Janssen Research & Development, LLC.
CITATION: “Human Umbilical Tissue-Derived Cells (hUTC) Promote Synapse Formation and Neurite Outgrowth via Thrombospondin Family Proteins,” Sehwon Koh, Namsoo Kim, Henry H. Yin, Ian R. Harris, Nadine S. Dejneka, and Cagla Eroglu. Journal of Neuroscience, November 25, 2015.    http://dx.doi.org:/10.1523/JNEUROSCI.1364-15.2015
ScienceDaily
Cells isolated from the human umbilical cord have been shown to produce molecules that help retinal neurons from the eyes of rats grow, connect and survive. The findings implicate one family of molecules in particular — thrombospondins – that may have therapeutic potential for the treatment of degenerative eye diseases.

The findings, which appear Nov. 25 in the Journal of Neuroscience, implicate one family of molecules in particular — thrombospondins — that may have therapeutic potential for the treatment of degenerative eye diseases.

“By learning more about how these cells work, we are one step closer to understanding the disease states in which these cells should be studied,” said Cagla Eroglu, an assistant professor of cell biology and neurobiology at the Duke University Medical Center, who led the research.

Umbilical cord tissue-derived cells (hUTC) differ from umbilical cord blood cells in that they are isolated from cord tissue itself, rather than the blood. The Duke team used an established cell culture system to determine whether and how the hUTCs might affect the growth of neurons isolated from the retinas of rat eyes.

Something present in the fluid surrounding the neurons in the bath with the hUTCs was apparently affecting the neurons. Through a series of experiments, the researchers determined that relatively large molecules, thrombospondin 1, 2 and 4, were primarily responsible for the effect.

Blocking thrombospondins was found to reduce new connections among neurons. By genetically inhibiting the individual members of the thrombospondin family, the researchers found that TSP1, TSP2, and TSP4 in particular were required to create both neurites and new connections.

However, blocking TSP1, 2 and 4 did not affect neuron survival, suggesting that there is some other factor in the UTC cells that promotes cell longevity. Her group is now searching for those molecules.

Golgi Cells Have Active Dendrites
Stephanie Rudolph, Court Hull, and Wade G. Regehr
The Journal of Neuroscience, Nov 25, 2015 • 35(47):i • i    (see pages 15492–15504)
The cerebellum coordinates multijoint movements and contributes to motor learning. These functions require precise spike timing in Purkinje cells, the cerebellar output neurons. Purkinje cell spiking is driven partly by granule cells, which receive information about ongoing movements from mossy fibers, and the timing and spatial extent of granule cell output is determined largely by inhibitory input from spontaneously active interneurons called Golgi cells.
Golgi cell spiking is modulated by excitatory input from both mossy fibers and granule cells. How these inputs are integrated in Golgi cell dendrites remains poorly understood. Finding no evidence for active conductances in Golgi cell dendrites, Vervaeke et al. (2012, Science 30: 1624) hypothesized that dendritic gap junctions enable granule cell inputs to influence Golgi cell activity. Although gap junctions likely do contribute to dendritic processing in Golgi cells, Rudolph et al. now show that Golgi cell dendrites also express voltage-gated channels.
If dendrites lacked active conductances, one would expect signals to decay with distance from the soma. But calcium imaging in rat cerebellar slices revealed that action potentials caused uniform calcium elevation throughout Golgi cell dendrites. Moreover, applying a voltage-gated sodium channel (VGSC) blocker selectively to dendrites reduced spike-associated calcium elevation in distal dendrites. In addition, blocking T- and R-type voltage-gated calcium channels (VGCCs) attenuated calcium elevation selectivelyin distal dendrites,while blocking N-type channels reduced calcium elevation only in proximal dendrites.
Blocking voltage-gated channels also had functional consequences. Blocking N-type channels decreased the amplitude of the spike afterhyperpolarization and increased the spike rate of Golgi cells. In contrast, T-type channel blockers had little effect on baseline firing frequency. Nonetheless, blocking T-type channels attenuated rebound spiking after hyperpolarization and reduced the amplitude of EPSPs evoked by stimulation of granule cell axons.
These experiments suggest that VGSCs help depolarize distal dendrites to enhance activation of T-type VGCCs, which in turn amplify responses to granule cells and promote rebound bursting. Meanwhile, N-type VGCCs located near the soma appear to be tightly coupled to calcium-activated potassium channels, which regulate the spontaneous spike rate of Golgi cells. Thus, Golgi cell dendrites have multiple types of voltage-sensitive channels that are differently distributed and serve distinct roles in ensuring the precise timing of cerebellar output.
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Blocking Differentiation to Produce Stem Cells

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Blocking Differentiation is Enough to Turn Mature Cells into Stem Cells

 

 

ID3 inhibitor of DNA binding 3, dominant negative helix-loop-helix protein [ Homo sapiens (human) ]

Gene ID: 3399, updated on 15-Nov-2015

http://www.ncbi.nlm.nih.gov/gene?Db=gene

 

Official Symbol ID3 provided by HGNC 

Official Full Name inhibitor of DNA binding 3, dominant negative helix-loop-helix protein provided by HGNC

Primary source HGNC:HGNC:5362 See related Ensembl:ENSG00000117318; HPRD:02608; MIM:600277; Vega:OTTHUMG00000003229

Gene type protein coding

RefSeq status REVIEWED

OrganismHomo sapiens

LineageEukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Primates; Haplorrhini; Catarrhini; Hominidae; Homo

Also known as HEIR-1; bHLHb25

Summary The protein encoded by this gene is a helix-loop-helix (HLH) protein that can form heterodimers with other HLH proteins. However, the encoded protein lacks a basic DNA-binding domain and therefore inhibits the DNA binding of any HLH protein with which it interacts. [provided by RefSeq, Aug 2011]

Orthologs mouse all

 

Location:
1p36.13-p36.12
Exon count:
3
Annotation release Status Assembly Chr Location
107 current GRCh38.p2 (GCF_000001405.28) 1 NC_000001.11 (23557930..23559794, complement)
105 previous assembly GRCh37.p13 (GCF_000001405.25) 1 NC_000001.10 (23884421..23886285, complement)

Chromosome 1 – NC_000001.11Genomic Context describing neighboring genes

Related articles in PubMed

 

Induced Developmental Arrest of Early Hematopoietic Progenitors Leads to the Generation of Leukocyte Stem Cells

Tomokatsu Ikawa, Kyoko Masuda, Mirelle J.A.J. Huijskens, Rumi Satoh, Kiyokazu Kakugawa, Yasutoshi Agata, Tomohiro Miyai, Wilfred T.V. Germeraad, Yoshimoto Katsura, Hiroshi Kawamoto
Stem Cell Reports Nov 10, 2015; Volume 5, Issue 5, 716–727.   DOI: http://dx.doi.org/10.1016/j.stemcr.2015.09.012
Highlights
  • Overexpression of ID3 endows hematopoietic progenitors with self-renewal activity
  • A simple block of cell differentiation is sufficient to induce stem cells
  • Induced leukocyte stem (iLS) cells exhibit robust multi-lineage reconstitution
  • Equivalent progenitors were produced from human cord blood hematopoietic stem cells

Self-renewal potential and multipotency are hallmarks of a stem cell. It is generally accepted that acquisition of such stemness requires rejuvenation of somatic cells through reprogramming of their genetic and epigenetic status. We show here that a simple block of cell differentiation is sufficient to induce and maintain stem cells. By overexpression of the transcriptional inhibitor ID3 in murine hematopoietic progenitor cells and cultivation under B cell induction conditions, the cells undergo developmental arrest and enter a self-renewal cycle. These cells can be maintained in vitro almost indefinitely, and the long-term cultured cells exhibit robust multi-lineage reconstitution when transferred into irradiated mice. These cells can be cloned and re-expanded with 50% plating efficiency, indicating that virtually all cells are self-renewing. Equivalent progenitors were produced from human cord blood stem cells, and these will ultimately be useful as a source of cells for immune cell therapy.

Figure thumbnail fx1

http://www.cell.com/cms/attachment/2040173852/2053709392/fx1.jpg

 

Somatic tissues with high turnover rates, such as skin, intestinal epithelium, and hematopoietic cells, are maintained by the activity of self-renewing stem cells, which are present in only limited numbers in each organ (Barker et al., 2012,Copley et al., 2012, Fuchs and Chen, 2013). For example, the frequency of hematopoietic stem cells (HSCs) in the mouse is about 1 in 105 of total bone marrow (BM) cells (Spangrude et al., 1988). Once HSCs begin the differentiation process, their progeny cells have hardly any self-renewal capacity, indicating that self-renewal is a special feature endowed only to stem cells.

Cells such as embryonic stem (ES) cells that retain self-renewal potential and multipotency only in vitro can also be included in the category of stem cells. Such stemness of ES cells is thought to be maintained by formation of a core transcriptional network and an epigenetic status unique to ES cells (Lund et al., 2012, Meissner, 2010, Ng and Surani, 2011). A stem cell equivalent to ES cells, called induced pluripotent stem (iPS) cells, can be produced from somatic cells by overexpression of only a few specific transcription factors (OCT3/4, SOX2, KLF4, and C-MYC), which are thought to be the essential components in forming the core network of transcriptional factors that define the status of ES cells (Takahashi et al., 2007, Takahashi and Yamanaka, 2006, Yamanaka, 2012). It is thus generally conceived that acquisition of such a network for a somatic cell depends on the reprogramming of the epigenetic status of that cell.

On the other hand, it could be envisioned that the self-renewing status of cells represents a state in which their further differentiation is inhibited. It is known, for example, that to maintain ES/iPS cells, factors such as leukemia inhibitory factor and basic fibroblast growth factor, for mouse and human cultures, respectively (Williams et al., 1988, Xu et al., 2005), are required, and these factors are thought to block further differentiation of the cells. In this context, it has previously been shown that systemic disruption of transcription factors essential for the B cell lineage, such as PAX5, E2A, and EBF1, leads to the emergence of self-renewing multipotent hematopoietic progenitors, which can be maintained under specific culture conditions (Ikawa et al., 2004a, Nutt et al., 1999, Pongubala et al., 2008). It has recently been shown that the suppression of lymphoid lineage priming promotes the expansion of both mouse and human hematopoietic progenitors (Mercer et al., 2011, van Galen et al., 2014). Therefore, it would seem theoretically possible to make a stem cell by inducing inactivation of these factors at particular developmental stages. Conditional depletion of PAX5 in B cell lineage committed progenitors, as well as mature B cells, resulted in the generation of T cells from the B lineage cells (Cobaleda et al., 2007, Nutt et al., 1999, Rolink et al., 1999). These studies, however, were mainly focused on the occurrence of cell-fate conversion by de-differentiation of target cells. Therefore, the minimal requirement for the acquisition of self-renewal potential remains undetermined.

Our ultimate goal is to obtain sufficient number of stem cells by expansion to overcome the limitation of cell numbers for immune therapies. We hypothesize that stem cells can be produced by simply blocking differentiation. As mentioned earlier, self-renewing multipotent progenitors (MPPs) can be produced by culturing E2A-deficient hematopoietic progenitors in B cell-inducing conditions (Ikawa et al., 2004a). Because it remains unclear at which developmental stage the acquisition of self-renewing potential has occurred in the case of such a systemic deletion, we thought to develop a method in which E2A function could be inactivated and reactivated in an inducible manner. We decided to use the ID3 protein for this purpose, because it is known that ID proteins serve as dominant-negative inhibitors of E proteins (Norton et al., 1998, Sayegh et al., 2003). Here we show that the overexpression of ID3 into HSCs or hematopoietic progenitor cells (HPCs) in both mouse and human induces the stemness of the progenitors and that the cells acquire the self-renewal activity. The ID3-expressing cells can be maintained in vitro as MPPs with T, B, and myeloid lineage potentials.

 

Results

Jump to Section
Introduction
Results
  Generation of ID3-Transduced Hematopoietic Progenitors
  IdHP Cells Are Multipotent, Maintaining T, B, and Myeloid Lineage Potentials
  IdHP Cells Are Multipotent at a Clonal Level
  Generation of IdHP Cells from Mouse BM
  Generation of Inducible IdHP Cells Using ID3-ER Retrovirus
  Generation of IdHP Cells from Human Cord Blood Hematopoietic Progenitors
Discussion
Experimental Procedures
  Mice
  Antibodies
  Growth Factors
  Isolation of Hematopoietic Progenitors
  Retroviral Constructs, Viral Supernatants, and Transduction
  In Vitro Differentiation Culture System
  Cloning of mIdHP Cells
  Colony-Forming Unit in Culture Assay
  Cell Cycle Assay
  Adoptive Transfer of mIdHP and hIdHP Cells
  PCR Analysis of Igh Gene Rearrangement
  RNA Extraction and qRT-PCR
  Microarray Analysis
Author Contributions
Supplemental Information
References

Generation of ID3-Transduced Hematopoietic Progenitors

IdHP Cells Are Multipotent, Maintaining T, B, and Myeloid Lineage Potentials

IdHP Cells Are Multipotent at a Clonal Level

Generation of IdHP Cells from Mouse BM

Generation of Inducible IdHP Cells Using ID3-ER Retrovirus

Generation of IdHP Cells from Human Cord Blood Hematopoietic Progenitors

Thumbnail image of Figure 1. Opens large image

http://www.cell.com/cms/attachment/2040173852/2053709390/gr1.jpg

 

Identification of cellular and molecular events regulating self-renewal or differentiation of the cells is a fundamental issue in the stem cell biology or developmental biology field. In the present study, we revealed that the simple inhibition of differentiation in HSCs or HPCs by overexpressing ID proteins and culturing them in suitable conditions induced the self-renewal of hematopoietic progenitors and allowed the extensive expansion of the multipotent cells. The reduction of ID proteins in MPPs resulted in the differentiation of the cells into lymphoid and myeloid lineage cells. Thus, it is possible to manipulate the cell fate by regulating E-protein or ID-protein activities. This inducible system will be a useful tool to figure out the genetic and epigenetic program controlling the self-renewal activity of multipotent stem cells.

Previous studies have shown that hematopoietic progenitors deficient for E2A, EBF1, and PAX5 maintain multilineage differentiation potential without losing their self-renewing capacity (Ikawa et al., 2004a, Nutt et al., 1999, Pongubala et al., 2008), indicating that the inhibition of the differentiation pathway at certain developmental stages leads to the expansion of multipotent stem cells. However, the MPPs were not able to differentiate into B cells because they lacked the activities of transcription factors essential for the initiation of the B lineage program. In addition, a restriction point regulating the lineage-specific patterns of gene expression during B cell specification remained to be determined because of the lack of an inducible system that regulates B cell differentiation. Here we have established the multipotent iLS cells using ID3-ER retrovirus, which can be maintained and differentiated into B cells in an inducible manner by simply adding or withdrawing 4-OHT. The data indicated the essential role of E2A for initiation of the B cell program that restricts other lineage potentials, because the depletion of 4-OHT from the culture immediately leads to the activation of E proteins, such as E2A, HEB, and E2-2, that promote B cell differentiation. This strategy is useful in understanding the cues regulating the self-renewal or differentiation of uncommitted progenitors to the B cell pathway. Analysis of genome-wide gene expression patterns and histone modifications will determine the exact mechanisms that underlie the B cell commitment process.

The iLS cells can also be generated from human CB hematopoietic progenitors. Human iLS cells exhibited differentiation potential and self-renewal activity similar to those of murine iLS cells, suggesting a similar developmental program during human B cell fate specification. Our data are consistent with a study demonstrating the critical role of the activity of ID and E proteins for controlling the status of human HSCs and progenitors (van Galen et al., 2014). This study reported that the overexpression of ID2 in human CB HSCs enhanced the myeloid and stem cell program at the expense of lymphoid commitment. Specifically, ID2 overexpression resulted in a 10-fold expansion of HSCs, suggesting that the inhibition of E-protein activities promotes the self-renewal of HSCs by antagonizing the differentiation. This raises a question about the functional differences between ID2 and ID3. Id3 seems to suppress the B cell program and promote the myeloid program more efficiently than does ID2, because the ID2-expressing HPCs appear to retain more B cell potential than ID3-expressing iLS cells (Mercer et al., 2011, van Galen et al., 2014). The self-renewal activity and differentiation potential of ID2-HPCs derived from murine HSCs in the BM seemed to be limited both in vivo and in vitro analysis (Mercer et al., 2011). In our study, the iLS cells retained more myeloid potential and self-renewal capacity during the culture. Strikingly, the multipotent iLS cells enormously proliferated (>103-fold in 1 month) as long as the cells were cultured in undifferentiated conditions. This could be due to the functional differences among Id family genes. Alternatively, combination with additional environmental signals, such as cytokines or chemokines, may affect the functional differences of ID proteins, although any ID proteins can repress the activation of the E2A targets, such as Ebf1 and Foxo1, that are essential for B cell differentiation. ID1 and ID3, but not ID2, are demonstrated to be negative regulators of the generation of hematopoietic progenitors from human pluripotent stem cells (Hong et al., 2011). Further analysis is required to determine the physiological role of ID proteins in regulating hematopoietic cell fate. It also remains to be determined whether the ID3-ER system can be applied to human progenitors. It would be informative to compare the regulatory networks that control B cell differentiation in mouse and human.

Immune cell therapy has become a major field of interest in the last two decades. However, the required high cell numbers restrain the application and success of immune reconstitution or anti-cancer treatment. For example, DCs are already being used in cell therapy against tumors. One of the major limitations of DC vaccine therapy is the difficulty in obtaining sufficient cell numbers, because DCs do not proliferate in the currently used systems. The method of making iLS cells could be applied to such cell therapies. Taken together, the simplicity of this method and the high expansion rate and retention of multilineage potential of the cells make this cell source appealing for regenerative medicine or immune cell therapy.

In summary, we showed that an artificially induced block of differentiation in uncommitted progenitors is sufficient to produce multipotent stem cells that retain self-renewal activity. Once the differentiation block is released, the cells start differentiating into mature cells both in vivo and in vitro. Thus, this method could be applicable for establishing somatic stem cells from other organs in a similar manner, which would be quite useful for regenerative medicine. The relative ease of making stem cells leads us to conceive that a block in differentiation is essential not only in other types of artificially engineered stem cells, such as ES cells and iPS cells, but also in any type of physiological somatic stem cell. In this context, it is tempting to speculate that it could have been easy for a multicellular organism to establish somatic stem cells by this mechanism during evolution.

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Stem Cell Therapy for Coronary Artery Disease (CAD)

Author and Curator: Larry H. Bernstein, MD, FCAP

and

Curator: Aviva Lev-Ari, PhD, RN

 

There is great interest and future promise for stem cell therapy in ischemic heart disease.  This is another report for the active work in cardiology with stem cell therapy by MA Gaballa and associates at University of Arizona.

Stem Cell Therapy for Coronary Heart Disease

Julia N. E. Sunkomat and Mohamed A. Gaballa

The University ofArizona Sarver Heart Center, Section of Cardiology, Tucson, Ar
Cardiovascular Drug Reviews 2003: 21(4): 327–342

Keywords: Angiogenesis — Cardiac therapy — Coronary heart disease — Heart failure — Myoblasts — Myocardial ischemia — Myocardial regenera­tion — Stem cells

ABSTRACT

Coronary artery disease (CAD) remains the leading cause of death in the Western world. The high impact of its main sequelae, acute myocardial infarction and congestive heart failure (CHF), on the quality of life of patients and the cost of health care drives the search for new therapies. The recent finding that

stem cells contribute to neovascularization and possibly improve cardiac function after myocardial infarction makes stem cell therapy the most highly active research area in cardiology. Although the concept of stem cell therapy may revolutionize heart failure treatment, several obstacles need to be ad­dressed. To name a few:

  1.  Which patient population should be considered for stem cell therapy?
  2.  What type of stem cell should be used?
  3.  What is the best route for cell de­livery?
  4.  What is the optimum number of cells that should be used to achieve functional effects?
  5.  Is stem cell therapy safer and more effective than conventional therapies?

The published studies vary significantly in design, making it difficult to draw conclusions on the efficacy of this treatment. For example, different models of

  1. ischemia,
  2. species of donors and recipients,
  3. techniques of cell delivery,
  4. cell types,
  5. cell numbers and
  6. timing of the experiments

have been used. However, these studies highlight the landmark concept that stem cell therapy may play a major role in treating cardiovascular diseases in the near future. It should be noted that stem cell therapy is not limited to the treatment of ischemic cardiac disease.

  • Non-ischemic cardiomyopathy,
  • peripheral vascular disease, and
  • aging may be treated by stem cells.

Stem cells could be used as vehicle for gene therapy and eliminate the use of viral vectors. Finally, stem cell therapy may be combined with phar­macological, surgical, and interventional therapy to improve outcome. Here we attempt a systematic overview of the science of stem cells and their effects when transplanted into ischemic myocardium.

INTRODUCTION

Background

Congestive heart failure (CHF) is the leading discharge diagnosis in patients over the age of 65 with estimates of $24 billion spent on health care in the US (1,11). The number one cause of CHF is coronary artery diseases (CAD). Coronary care units, reperfusion therapy (lytic and percutaneous coronary intervention) and medical therapy with anti-pla­telet agents, statins, ACE-inhibitors and â-adrenoceptor antagonists all significantly reduce morbidity and mortality of CAD and CHF (9), but it is very difficult to regenerate new viable myocardium and new blood vessels.

Identification of circulating endothelial progenitor cells in peripheral blood that incor­porated into foci of neovascularization in hindlimb ischemia (4) and the successful engraftment of embryonic stem cells into myocardium of adult dystrophic mice (31) intro­duced a new therapeutic strategy to the field of cardiovascular diseases: tissue regeneration. This approach is supported by the discovery of primitive cells of extracardiac origin in cardiac tissues after sex-mismatched transplants suggesting that an endogenous repair mechanism may exist in the heart (35,45,54). The number of recruited cells varied significantly from 0 (19) to 18% (54), but the natural course of ischemic cardiomyopathy implies that cell recruitment for tissue repair in most cases is insufficient to prevent heart failure. Therefore, investigational efforts are geared towards

  • augmenting the number of multipotent stem cells and endothelial and myocardial progenitor cells at the site of ischemia to induce clinically significant angiogenesis and potentially myogenesis.

Stem and Progenitor Cells

Stem cells are defined by their ability to give rise to identical stem cells and progenitor cells that continue to differentiate into a specific tissue cell phenotype (23,33). The po­tential of mammalian stem cells varies with stage of development and age (Table 1).

In mammals, the fertilized oocyte and blastomere cells of embryos of the two to eight cell stage can generate a complete organism when implanted into the uterus; they are called totipotent stem cells. After the blastocyst stage, embryonic stem cells retain the ability to differentiate into all cell types, but

  • cannot generate a complete organism and thus are denoted pluripotent stem cells.

Other examples of pluripotent stem cells are embryo­nic germ cells that are derived from the gonadal ridge of aborted embryos and embryonic carcinoma cells that are found in gonadal tumors (teratocarcinomas) (23,33). Both these cell types can also differentiate into cells of all three germ layers, but are not as well inves­tigated as embryonic stem cells.

It is well established that embryonic stem cells can differentiate into cardiomyocytes (7,10,13,14,31,37,76), endothelial cells (55), and smooth muscle cells (5,22,78) in vitro, but it is unclear whether

  • pure populations of embryonic stem cell-derived cardiomyocytes can integrate and function appropriately in the heart after transplantation.
  • one study reported arrhythmogenic potential of embryonic stem cell-derived cardiomyocytes in vitro (80).

Adult somatic stem cells are cells that have already committed to one of the three germ layers: endoderm, ectoderm, or mesoderm (76). While embryonic stem cells are defined by their origin (the inner cell mass of the blastocyst), the origin of adult stem cells in mature tissues is still unknown. The primary role of adult stem cells in a living organism is thought to be maintaining and repairing the tissue in which they reside. They are the source of more identical stem cells and cells with a progressively more distinct phenotype of specialized tissue cells (progenitor and precursor cells) (Fig. 1). Until recently adult stem cells were thought to be lineage-specific, meaning that they can only differentiate into the cell-type of their original tissue. This concept has now been challenged with the discovery of multipotent stem and progenitor cells (26, 50, 51).

The presence of multipotent stem and progenitor cells in adult mammals has vast im­plications on the availability of stem cells to research and clinical medicine. Recent publi­cations, however, have questioned whether the adaptation of a phenotype in those dogma-challenging studies is really a result of trans-differentiation or rather a result of cell and nuclear fusion (60,68,75,79). Spontaneous fusion between mammalian cells was first re­ported in 1961 (8), but how frequently fusion occurs and whether it occurs in vivo is not clear.

The bone marrow is a known source of stem cells. Hematopoietic stem cells are fre­quently used in the field of hematology. Surface receptors are used to differentiate hematopoietic stem and progenitor cells from mature cells. For example, virtually all

  • hematopoietic stem and progenitor cells express the CD34+ glycoprotein antigen on their cell membrane (73),

though a small proportion of primitive cells have been shown to be CD34 negative (58).

The function of the CD34+ receptor is not yet fully understood. It has been suggested that it may act as a regulator of hematopoietic cell adhesion in the bone marrow microenvironment. It also appears to be involved in the maintenance of the hematopoietic stem/progenitor cell phenotype and function (16,21). The frequency of immature CD34+ cells in peripheral circulation diminishes with age.

  • It is the highest (up to 11%) in utero (69) and decreases to 1% of nucleated cells in term cord blood (63).
  • This equals the per­centage of CD34+ cells in adult bone marrow.
  • The number of circulating stem cells in adult peripheral blood is even lower at 0.1% of nucleated cells.

Since hematopoietic stem cells have been identified as endothelial progenitor cells (29,30,32) their low density in adult bone marrow and blood could explain the inadequacy of endogenous recruitment of cells to injured organs such as an ischemic heart. The bone marrow is also home to another stem cell population the so-called mesenchymal stem cells. These may constitute a subset of the bone marrow stromal cells (2,43). Bone marrow stromal cells are a mixed cell popu­lation that generates

  1. bone,
  2. cartilage,
  3. fat,
  4. connective tissue, and
  5. reticular network that sup­ports cell formation (23).

Mesenchymal stem cells have been described as multipotent (51,52) and as a source of myocardial progenitor cells (41,59). They are, however, much less defined than the hematopoietic stem cells and a characteristic antigen constellation has not yet been identified (44).

Another example of an adult tissue containing stem cells is the skeletal muscle. The cells responsible for renewal and growth of the skeletal muscle are called satellite cells or myoblasts and are located between the sarcolemma and the basal lamina of the muscle fiber (5). Since skeletal muscle and cardiac muscle share similar characteristics such as they both are striated muscle cells, satellite cells are considered good candidates for the repair of damaged myocardium and have been extensively studied (20,25,38–40,48,56, 64–67). Myoblasts are particularly attractive, because they can be autotransplanted, so that issues of donor availability, ethics, tumorigenesis and immunological compatibility can be avoided. They also have been shown to have a high growth potential in vitro and a strong resistance to ischemia in vivo (20). On the down side

  • they may have more arrhythmogenic potential when transplanted into myocardium than bone marrow or peripheral blood de­rived stem cells and progenitor cells (40).

Isolation of Cells Prior to Transplantation

Hematopoietic stem and progenitor cells are commonly identified by the expression of a profile of surface receptors (cell antigens). For example, human hematopoietic stem cells are defined as CD34+/CD59+/Thy-1+/CD38low//c-kit/low/lin, while mouse hema-topoietic stem cells are defined as CD34low//Sca-1+/Thy-1+/low/CD38+/c-kit+/lin (23). Additional cell surface receptors have been identified as markers for subgroups of hema-topoietic stem cells with the ability to differentiate into non-hematopoetic tissues, such as endothelial cells (57,78). These can be specifically targeted by isolation methods that use the receptors for cell selection (positive selection with antibody coated magnetic beads or fluorescence-activated cell sorting, FACS). Other stem cell populations are identified by their behavior in cell culture (mesenchymal stem cells) or dye exclusion (SP cells). Finally, embryonic stem cells are isolated from the inner cell mass of the blastocyst and skeletal myoblasts are mechanically and enzymatically dissociated from an easily acces­sible skeletal muscle and expanded in cell culture.

FIG. 1. Maturation process of adult stem cells: with acquisition of a certain phenotype the cell gradually loses its self-renewal capability.  (unable to transfer)

METHODICAL APPROACHES 

j.1527-3466.2003.tb00125.x  fig stem cell

FIG. 2. Intramyocardial injection:

the cells are injected directly into the myocardium through the epicardium. Usually a thoracotomy or sternotomy is required. Transendocardial injection: access can be gained from the ar­terial vasculature. Cells are injected through the endocardium into the myocardium, ideally after identifying the ischemic myocardium by perfusion studies and/or electromechanical mapping. Intracoronary injection: the coronary artery is accessed from the arterial vasculature. Stem cells are injected into the lumen of the coronary artery. Proximal washout is prevented by inflation of a balloon. Cells are then distributed through the capillary system. They eventually cross the endothelium and migrate towards ischemic areas.

The intracoronary delivery of stem cells (Fig. 2) and distribution through the coronary system has also been explored (6,62,74). This approach was pioneered by Robinson et al. (56), who demonstrated successful engraftment within the coronary distribution after intracoronary delivery of genetically labeled skeletal myoblasts. The risk of intracoronary injection is comparable to that of a coronary angiogram and percutaneous transluminal coronary angioplasty (PTCA) (62), which are safe and clinically well established.

RESULTS IN ANIMAL STUDIES AND HUMAN TRIALS

Dif­ferentiation into cardiomyocytes was observed after transplantation of embryonic stem cells, mesenchymal stem cells, lin/c-kit+ and SP cells. The induction of angiogenesis was observed after transplantation of embryonic stem cells, mesenchymal stem cells, bone marrow-derived mononuclear cells, circulating endothelial progenitor cells, SP cells and lin/c-kit+ cells.

The use of embryonic stem cells in ischemia was examined in two studies (42,43). These studies demonstrated that mice embryonic stem cells transplanted into rat myo­cardium exhibited cardiomyocyte phenotype at 6 weeks after transplantation. In addition, generation of myocardium and angiogenesis were observed at 32 weeks after allogenic transplantation in rats. In these two studies no arrhythmias or cardiac tumors were reported.

Several studies have shown retardation of LV remodeling and improvement of cardiac function after administration of bone marrow-derived mononuclear cells. For example, decreases in infarct size, and increase in ejection fraction (EF), and left ventricular (LV) time rate change of pressure (dP/dtmax) were observed after direct injection of bone marrow-derived mononuclear cells 60 min after ischemia in swine (28). In humans, intra-coronary delivery and transendocardial injection of mononuclear cells leads to a decrease in LV dimensions and improvement of cardiac function and perfusion (49,62). A decrease in end systolic volume (ESV) and an increase in EF as well as regional wall motion were observed following intracoronary administration of CD34+/CD45+ human circulating en­dothelial cells (6). Injection of circulating human CD34+/CD117+ cells into infarcted rat myocardium induced neoangiogenesis and improved cardiac function (32). This study suggests that the improvement in LV remodeling after infarction appears to be in part me­diated by a decrease in apoptosis within the noninfarcted myocardium. Two other studies reported increased fractional shortening, improved regional wall motion and decreased left ventricular dimensions after transplantation of human CD34+ cells (29,30). Improved global left ventricular function and infarct perfusion was demonstrated after intramyo-cardial injection of autologous endothelial progenitor cells in humans (61).

DISCUSSION AND OUTLOOK

The idea of replacing damaged myocardium by healthy cardiac tissue is exciting and has received much attention in the medical field and the media. Therefore, it is important for the scientist to know what is established and what is based on premature conclusions. Currently, there are data from animal studies and human trials (Table 2). However, some of these data are not very concrete. For example,

  • many animal studies do not report the level of achieved neoangiogenesis and/or regeneration of myocardium.
  • In studies where the numbers of neovessels and new cardiomyocytes are specified, these numbers are often very low.

While these experiments confirm the concept that bone marrow and peripheral blood-derived stem and progenitor cells can differentiate into cardiomyocytes and endo­thelial cells when transplanted into ischemic myocardium, they also raise the question how effective this treatment is.

The results of the clinical trials that have been conducted are encouraging, but they need to be interpreted with caution. The common endpoints of these studies include left ventricular dimensions, perfusion, wall motion and hemodynamic function. While all studies report improvement after mononuclear cell, myoblast or endothelial progenitor cell transplantation, it is difficult to separate the effects of stem cell transplantation from the effects of the state-of-the art medical care that the patients typically received.

CONCLUSION

While the majority of studies demonstrate neoangiogenesis and some studies also show regeneration of myocardium after stem/progenitor cell transplantation, it remains unclear whether the currently achieved level of tissue regeneration is sufficient to affect clinical outcome. Long-term follow-up of patients that received stem/progenitor cells in clinical trials will provide important information on the potential risks of neoplasm and arrhythmias and, therefore, safety of this treatment. Ultimately, postmortem histological confirmation of scar tissue repair by transplanted cells and randomized placebo control trials with long-term follow-up are required to prove efficacy of this treatment.

REFERENCES (10)

1. American Heart Association Disease and Stroke Statistics-2003 Update, Dallas TX, American Heart Associ­ation; 2002 http://http://www.americanheart.org/downloadable/heart/10461207852142003HDSStatsBook.pdf

2. Arai A, Sheikh F, Agyeman K, et al. Lack of benefit from cytokine mobilized stem cell therapy for acute myocardial infarction in nonhuman primates. J Am Coll Cardiol 2003;41(Suppl 6A):371.

3. Asahara T, Masuda H, Takahashi T, et al. Bone marrow origin of endothelial progenitor cells responsible for postnatal vasculogenesis in physiological and pathological neovascularization. Circ Res 1999;85:221–228.

4. Asahara T, Murohara T, Sullivan A, et al. Isolation of putative progenitor endothelial cells for angiogenesis. Science 1997;275:964–967.

5. Asakura A, Seale P, Girgis-Gabardo A, Rudnicki M. Myogenic specification of side population cells in skeletal muscle. J Cell Biol 2002;159(1):123–134.

6. Assmus B, Schaechinger V, Teupe C, et al. Transplantation of progenitor cells and regeneration en­hancement in acute myocardial infarction (TOPCARE-AMI). Circulation 2002;106:r53–r61.

7. Bader A, Al-Dubai H, Weitzer G. Leukemia inhibitory factor modulates cardiogenesis in embryoid bodies in opposite fashions. Circ Res 2000;86(7):787–794.

8. Barski G, Sorieul S, Cornefert F. “Hybrid” type cells in combined cultures of two different mammalian cell strains. J Natl Cancer Inst 1961;26:1269–1291.

9. Boersma E, Mercado N, Poldermans D, Gardien M, Vos J, Simoons M. Acute myocardial infarction. Lancet 2003;361:847–58.

  1. 10.          Boheler K, Czyz J, Tweedie D, Yang H, Anisimov S, Wobus A. Differentiation of pluripotent embryonic stem cells into cardiomyocytes. Circ Res 2002;91:189–201.

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Source of Stem Cells to Ameliorate Damaged Myocardium (Part 2)

Author and Curator: Larry H. Bernstein, MD, FCAP

and

Curator: Aviva Lev-Ari, PhD, RN

 

This is Part 2 of a 3 part series of perspectives on stem cell applications to regenerating damaged myocardium.

Progenitor Cell Transplant for MI and Cardiogenesis  (Part 1)
Larry H. Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN
https://pharmaceuticalintelligence.com/2013-10-27/larryhbern/Progenitor-Cell-Transplant-for-MI,-and-cardiogenesis/

Source of Stem Cells to Ameliorate Damage Myocardium (Part 2)
Larry H. Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN
https://pharmaceuticalintelligence.com/2013-10-29/larryhbern/Source_of_Stem_Cells_to_Ameliorate_ Damaged_Myocardium/

An Acellular 3-Dimensional Collagen Scaffold  Induces Neo-angiogenesis (Part 3)
Larry H. Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN
https://pharmaceuticalintelligence.com/2013-10-29/larryhbern/An_Acellular_3-Dimensional_Collagen_Scaffold _Induces_Neo-angiogenesis/

This series of articles discusses the difficulties we have encountered in adopting stem cell research to clinical therapeutics in regeneration of cardiac tissue damaged post myocardial infarct.  Enormous problems have been encountered in the selection of progenitor cells, the growth into compatible and functional myocardial tissue, and the survival of the myocardium.  Part I went into some detail on a method of obtaining suitable cells, growing them in sheets, and transferring the sheets to the surface for regeneration and repair, which is now going into clinical trials.  Part I will be confined to the importance of source of progenitor cells, whether adult stem cells or umbilical cord blood.

Do Adult Stem Cells Ameliorate the Damaged Myocardium? Human Cord Blood as a Potential Source of Stem Cells

Elise M.K. Furfaro and Mohamed A. Gaballa
Dept Internal Med, Sarver Heart Center, University of Arizona, College of Medicine, Tucson, AZ
Current Vascular Pharmacology, 2007, 5, 27-44  © 2007 Bentham Science Publishers Ltd.

Abstract: The heart does not mend itself after infarction. Cell-based strategies have promising therapeutic potential. Recent clinical and pre-clinical studies demonstrate varying degrees of improvement in cardiac function using different adult stem cell types such as bone marrow (BM)-derived progenitor cells and skeletal myoblasts. However, the efficacy of cell therapy after myocardial infarction (MI) is inconclusive and the cellular source with the highest potential for regeneration is unclear. Clinically, BM and skeletal muscle are the most commonly used sources of autologous stem cells. One major pitfall of using autologous stem cells is that the number of functional cells is generally depleted in the elderly and chronically ill. Therefore, there is an urgent need for a new source of adult stem cells. Human umbilical cord blood (CB) is a candidate and appears to have several key advantages. CB is a viable and practical source of progenitor cells. The cells are naïve and what’s more, CB contains a higher number of immature stem/progenitor cells than BM.

We review recent clinical experience with adult stem cells and explore the potential of CB as a source of cells for cardiac repair following MI. We conclude that there is a conspicuous absence of clinical studies utilizing CB-derived cells and there is a pressing need for large randomized double-blinded clinical trials to assess the overall efficacy of cell-based therapy.

Keywords: Umbilical cord blood, adult stem cell, myocardial infarction, congestive heart failure, human bone marrow, skeletal muscle, angiogenesis

 INTRODUCTION

There is an urgent need for new and effective therapy for congestive heart failure (CHF). Heart cells may have a limited capacity to regenerate after myocardial infarction (MI), therefore the use of stem cells for cardiac repair is a logical option. In the past three years, clinical and pre-clinical stud-ies examined the potential of a variety of adult stem cells from different sources as therapy for cardiac disease [1-40]. Adult stem cells are typically chosen in clinical studies be-cause their use avoids the ethical problems associated with embryonic cells. Furthermore, adult stem cells were reported to be pluripotent, capable of differentiating to different cell types [41-45]. Bone marrow-derived hematopoietic stem cells, for example, appear to differentiate into brain cells, skeletal muscle cells, liver cells and cardiomyocytes [42-45]. However, the conclusions of the studies have been recently challenged [10-21, 45].

Regardless of the source, stem cells are difficult to iden-tify because they are hard to distinguish from other cells. No techniques are available to reliably identify stem cells other than surface markers. However, cell surface markers are fickle in that none of them appear to be unique to stem cells. For example, stem and progenitor cells of a varying degree of maturity all express the CD34+ surface marker.. Stem cells are typically recovered by isolation of mononuclear cells (MNCs) and subsequent enrichment for a subset of cells that express certain surface markers such as CD34+ or CD133+, etc. These precursors are commonly sorted using the fluores-cence activated sorting system [1-45].

Direct intramyocardial injection of stem cells into the myocardium is the common route of delivery during surgical intervention. This technique of local delivery of stem/ pro-genitor cells to the myocardium has been shown to be feasi-ble and safe in patients with heart disease [1-4, 10-12, 13, 20, 22, 28]. Other than open-heart surgery, the intra-coronary route appears to be the preferred approach in clinical studies because the stem cells are delivered directly to the affected area without traumatizing the myocardium or submitting the body to the systemic side effects of stem cell mobilization [5-9, 14-19, 21]. A complementary approach to increase the efficiency of progenitor cell transplantation is to enhance cell recruitment and retention in the infarcted heart. For example, stromal cell-derived factor (SDF-1α) has recently been shown to play a critical role in stem cell recruitment to the heart after MI [46].

Although there are other sources of adult stem cells such as adipose tissue [47, 48] and cardiac tissue [49, 50], this review briefly discusses clinical trials using BM stem cells and skeletal muscle myoblasts and pre-clinical studies that used cord blood (CB) cells for heart repair carried out during the past three years. This time period was chosen due to the plethora of excellent published reviews that serve as a foun-dation for this work [51-54]. In addition, the reader may re-fer to several recently published reviews [55-63]. Current clinical experience purports the safety and feasibility of BM stem cells and skeletal muscle myoblasts as autologous cell-therapy for cardiac disease [1-20, 22-30]. However, these cell sources have limitations. For example, recovering sufficient numbers of functional BM progenitor cells is a problem in the elderly and ill [64]. Cardiovascular diseases such as diabetes are associated with BM cell dysfunction [64]. Cardiac calcifications were reported in patients following BM stem cell transplantation [64]. Bone marrow-derived mesenchymal cells (MSCs) have been suggested to play a role in myocardial scarring [64]. Skeletal myoblasts have been associated with arrhythmias and have failed to establish gap junctions with native myocardial cells [64]. Furthermore, the efficacy of these cells in repairing damaged myocardium in clinical settings is still not clear partially due to the lack of protocol standardization as well as the use of adjunct treatment. Different diseases, cell types, cell numbers, routes of cell delivery, end point measurements, and the small number of patients included in these studies make it difficult to draw conclusions about the efficacy of stem cell therapy. Larger clinical trials are now underway to assess the risks and benefits of cell-transplantation using stem cells from BM and skeletal muscle [65].

Another emerging source of stem cells is human umbilical CB. CB has the advantage of being readily available. Numerous CB banks already exist and their number is on the rise [64, 66]. CB is obtained by a non-invasive procedure, and contains a larger portion of immature and non-committed cells than BM. Stem cells derived from CB are expandable ex vivo, appear to be more resistant to apoptosis and the risk of transmission of infection is low [64, 67]. In addition, transplantation of CB cells is associated with a lower incidence and risk of graft-versus-host disease [68, 69]. Similar to previous studies that reported beneficial effects of stem cells isolated from BM and skeletal muscle, CB stem cells also show promise for cardiac repair [1, 3-9, 10¬12, 14, 15, 17-23, 25, 27-29]. Over four thousand CB transplants worldwide have been performed for the treatment of other diseases such as leukemia and immune deficiencies [70]. In contrast, to date, no clinical trials using CB-derived stem cells for transplant after MI have been reported.

The following is an update on recent clinical trials that used BM and skeletal muscle stem cells and preclinical studies that used CB cells to repair the injured myocardium. The emphasis is to evaluate CB as a potential and practical source of stem cells for heart repair after MI.

SKELETAL STEM CELLS

Being the first cell type used clinically, it seems logical to start by discussing the use of skeletal myoblasts, or skeletal muscle satellite cells, as cell therapy after MI. The advantages of these cells are that they are readily available from muscle biopsies, they are contractile cells, and they can be expanded ex vivo before delivery into the myocardium. Moreover, they appear to have an increased resistance to ischemia [55, 71]. Cell transplantation was usually performed concomitant to revascularization or in patients with previous revascularization [1, 2, 4-6]. Most of the studies used direct injection as the delivery route [1-4]. The number of patients in each study ranged from five to 30 and patients were followed up from 68 days to four years. Except for one study, transplantation of satellite cells was shown to improve left ventricular ejection fraction (LVEF) in all recent clinical studies [1, 3-6].
Several of these studies showed improvement in New York Heart Association (NYHA) class. Interestingly, Pagani et al. showed enhanced angiogenesis after cell transplantation, but they did not measure cardiac function or ventricular remodeling. Unfortunately, it appears that the incidence of arrhythmia and ventricular tachycardia, necessitating the implementation of prophylactic amiodarone or implanted cardioverter defibrillator as an adjunct treatment, is commonplace among these trials [2-6]. Further undermining the clinical use of skeletal myoblasts is the reported lack of cardiomyogenesis and electrical coupling with native cardiac cells that would be necessary to maintain a healthy and functioning heart [55, 72]. Detailed descriptions of these most recent clinical studies using skeletal muscle satellite cells are included in Table 1 (not shown).

[It is not surprising to this reader that the inadequacy of skeletal muscle donor cells is found to be inadequate for maintaining normal cardiac contractility.  Even though contraction of skeletal muscle, smooth muscle, and heart muscle share a basic motif involving CaMKII, the generation of a calcium spark triggering contraction involves a specific relationship between CaMKIIδ and the RyR2 receptor.   CaMKIIδ is specific to the cardiomyocyte.  The other consideration is that the heart is a syncytium, and it has a relationship to neurohumoral control, distinctly different than that in skeletal muscle  This is perhaps the most telling observation in the observed lack of cardiomyogenesis and electrical coupling with native cardiac cells that would be necessary to maintain a healthy and functioning heart [55, 72]].

BM CLINICAL TRIALS

To date, only small-scale clinical trials, including five to 69 patients, have been performed using bone marrow-derived stem cells (BM-SCs) for transplantation. Three different types of BM-SCs are typically used in recent clinical trials, namely un-fractionated MNCs, CD34+ cells and MSCs. These cells were proposed to treat acute or old MI as well as heart failure [7-21]. Intracoronary injection is the delivery route of choice for these cells [7-9, 14-21]. Revascularization with percutaneous coronary intervention (PCI) or coronary artery bypass graft (CABG) is commonly used concomitant to cell treatment [13, 15, 16, 18-21]. Several recent trials purported improvement in cardiac function and/or ventricular remodeling three to 12 months after cell treatment [7-11, 15, 17, 18-21]. Some of these studies reported additional enhancement in clinical parameters such as

  • end diastolic (EDV),
  • end systolic volume (ESV)
  • and/or myocardial perfusion [7-9, 10, 17-20].

A small number of studies reported no benefits from BM transplantation [12-14, 16]. In one study, bone marrow transplantation was complicated by coronary artery re-occlusion [21]. The primary endpoint of most of these trials was to assess the safety and feasibility of BM-SC transplantation as a treatment for ischemic heart disease, however these studies are underpowered. In addition, the efficacy of bone marrow cell therapy is difficult to ascertain from clinical studies, at least in part, due to common utilization of adjunct therapy such as revascularization. More detailed descriptions of bone marrow clinical studies are found in Tables 2-5 (not shown).

MOBILIZATION OF BM-DERIVED CELLS

Since transplantation of autologous BM-SCs leads to improvement in cardiac function, mobilization of BM-SCs using cytokines to increase the number of circulating cells was utilized in succeeding studies. Granulocyte colony stimulating factor (G-CSF) is the most common cytokine used to mobilize BM-SCs in clinical studies [22-31]. The feasibility and safety of G-CSF has been reported by several investigators. The number of patients in the G-CSF studies ranged from five to 114 and they were followed for up to 52 weeks. Clinical studies in the last three years have shown that cardiac function improved in about half of the trials using G-CSF to mobilize BM-SCs [22, 23, 27-29]. The remaining half of G-CSF studies reported no effects on cardiac function [24-26, 30, 31]. In one study, an unexpected reduction in LVEF was reported [31]. Adverse effects of G-CSF treatment were reported in almost all the recent clinical studies [22, 24-27, 29, 31]. Detailed descriptions of G-CSF stud¬ies are shown in Tables 6-7.

HUMAN UMBILICAL CORD BLOOD: NO LONGER A WASTE PRODUCT

Amidst the flurry of clinical studies utilizing BM and skeletal muscle SCs, it is a wonder why no trials are reported using CB cell transplantation in humans. However, several pre-clinical studies using various animal models demonstrated the potential use of CB stem cells for cardiac repair after MI [32-40]. Conserved commonalities of cardiac function improvement exist in these studies despite dissimilarity of protocols used [32-40]. The following is a description of the pre-clinical studies which used different subsets of CB-derived stem cells to treat MI. In this review, the pre-clinical studies are categorized according to the type of stem cell administered.

We first start with studies that used CB-derived MNCs. Ma et al. reported that intravenous injection of six million CB-MNCs into non-obese diabetic severe combined immunodeficiency (NOD/SCID) mice 24 h post-MI resulted in an increase in capillary density and decrease in both infarct size and collagen deposition three weeks after treatment [38]. No myogenesis was observed. Human DNA was identified in 10 out of the 19 mice that underwent induction of MI. Direct myocardial injection of one-sixth of the amount of cells used in the above study in rats also reduced infarct size and increased both ventricular wall thickness and LVdP/dt and ejection fraction up to six months after treatment [34].

Similar to CB-MNCs, transplantation of two hundred thousand CD34+ cells, a subset of MNCs, within 20 min after MI

  • increased vascular density,
  • reduced LV dilation, and
  • improved cardiac function four weeks after treatment [35].

However, only about one percent of the injected cells were incorporated into the vessels of the rat myocardium, which suggests that angiogenic factors released by these cells may contribute to the observed angiogenesis [35]. A subset of CD34+, CD34+ KDR+ cell fraction, was proposed to be the subset of cells responsible for angiogenesis induction and improvement in cardiac function after treatment with either MNCs or CD34+ cells [32]. Two hundred thousand of either CD34+ or MNCs, or two thousand of either CD34+ KDR+ or CD34+ KDR- cells were injected in a NOD/SCID mouse model of MI. Compared to transplantation of MNCs or PBS, CD34+ cells

  • increased LVdP/dt,
  • decreased LV end diastolic pressure and
  • infarct size up to five months after MI.

Treatment with two thousand CD34+KDR+ cells, which is two log less than the number of CD34+ cells, resulted in more
angiogenesis compared to either MNC or CD34+ [32].

An immature subset of CB-MNCs, CD133+ cells, were also reported to improve cardiac function after transplantation into MI mice [37]. One to two million CD133+ cells were intravenously injected into athymic nude rats seven days after MI. Four weeks after transplantation,

  • reduction in both scar thinning and
  • LV systolic dilation, and
  • increase in LV fractional shortening were observed.

In contrast to other studies, vessel density did not differ between the cell-treated and control rats [37]. Similarly, transplantation of a subset of these immature CD133+ cells, CD34+ CD133+ cells, into a mouse model of hindlimb ischemia resulted in angiogenesis induction [40]. Transplantation of one hundred thousand CD34+ CD133+ cells into ischemic limbs of immunosup-pressed mice increased both vessel and muscle fiber densities fourteen days after injection. In contrast, administration of CD34+ cells resulted in increased vessel density only. Neither of these findings was observed after administration of CD34- cells [40].

An alternative subset of progenitor cells, called endothelial progenitor cells (EPCs) from either CB or adult peripheral blood (PB), was also shown to induce angiogenesis in ischemic hindlimb [39]. EPCs were derived from MNC CD34+ cells and identified in culture as attaching cells that exhibit spindle-shape. These cells

  1. incorporated acetylated-low density lipoprotein,
  2. released nitric oxide, and
  3. expressed KDR, VE-cadherin, CD31, and vW factor and CD45-.

Not only were the CB-derived EPCs more abundant (10 fold increase) than those derived from PB, they also further in-creased capillary density when injected into ischemic tissue [39].

Finally, another CB-derived cell subset, denoted as human unrestricted somatic stem cells (USSCs), was shown to engraft in the infarcted heart and improve cardiac perfusion [36]. USSCs were defined as negative for the following surface markers:

  • CD14, CD31, CD33, CD34, CD45, CD56, CD133 and human leukocyte antigen class II and
  • positive for CD13, CD29, CD44, and CD49e.

In a porcine model of MI, one hundred million USSCs were directly injected into the infarcted heart four weeks after MI.

  1. Regional perfusion,
  2. LVEF,
  3. scar thickness, and
  4. wall motion increased four weeks after transplantation [36].

In addition to cell transplantation alone, the combination of gene and cell therapy was shown to be a potential treatment for MI [33]. For example,

CD34+ cells transduced with the adeno associated viral vector that encoded either human angiopieotin-1 or vascular endothelial growth factor (VEGF) were intramyocardially injected in a mouse model of MI. Improved cardiac function and increased capillary density were observed with CD34+ cells alone.

However, exaggerated improvements were obtained with the combined therapy of CD34+ cells transfected with Angiopieotin-1 and or VEGF. Compared with CD34+ treatment alone,

  • the combined therapy further increased capillary density and decreased infarct size [33].

Taken together, based on the pre-clinical studies, a common feature of transplantation of human CB-derived cells is

  • induction of angiogenesis and cardiac function improvement in animal models of ischemia.

Myogenesis does not seem to be a mechanism of the beneficial effects of CB transplantation.

Compared with adult stem cells, CB cell treatment has limitations. The practical and crucial difference between stem cells obtained from adult human donors and from CB is quantitatively, not qualitatively based. It is uncommon that more than several million stem cells can be isolated from CB. That amount may be too small for transplantation to an adult. Children appear to be ideal recipients when utilizing this source of stem cells since they are smaller patients and require fewer cells per kilogram of body weight [71]. However, ex vivo expansion of these cells may overcome this limitation [73, 74]. There is another concern that the use of CB for transplantation presents a higher risk of transmitting opportunistic infections [75]. The human herpes viruses are common pathogens found in transplant recipients. Currently, it is routine to test for the presence of anti-cytomegalovirus immunoglobin M. However, screening prospective CB donors for these pathogens reduces the risk of transmission of infection [75].

(Tables from published document are to be viewed in that document.)

CONCLUSIONS

Although early clinical studies suggest that bone marrow and skeletal myoblast transplantation into the infarcted heart improves cardiac perfusion and function, there is an urgent need for large randomized double-blinded clinical trials that assess the overall efficacy of cell-based therapy. In addition, little is known about the mechanisms by which stem cells render their positive effects. Cardiac regeneration by bone marrow cells is an obvious mechanism. However, a small number of experimental studies have purported the occurrence of myocardial regeneration by bone marrow cells. Furthermore, substantial evidence demonstrates that cell types other than cardiomyocytes improve cardiac function, suggesting that the beneficial effects of cell therapy may be independent of cardiac regeneration [76-89]. Enhanced vascularization, on the other hand, is a common finding after bone marrow cell transplantation. Cell engrafment to the vascular wall as well as angiogenic factors released by transplanted cells may be responsible for the enhanced vascularization. Obviously, there remain a considerable number of unanswered questions that must be addressed in basic science laboratories before stem cell therapy becomes standard practice. For example, what are the mechanisms of improvement in cardiac function? Which cell type is best-suited for transplantation? What is the optimal cell concentration that should be used for transplant and what is the most effective route of delivery?

The target patient population which would draw clinical benefit from cell-based therapy must also be defined and the optimal time of injection after the onset of infarction has to be determined. Currently, it is difficult to assess the efficacy of stem cell treatment of MI. This is in part due to lack of standardization among clinical as well as pre-clinical studies. Therefore, in order to accomplish these objectives, there is great need for communication among the various research groups concerned with stem cells and clinical studies.

Here we add yet another source of stem cells, namely the umbilical CB. This source of stem cells had many advantages mentioned in the preceding sections. In addition, pre-clinical studies indicate the efficacy of CB cells in myocardial repair. However, the fate and benefits of these cells need to be tested in clinical settings.

REFERENCES

[1]  Herreros J, Prosper F, Perez A, Gavira JJ, Garcia-Velloso MJ, Barba J, et al. Autologous intramyocardial injection of cultured skeletal muscle-derived stem cells in patients with non-acute myocardial infarction. Eur Heart J 2003; 24: 2012-20.

[2]  Pagani FD, DerSimonian H, Zawadzka A, Wetzel K, Edge AS, Jacoby DB, et al. Autologous skeletal myoblasts transplanted to ischemia-damaged myocardium in humans. Histological analysis of cell survival and differentiation. J Am Coll Cardiol 2003; 41: 879-88.

[3]  Smits PC, van Geuns RJ, Poldermans D, Bountioukos M, Onder-water EE, Lee CH, et al. Catheter-based intramyocardial injection of autologous skeletal myoblasts as a primary treatment of ischemic heart failure: clinical experience with six-month follow-up. J Am Coll Cardiol 2003; 42: 2063-9.

[4]  Dib N, Michler RE, Pagani FD, Wright S, Kereiakes DJ, Lengerich R, et al. Safety and feasibility of autologous myoblast transplantation in patients with ischemic cardiomyopathy: four-year follow-up. Circulation 2005; 112: 1748-55.

[5]  Siminiak T, Kalawski R, Fiszer D, Jerzykowska O, Rzezniczak J, Rozwadowska N, et al. Autologous skeletal myoblast transplantation for the treatment of postinfarction myocardial injury: phase I clinical study with 12 months of follow-up. Am Heart J 2004; 148: 531-7.

[6]  Siminiak T, Fiszer D, Jerzykowska O, Grygielska B, Rozwadowska N, Kalmucki P, et al. Percutaneous trans-coronary-venous trans-plantation of autologous skeletal myoblasts in the treatment of post-infarction myocardial contractility impairment: the POZNAN trial. Eur Heart J 2005; 26: 1188-95.

[7]  Assmus B, Schachinger V, Teupe C, Britten M, Lehmann R, Dobert N, et al. Transplantation of Progenitor Cells and Regeneration Enhancement in Acute Myocardial Infarction (TOPCARE-AMI). Circulation 2002; 106: 3009-17.

[8]  Schachinger V, Assmus B, Britten MB, Honold J, Lehmann R, Teupe C, et al. Transplantation of progenitor cells and regeneration enhancement in acute myocardial infarction: final one-year results of the TOPCARE-AMI Trial. J Am Coll Cardiol 2004; 44: 1690-9.

[9]  Britten MB, Abolmaali ND, Assmus B, Lehmann R, Honold J, Schmitt J, et al. Infarct remodeling after intracoronary progenitor cell treatment in patients with acute myocardial infarction (TOPCARE-AMI): mechanistic insights from serial contrast-enhanced magnetic resonance imaging. Circulation 2003; 108: 2212-8.

[10]  Perin EC, Dohmann HFR, Borojevic R, Silva SA, Sousa ALS, Mesquita CT, et al. Transendocardial, autologous bone marrow cell transplantation for severe, chronic ischemic heart failure. Circulation 2003; 107: 2294–302.

Human umbilical cord blood stem cells, myocardial infarction (and stroke)

Nathan Copeland, David Harris and Mohamed A Gaballa
Nathan Copeland, Research Associate and Medical Student, University of Arizona Medical School, Tucson, Arizona; David Harris, Professor of Microbiology and Immunology, University of Arizona, Tucson, Arizona; Mohamed A Gaballa, Director, Center for Cardiovascular Research, Sun Health Research Institute, Sun City, Arizona; Section Chief of Basic Science, Cardiology Section, Banner GoodSam Medical Center, Phoenix, Arizona
Clinical Medicine 2009, Vol 9, No 4: 342–5

ABSTRACT – Myocardial infarction (MI) and stroke are the first and third leading causes of death in the USA accounting for more than 1 in 3 deaths per annum. Despite interventional and pharmaceutical advances, the number of people diagnosed with heart disease is on the rise. Therefore, new clinical strategies are needed. Cell-based therapy holds great promise for treatment of these diseases and is currently under extensive preclinical as well as clinical trials. The source and types of stem cells for these clinical applications are questions of great interest. Human umbilical cord blood (hUCB) appears to be a logical candidate as a source of cells. hUCB is readily available, and presents little ethical challenges. Stem cells derived from hUCB are multipotent and immunologically naive. Here is a critical literature review of the beneficial effects of hUCB cell therapy in preclinical trials.
KEY WORDS: animal models, cerebral infarction, myocardial infarction, stem cells, umbilical cord blood

Introduction

The study of stem cell therapies to address some of the most daunting medical challenges, including heart disease and stroke, has advanced steadily over the last three years. The majority of preclinical studies of stem cells as a potential therapy for either myocardial or cerebral ischaemia were positive on average. Small clinical trials, however, show either no or modest improvement in cardiac function after myocardial infarction (MI). Currently, there are two major types of autologous cells that are clinically used for MI and stroke. The first is skeletal myoblasts, harvested from skeletal muscle. These cells can be expanded in culture. Positive outcomes were recently reported in a phase 1 clinical trial using catheter-based injection of myoblasts to the endocardium (CAUSMIC, American Heart Association (AHA) Scientific Sessions 2007). The second is bone marrow cells (BMCs). Intracoronary injection of BMCs improve global left ventricular function (IC-BMC, AHA Scientific Sessions 2007). However, direct injection of BMC administration into scarred myocardium does not alter cardiac contractility of the injured area (IC/IM-BMC, AHA Scientific Sessions 2007). The effects of stem cell therapy can only be addressed using clinical trials that:

•             are randomised, blinded, placebo controlled and adequately sized

•             use standardisation of autologous stem cell processing protocols

•             use robust endpoints of efficacy and safety

•             ensure that follow-up is complete and of adequate duration.

It is becoming clear that realisation of the full potential of the therapeutic benefit of stem cells will require understanding the biology of these undifferentiated cells. A successful therapy will require a source with plentiful supply of multipotent stem cells with minimal or no immune rejection. Several sources of stem cells were explored such as

  • adipose tissue,1–3
  • cardiac tissue,4
  • skeletal muscle biopsies,5,6 and
  • hUCB.

Whether these subpopulations of cells are best suited to treat a disease is still unanswered.

Currently, the only confirmed source for totipotential cells is embryonic. However, there are ethical and scientific obstacles to unbridled use of such cells. For clinical application, autologous adult stem cells are the obvious choice. To date, only adult stem cells derived from a patient’s own bone marrow are being used in clinical trials.

Autologous BMC therapy is not without problems. The majority of instances of MI and cerebral ischaemia (CI) occur in the elderly. Since the quantity and function of BMCs decrease with age, an allogeneic younger donor may be used to source BMCs. This may hinder the efficiency of such a treatment and suffer rejection, therefore another source of stem cells is needed.

Cryopreserved stem cells derived from human leukocyte antigen (HLA)-matched and unmatched unrelated donor hUCB were realised as a sufficient source of transplantable hematopoietic stem cells with high donor-derived engraftment and low risk of refractory acute graft-versus-host disease. However, the use of hUCB cells as treatment for either MI or CI has only been recently investigated in preclinical models.

There are several outstanding review articles on stem cells derived from cord blood in MI7–11 and stroke.12–17 This article adds depth to the debate by providing an updated review as well as presenting an integrated overview of studies involving MI and CI cell-based therapy. In the preparation of this review, every effort was made to include all relevant publications since 2005. Due to space limitations, the number of articles cited has been limited.

Cardiovascular disease

Since 2005, several studies have explored the use of various sub-populations of hUCB stem cells for regenerative therapy. Five types of UCB-derived stem cells were investigated: umbilical cord derived stem (UCDS), unrestricted somatic stem cells (USSC), mononuclear progenitor cells (MNCs), CD133+ and CD34+ subpopulations. The experimental parameters of the studies varied. The majority of studies, however, were performed using the rat animal model and utilising the left antero-lateral descending (LAD) coronary artery ligation model of MI with intramyocardial injection of the stem cells. The laboratory used a similar model to determine the efficacy of stem cell derived from hUCB to improve cardiac function after ischemia and reperfusion. The data indicated that intracoronary administration of mononuclear or CD34+ cells derived from hUCB improved cardiac function after MI by inducing neovascularisation and retarding left ventricular (LV) remodelling.37

The majority of reported studies using hUCB cells showed improvement in the outcomes.18–25 Cardiac functional improvements were almost universally reported as evaluated by:

  • increased ejection fraction;
  • improved wall motion;
  • lowered LV end-diastolic pressure; and
  • increased cardiac contraction as determined by the maximum slope of LV pressure.18–21,23–25

There were conflicting reports on the effects of stem cells on LV fractional shorting. One study reported improved shortening while another reported that BM but not UCB cells produced improved shortening.22,23 Improvements in

  • myocardial perfusion, evaluated by increased capillary density, were repeatedly demonstrated as were
  • reductions in infarct size and the number of apoptotic cells.18–25

Retardation or reduction in LV remodelling were also reported.18,21,22 Although the vast majority of studies showed positive outcomes, HLA matching and further study are still needed before UCB stem cell therapies can become safe and effective treatments in humans. A prime example of the need for further elucidation of these emerging therapies can be illustrated by the findings in a study by Moelker.26 This study used intracoronary administration of unrestricted somatic stem cells (USSCs) in a balloon left circumflex artery (LCX) occlusion ischaemia-reperfusion porcine model of MI. They found that treatment did not improve outcome and actually increased infarct size. Their histological analysis revealed that the injected cells worsened the infarct by obstructing vessels downstream.

Furthermore, the mechanisms of the observed benefits of UCB stem cell therapy in MI are under investigation:

  • improved myocardial perfusion,
  • attenuation of cardiac remodelling,
  • reduction of inflammatory responses by
    • limiting expression of TNF-a, MCP-1, MIP and INF–y, and cardiac regeneration.18–5

Tissue regeneration may be mediated by incorporation of delivered cells in the target tissue.18–21,23 An in vitro study confirmed that mononuclear cells were migrated toward homogenised infarcted myocardium and that the greatest migration occurred at two and 24 hours post-MI.20 Paracrine effect, ie the delivered cells release factors that promote neovascularisation, was also reported. Indeed, the study laboratory has shown that hUCB cells release angiogenic factors in vitro under hypoxic conditions. The data are consistent with a previous report that showed

  • increased expression of VEGF 164 and 188 accompanied by
  • angiogenesis and improved remodelling after administration of hUCB mononuclear cells into the myocardium.21

Identifying subpopulations of progenitor cells with the highest potential for tissue repair is another unanswered ques¬tion prior to widespread application of this therapy in clinical settings. Previous studies showed that UCB-derived endothe¬lial progenitor cells (EPC) to be a promising subset of stem cells for treatment of MI; however their number may be insufficient to treat adult patients. This problem can be addressed by expanding these cells in culture prior to transplant. Techniques are being developed to culture clinically significant quantities (60 population doublings) of EPCs from UBC CD.25 Transplantation of these expanded cells improved ejection fraction (EF) and vascular density in vivo, demonstrating that such a culture method may be a viable option to produce EPCs for future use in humans. Another study evaluated the use of gene therapies in conjunction with UCB stem cell therapy.24 CD34+ cells were transfected with AAV-Ang1 and/or AAV-VEGF 165. The gene-modified stem cells resulted in greater increases in capillary density and cardiac performance along with larger reduction in infarct size compared to CD34+ cell therapy alone.

References

1 Valina C, Pinkernell K, Song YH et al. Intracoronary administration of autologous adipose tissue-derived stem cells improves left ventricular function, perfusion, and remodelling after acute myocardial infarction. Eur Heart J 2007;28:2667–77.

2 Zhang DZ, Gai LY, Liu HW et al. Transplantation of autologous adipose-derived stem cells ameliorates cardiac function in rabbits with myocardial infarction. Chin Med J (Engl) 2007;120:300–7.

4 Hoogduijn MJ, Crop MJ, Peeters AM et al. Human heart, spleen, and perirenal fat-derived mesenchymal stem cells have immunomodulatory capacities. Stem Cells Dev 2007;16:597–604.

5 Payne TR, Oshima H, Okada M et al. A relationship between vascular endothelial growth factor, angiogenesis, and cardiac repair after muscle stem cell transplantation into ischemic hearts. J Am Coll Cardiol 2007;50:1677–84.

6 Herreros J, Prósper F, Perez A et al. Autologous intramyocardial injection of cultured skeletal muscle-derived stem cells in patients with non-acute myocardial infarction. Eur Heart J 2003;24:2012–20.

7 Goldberg JL, Laughlin MJ, Pompili VJ. Umbilical cord blood stem cells: implications for cardiovascular regenerative medicine. J Mol Cell Cardiol 2007;42:912–20.

8  Wu KH, Yang SG, Zhou B et al. Human umbilical cord derived stem cells for the injured heart. Med Hypotheses 2007;68:94–7.

9 Zhang L, Yang R, Han ZC. Transplantation of umbilical cord blood-derived endothelial progenitor cells: a promising method of therapeutic revascularisation. Eur J Haematol 2006;76:1–8.

18 Wu KH, Zhou B, Yu CT et al. Therapeutic potential of human umbil¬ical cord derived stem cells in a rat myocardial infarction model. Ann Thorac Surg 2007;83:1491–8.

19 Kim BO, Tian H, Prasongsukarn K et al. Cell transplantation improves ventricular function after a myocardial infarction: a preclinical study of human unrestricted somatic stem cells in a porcine model. Circulation 2005;112:I96–104.

20 Henning RJ, Burgos JD, Ondrovic L et al. Human umbilical cord blood progenitor cells are attracted to infarcted myocardium and sig-nificantly reduce myocardial infarction size. Cell Transplant 2006;15:647–58.

21 Hu CH, Wu GF, Wang XQ et al. Transplanted human umbilical cord blood mononuclear cells improve left ventricular function through angiogenesis in myocardial infarction. Chin Med J (Engl)  2006;119:1499–506.

22 Ma N, Ladilov Y, Moebius JM et al. Intramyocardial delivery of human CD133+ cells in a SCID mouse cryoinjury model: Bone marrow vs. cord blood-derived cells. Cardiovasc Res 2006;71:158–69.

23 Leor J, Guetta E, Feinberg MS et al. Human umbilical cord blood-derived CD133+ cells enhance function and repair of the infarcted myocardium. Stem Cells 2006;24:772–80.

24 Chen HK, Hung HF, Shyu KG et al. Combined cord blood stem cells and gene therapy enhances angiogenesis and improves cardiac perfor-mance in mouse after acute myocardial infarction. Eur J Clin Invest 2005;35:677–86.

 

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Progenitor Cell Transplant for MI and Cardiogenesis  (Part 1

Author and Curator: Larry H. Bernstein, MD, FCAP
and
Curator: Aviva Lev-Ari, PhD, RN
This article is Part I of a review of three perspectives on stem cell transplantation onto a substantial size of infarcted myocardium to generate cardiogenesis in tissue that is composed of both repair fibroblasts and cardiomyocytes, after essentially nontransmural myocardial infarct.

Progenitor Cell Transplant for MI and Cardiogenesis (Part 1)

Larry H. Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/10/28/progenitor-cell-transplant-for-mi-and-cardiogenesis/

Source of Stem Cells to Ameliorate Damage Myocardium (Part 2)

Larry H. Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013-10-29/larryhbern/Source_of_Stem_Cells_to_Ameliorate_ Damaged_Myocardium/

An Acellular 3-Dimensional Collagen Scaffold Induces Neo-angiogenesis
 (Part 3)

Larry H. Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013-10-29/larryhbern/An_Acellular_3-Dimensional_Collagen_Scaffold _Induces_Neo-angiogenesis/

The same approach is considered for stroke in one of these studies.  These are issues that need to be considered
  1. Adult stem cells
  2. Umbilical cord tissue sourced cells
  3. Sheets of stem cells
  4. Available arterial supply at the margins
  5. Infarct diameter
  6. Depth of ischemic necrosis
  7. Distribution of stroke pressure
  8. Stroke volume
  9. Mean Arterial Pressure (MAP)
  10. Location of infarct
  11. Ratio of myocytes to fibrocytes
  12. Coexisting heart disease and, or
  13. Comorbidities predisposing to cardiovascular disease, hypertension
  14. Inflammatory reaction against the graft

Transplantation of cardiac progenitor cell sheet onto infarcted heart promotes cardiogenesis and improves function

L Zakharova1, D Mastroeni1, N Mutlu1, M Molina1, S Goldman2,3, E Diethrich4, and MA Gaballa1*
1Center for Cardiovascular Research, Banner Sun Health Research Institute, Sun City, AZ; 2Cardiology Section, Southern Arizona VA Health Care System, and 3Department of Internal Medicine, The University of Arizona, Tucson, AZ; and 4Arizona Heart Institute, Phoenix, AZ
Cardiovascular Research (2010) 87, 40–49   http://dx.doi.org/10.1093/cvr/cvq027

Abstract

Aims

Cell-based therapy for myocardial infarction (MI) holds great promise; however, the ideal cell type and delivery system have not been established. Obstacles in the field are the massive cell death after direct injection and the small percentage of surviving cells differentiating into cardiomyocytes. To overcome these challenges we designed a novel study to deliver cardiac progenitor cells as a cell sheet.

Methods and results

Cell sheets composed of rat or human cardiac progenitor cells (cardiospheres), and cardiac stromal cells were transplanted onto the infarcted myocardium after coronary artery ligation in rats. Three weeks later, transplanted cells survived, proliferated, and differentiated into cardiomyocytes (14.6 ± 4.7%). Cell sheet transplantation suppressed cardiac wall thinning and increased capillary density (194 ± 20 vs. 97 ± 24 per mm2, P < 0.05) compared with the untreated MI. Cell migration from the sheet was observed along the necrotic trails within the infarcted area. The migrated cells were located in the vicinity of stromal-derived factor (SDF-1) released from the injured myocardium, and about 20% of these cells expressed CXCR4, suggesting that the SDF-1/CXCR4 axis plays, at least, a role in cell migration. Transplantation of cell sheets resulted in a preservation of cardiac contractile function after MI, as was shown by a greater ejection fraction and lower left ventricular end diastolic pressure compared with untreated MI.

Conclusion

The scaffold-free cardiosphere-derived cell sheet approach seeks to efficiently deliver cells and increase cell survival.These transplanted cells effectively rescue myocardium function after infarction by promoting not only neovascular-ization but also inducing a significant level of cardiomyogenesis
Keywords  Myocardial infarction • Cardiac progenitor cells • Cardiospheres • Cardiac regeneration • Contractility

Introduction

Despite advances in cardiac treatment after myocardial infarction (MI), congestive heart failure remains the number one killer world-wide. MI results in an irreversible loss of functional cardiomyocytes followed by scar tissue formation. To date, heart transplant remains the gold standard for treatment of end-stage heart failure, a procedure which will always be limited by the availability of a donor heart. Hence, developing a new form of therapy is vital.
A number of adult non-cardiac progenitor cells have been tested for myocardial regeneration, including skeletal myoblasts,1 bone-marrow2, and endothelial progenitor cells.3,4 In addition, several cardiac resident stem cell populations have been characterized based on the expression of stem cell marker proteins.5–8 Among these, the c-Kit+ population has been reported to promote myocardial repair.5,9 Recently, an ex vivo method to expand cardiac-derived progenitor cells from human myocardial biopsies and murine hearts was developed.10 Using this approach, undifferentiated cells (or cardiospheres) grow as self-adherent clusters from postnatal atrium or ventricular biopsy specimens.11
To date, the most common technique for cell delivery is direct injection into the infarcted myocardium.12 This approach is inefficient because more than 90% of the delivered cells die by apoptosis and only a small number of the survived cells differentiated into cardiomyocytes.13 An alternative approach to cell delivery is a biodegradable scaffold-based engineered tissue.14,15 This approach has the clear advantage in creating tissue patches of different shapes and sizes and in creating a beating heart by decellularization technology.16 Advances are being made to overcome the issue of small patch thickness and to minimize possible toxicity of the degraded substances from the scaffold.15 Recently, scaffold-free cell sheets were created from fibroblasts, mesenchymal cells, or neonatal myocytes.17,18 Transplantation of these sheets resulted in a limited improvement in cardiac function due to induced neovascularization and angiogenesis through secretion of angiogenic factors.17–19 However, few of those progenitor cells have differentiated into cardiomyocytes.17 The need to improve cardiac contractile function suggests focusing on cells with higher potential to differentiate to cardiomyocytes with an improved delivery method.
In the present study, we report a cell-based therapeutic strategy that surpasses limitation inherent in previously used methodologies. We have created a scaffold-free sheet composed of cardiac progenitor cells (cardiospheres) incorporated into a layer of cardiac stromal cells. The progenitor cells survived when transplanted as a cell sheet onto the infarcted area, improved cardiac contractile functions, and supported recovery of damaged myocardium by promoting not only vascularization but also a significant level of cardiomyogenesis. We also showed that cells from a sheet can be recruited to the site of injury driven, at least partially, by the stromal-derived factor (SDF-1) gradient.

Methods

Detailed methods are provided in the Supplementary Methods

Animals

Three-month-old Sprague Dawley male rats were used. Rats were randomly placed into four groups:
(1) sham-operated rats, n = 12;
(2) MI, n = 12;
(3) MI treated with rat sheet, n = 10; and
(4) MI treated with human sheet, n = 10.

Myocardial infarction

MI was created by the ligation of the left coronary artery.20 Animals were intubated and ventilated using a small animal ventilator (Harvard Apparatus). A left thoracotomy was performed via the third intercostal rib, and the left coronary artery was ligated. The extent of infarct was verified by measuring the area at risk: heart was perfused with PBS containing 4 mg/mL Evans Blue as previously described by our laboratory.20 The area at risk was estimated by recording the size of the under-perfused (pale-colored) area of myocardium (see Supplementary material online, Figure S1). Only animals with an area at risk >30% were used in the present study. Post-mortem infarct size was measured using triphenyl tetrazolium chloride staining as previously described by our laboratory.20

Isolation of cardiosphere-forming cells

Cardiospheres were generated as described10 from atrial tissues obtained from:
(1) human atrial resection samples obtained from patients (aged from 53 to 73 years old) undergoing cardiac bypass surgery at Arizonam Heart Hospital (Phoenix, AZ) in compliance with Institutional Review Board protocol (n = 10),
(2) 3-month-old SD rats (n = 10). Briefly, tissues were cut into 1–2 mm3 pieces and tissue fragments were cultured ‘as explants’ in a complete explants medium for 4 weeks (Supplementary Methods).
Cell sheet preparation, labelling, handling, and transplantation
Cardiosphere-forming cells (CFCs) combined with cardiac stromal cells were seeded on double-coated plates (poly-L-lysine and collagen type IV from human placenta) in cardiosphere growing medium (Supplementary Methods). The sheets created from the same cell donors were divided into two groups,
one for transplantation and the other for characterization by immunostaining and RT–PCR (Supplementary Methods).
Prior to transplantation, rat cell sheets were labelled with 2 mM 1,1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine, DiI, for tracking transplanted cells in rat host myocardium (Molecular Probes, Eugene, OR). Sheets created using human cells were transplanted unlabelled. Sheets were gently peeled off the collagen-coated plate and folded twice to form four layers. The entire sheet with 200 ml of media was
  • gently aspirated into the pipette tip,
  • transferred to the supporting polycarbonate filter (Costar) and
  • spread off by adding media drops on the sheet (Figure 2A).
Polycarbonate filter was used as a flexible mechanical support for cell sheet to facilitate handling during the transplantation. Immediately after LAD occlusion, the cell sheet was transplanted onto the infarcted area, allowed to adhere to the ventricle for 5–7 min, and the filter was removed before closing the chest (Figure 2A).

Cardiac function

Three weeks after MI, closed-chest in vivo cardiac function was measured using a Millar pressure conductance catheter system (Millar Instruments, Houston, TX) (Supplementary Methods).

Cell sheet survival, engraftment, and cell migration

Rat host myocardium and cell sheet composition after transplantation were characterized by immunostaining (Supplementary Methods). Rat-originated cells were traced by DiI, while human-originated cells were identified by immunostaining with anti-human nuclei or human lamin antibodies.
  1. To assess sheet-originated cardiomyocytes within the host myocardium, the number of cells positive for both human nuclei and myosin heavy chain (MHC) (human sheet); or both DiI and MHC (rat sheet) were counted.
  2. To assess sheet-originated capillaries within the rat host myocardium, the number of cells positive for both human nuclei and von Willebrand factor (vWf) (human sheet); or both DiI and vWf (rat sheet) were counted. Cells were counted in five microscopic fields within cell sheet and area of infarct (n = 5). The number of cells expressing specific markers was normalized to the total number of cells determined by 40,6-diamidino-2-phenylindole staining of the nuclei DNA.
  3. To assess the survival of transplanted cells, sections were stained with Ki-67 antibody followed by fluorescent detection and caspase 3 primary antibodies followed by DAB detection (Supplementary Methods).
  4. To evaluate human sheet engraftment, sections were stained with human lamin antibody followed by fluorescent detection (Supplementary Methods).
  5. Rat host inflammatory response to the transplanted human cell sheet 21 days after transplantation was evaluated by counting tissue mononuclear phagocytes and neutrophils (Supplementary Methods).

Imaging

Images were captured using Olympus IX70 confocal microscope (Olympus Corp, Tokyo, Japan) equipped with argon and krypton lasers or Olympus IX-51 epifluorescence microscope using excitation/emission maximum filters: 490/520 nm, 570 /595 nm, and 355 /465 nm. Images were processed using DP2-BSW software (Olympus Corp).

Statistics

All data are represented as mean ± SE Significance (P < 0.05) was deter-mined using ANOVA (StatView).

Results

Generation of cardiospheres

Cardiospheres were generated from atrial tissue explants. After 7–14 days in culture, a layer of stromal cells arose from the attached explants (Supplementary material online, Figure S2a). CFCs, small phase-bright single cells, emerged from explants and bedded down on the stromal cell layer (Supplementary material online, Figure S2b).
  • After 4 weeks, single CFCs, as well as cardiospheres (spherical colonies generated from CFCs) were observed (Supplementary material online, Figure S2c).
Cellular characteristics of cardiospheres in vitro
Immunocytochemical analysis of dissociated cardiospheres revealed that
  • 30% of cells were c-Kitþ indicating that the CFCs maintain multi-potency. About
  • 22 and 28% of cells expressed a, b-MHC and cardiac troponin I, respectively.
These cells represent an immature cardiomyocyte population because they were smaller (10–15 pm in length vs. 60–80 pm for mature cardiomyocytes) and no organized structure of MHC was detected. Furthermore
  • 17% of the cells expressed a-smooth muscle actin (SMA) and
  • 6% were positive for vimentin,
    • both are mesenchymal cell markers (Supplementary material online, Figure S3a and b).
  • Less then 5% of cells were positive for endothelial cell marker; vWf.
Cell characteristics of human cardiospheres are similar to those from rat tissues (Supplementary material online, Figure S3c).
Cardiospheres were further characterized based on the expression of c-Kit antigen. RT–PCR analysis was performed on both c-Kitþ and c-Kit2 subsets isolated from re-suspended cardiospheres. KDR, kinase domain protein receptor, was recently identified as a marker for cardiovascular lineage progenitors in differentiating embryonic stem cells.21 Here, we found that
  • the c-Kitþ cells were also Nkx2.5 and GATA4-positive, but were low or negative for KDR (Supplementary material online, Figure S3d). In contrast,
  • c-Kit2 cells strongly expressed KDR and GATA4, but were negative for Nkx2.5.
  • Both c-Kitþ and c-Kit2 subsets did not express Isl1, a marker for multipotent secondary heart field progenitors.22
Characteristics of cell sheet prior to transplantation
The cell sheet is a layer of cardiac stromal cells in which the cardiospheres were incorporated at a frequency of 21 ± 0.5 spheres per 100,000 viable cells (Figure 1A). The average diameter of cardiospheres within a sheet was 0.13 ± 0.02 mm and their average area was 0.2 ± 0.06 mm2 (Figure 1A). After sheets were peeled off the plate, it exhibited a heterogeneous thickness ranging from 0.05– 0.1 mm (n 1/4 10), H&E staining (Figure1B) and Masson’s Trichrome staining (Figure 1C) of the sheet sections revealed tissue-like organized structures composed of muscle tissue intertwined with streaks of collagen with no necrotic core. Based on the immunostaining results, sheet compiled of several cell types including
  • SMAþ cardiac stromal cells (50%),
  • MHCþ cardiomyocytes (20%), and
  • vWfþ endothelial cells (10%) (Figure 1D and E).
  • 15% of the sheet-forming cells were c-Kitþ suggesting the cells multipotency (Figure 1E).
  • Cells within the sheet expressed gap-junction protein C43, an indicator of electromechanical coupling between cells (Figure 1D).
  • 40% of cells were positive for the proliferation marker Ki-67 suggesting an active cell cycle state (Figure 1D, middle panel).
Human sheet expressed genes
  1. known to be upregulated in undifferentiated cardiovascular progenitors such as c-Kit and KDR;
  2. cardiac transcription factors Nkx2.5 and GATA4; genes related to adhesion, cell homing, and
  3. migration such as ICAM (intercellular adhesion molecule), CXCR4 (receptor for SDF-1), and
  4. matrix metalloprotease 2 (MMP2).
No expression of Isl1 was detected in human sheet (Figure 1F).
sheet transplant on MI_Image_2
Figure 1 Cell sheet characteristics. (A) Fully formed cell sheet. Arrow indicates integrated cardiosphere. (B) H&E staining; pink colour (arrowhead) indicates cytosol and blue (arrows) indicates nuclear stain. Note that there is no necrotic core within the cell sheet. (C) Masson’s Trichrome staining of sheet section. Arrowhead indicates collagen deposition within the sheet. (D and E) Sheet sections were labelled with antibodies against following markers: (D) vWf (green), Ki-67 (green), C43 (green); (E) c-Kit (green), MHC (red), SMA (red) as indicated on top of each panel. Nuclei were labelled with blue fluorescence of 40,6-diamidino-2-phenylindole (DAPI). (F) Gene expression analysis of the cell sheet. Scale bars, 200 pm (A) or 50 pm (B–E).

Cell sheet survival and proliferation

Two approaches were used to track transplanted cells in the host myocardium.
  • rat cell sheets were labelled with red fluorescent dye, DiI, prior to the transplantation.
  • the sheet created from human cells (human sheet) were identified in rat host myocardium by immunostaining with human nuclei antibodies.
DiI-labelling together with trichrome staining showed engraftment of the cardiosphere-derived cell sheet to the infarcted myocardium (Figure 2B–D). In vivo sheets grew into a stratum with heterogeneous thickness ranging from 0.1–0.5 mm over native tissue. The percentage of Ki-67þ cells within the sheet was 37.5 ± 6.5 (Figure 2F) whereas host tissue was mostly negative (except for the vasculature).
To assess the viability of transplanted cells, the heart sections were stained with the apoptosis marker, caspase 3. A low level of caspase 3 was detected within the sheet, suggesting that the majority of transplanted cells survived after transplantation (Figure 2G).
sheet transplant on MI_Image_3
Figure 2 Transplantation and growth of cell sheet after transplantation.
(A) Sheet transplantation onto infarcted heart. Detached cell sheet on six-well plate (left); cell sheet folded on filter (middle); and transplanted onto left ventricle (right). Scale bar 2 mm. DiI-labelled cell sheets grafted above MI area at day 3
(B) and day 21
(C) after transplantation.
(D) LV section of untreated MI rat at day 21 showing no significant red fluorescence background.
Bottom row (B–D) demonstrates the enlargement of box-selected area of corresponding top panels.
(E) Similar sections stained with Masson’s Trichrome. Section of rat (F) or human (G) sheet treated rat at day 21 after MI.
(F) Section was stained with antibody against Ki-67 (green). Cell sheet was pre-labelled with DiI (red). Nuclei stained with blue fluorescence of DAPI.
(G) Section was double stained with human nuclei (blue) and caspase 3 (brown, arrows) antibodies and counterstained with eosin.
Asterisks (**) indicate cell sheet area. Scale bars 200 mm (B–D, top row), 100 mm (B–D, bottom row, and E) or 50 mm (F, G).
Identification of inflammatory response
Twenty-one days after transplantation of human cell sheet, inflammatory response of rat host was examined. Transplantation of human sheet on infarcted rats reduced the number of mononuclear phagocytes (ED1-like positive cells) compared with untreated MI control (Supplementary material online, Figure S4a–e and l). In addition, the number of neutrophils was similar in both control untreated MI and sheet-treated sections (Supplementary material online, Figure S4f–k and m). These data suggest that at 21 days post transplantation, human cell sheet was not associated with significant infiltration of host immune cells.

Cell sheet engraftment and migration

Development of new vasculature was determined in cardiac tissue sections by co-localization of DiI labelling and vWf staining (Figure 3C). Three weeks after transplantation, the capillary density of ischaemic myocardium in the sheet-treated group significantly increased compared with MI animals (194 ± 20 vs. 97 ± 24 per mm2, P < 0.05, Figure 3A and B). The capillaries originated from the sheet ranged in diameter from 10 to 40 jim (n 1/4 30). A gradient in capillary density was observed with higher density in the sheet area which was decreased towards underlying infarcted myocardium. Mature blood vessels were identified within the sheet area and in the underlying myocardium in close proximity to the sheet evident by vWf and SMA double staining (Figure 3D).
sheet transplant on MI_Image_4
Figure 3 Neovascularization of infarcted wall. (A) Frozen tissue sections stained with vWf antibody (green). LV section of control (sham), infarcted (MI), and MI treated with cell sheet (sheet) rats. Scale bar, 100 jim. (B) Capillary density decreased in the MI compared with sham (*P < 0.05) and improved after cell sheet treatment (#P < 0.05). (C) Neovascularization within cell sheet area was recognized by co-localization of DiI- (red) and vWf (green) staining. Scale bar 100 jim. (D) Mature blood vessels (arrows) were identified by co-localization of SMA (red) and vWf (green) staining. Scale bar 50 jim.
Furthermore, 3 weeks after transplantation, a large number of labelled human nuclei positive or DiI-labelled cells were detected deep within the infarcted area indicating cell migration from the epicardial surface to the infarct (Figure 4A, B, and D). Minor or no migration was detected when the cell sheet was transplanted onto non-infarcted myocardium, sham control (Figure 4C). To evaluate engraftment of sheet-originated cells, sections were labelled with anti-human nuclear lamin antibody. Quantification of engraftment was performed using two approaches: fluorescence intensity and cell counting. Fluorescence intensity of the signal was analysed and compared for different areas of myocardium (Figure 4E–J). Since the transplanted sheets are created by human cells and are stained with human nuclear lamin-labelled with green fluorescence, the signal intensity of the sheet is set to 100% (100% of cells are lamin-positive). Myocardial area with no or limited number of labelled cells had the lowest level of fluorescence signal (13%, or 3.2 ± 1.4% of total number of cells), while
  1. the area where the cell migrated from the sheet to the infarcted myocardium had higher signal intensity (47%, or 11.9 ± 1.7% of total number of cells), indicating a higher number of sheet-originated cells are engrafted in the infarcted area.) (Figure 4K and L).
  2. Migrated cells were positive for KDR (Supplementary material online, Figure S5).
sheet transplant on MI_Image_5
Figure 4 Engraftment quantification of cells migrated from the sheet into the infarcted area of MI. Animals were treated with rat (A) or human (B–F) sheets. Cardiomyocytes were labelled with MHC antibody (A, green or B, red). Rat sheet-originated cells were identified with DiI-labelling, red (A). Arrows indicate the track of migrating cells. Human sheet-originated cells were identified by immunostaining with human nuclei antibody followed by secondary antibodies conjugated with either Alexa 488 (B, E and F, green) or AP (C, D, blue). No migration was detected when the cell sheet was transplanted onto non-infarcted myocardium (C). Heart sections were counterstained with eosin, pink (C–D). Higher magnification of area selected in the box is presented (D, right). Immunofluorescence of sheet (green) grafted to the myocardium surface (E) or cells migrated to the infarction area (F). Fluorescence profiles acrossthe cell sheet itself(G, box 1), area underlying cell sheet (I, box 2) and infarction areawith migrated cells (F, box 3). Mean fluorescence intensityofthe grafted human (K) cells was determined by outlining the region of interest (ROI) and subtracting the background fluorescence for the same region. Fluorescence intensity was normalized to the area of ROI (ii 1/4 6). (L) Percent engraftment was defined as number of lamin-positive cells divided by total number of cells per ROI. ‘M’, myocardium,’S’ sheet, ‘I’ infarction. Scale bars 100 mm (A–C, D, left, E and F), or 50 mm (D, right).
To elucidate a possible mechanism of cell migration, sections were stained to detect SDF1 and its unique receptor CXCR4. The migration patterns of cells from the sheet coincided with SDF-1 expression. Within 3 days after MI, SDF-1 was expressed in the injured myocardium (Figure 5A). At 3 weeks after MI and sheet transplantation, SDF-1 was co-localized with the migrated labelled cells (Figure 5B). PCR analysis revealed CXCR4 expression in cell sheet before transplantation (Figure 1F). However, after transplantation only a fraction of migrated cells expressed CXCR4 (Figure 5C).
sheet transplant on MI_Image_6
Figure 5 Migration of sheet-originated cells into the infarcted area. Confocal images of MI animals treated with sheets from rats (A and B) or human (C). SDF1 (green) was detected at border zone of the infarct at day 3 (A) and day 21 (B). Rat sheet-originated cells were identified with DiI-labelling (red). Note co-localization of DiI-positive sheet-originated cells with SDF1 at 21 days after MI (B). Human cells were identified by immunostaining with human nuclei antibody, red, (C). Note human cells that migrated to the area of infarct express CXCR4 (green) (C). Scale bar, 200 mm (A, B) or 50 mm (C). ‘M’, myocardium, ‘S’ sheet, ‘I’ infarct.

3.7 Cardiac regeneration

The differentiation of migrating cells into cardiomyocytes was evident by the co-localization of MHC staining with either human nuclei (Figure 6A) or DiI (Figure 6B and C). In contrast to the immature cardiomyocyte-like cells within the pre-transplanted cell sheet, the migrated and newly differentiated cells within the myocardium were about 30–50 mm in size and co-expressed C43 (see Supplementary material online, Figure S6). Cardiomyogenesis within the infarcted myocardium was observed in the sheets created from either rat or human cells.
sheet transplant on MI_Image_6
Figure 6 Cardiac regeneration. Sections of MI animals treated with human (A) or rat (B, C) sheets. Human sheet was identified by immunostaining with human nuclei antibody (green). Section was double-stained with MHC (red) antibody. Newly formed cardiomyocytes was identified by co-localization of human nuclei and MHC (yellow, arrow). (B) Rat sheet-originated cells were identified by DiI labelling (red). Section was double-stained with MHC (green) antibody. Newly formed cardiomyocytes were detected by co-localization of DiI with MHC (yellow, arrows). (C) Higher magnification of area selected in the boxes (B). Scale bars 200 mm (B), or 20 mm (A, C). ‘M’, myocardium, ‘S’ sheet, ‘I’ infarct.

Cell sheet improved cardiac contractile function and retarded LV remodelling after MI

Closed-chest in vivo cardiac function was derived from left ventricle (LV) pressure–volume loops (PV loops), which were measured using a solid-state Millar conductance catheter system. MI resulted in a characteristic decline in LV systolic parameters and an increase in diastolic parameters (Table 1). Cell sheet treatment improved both systolic and diastolic parameters (Table 1). Specifically, load-dependent parameters of systolic function: ejection fraction (EF), dP/dTmax, and cardiac index (CI) were decreased in MI rats and increased towards sham control with the cell sheet treatment (Table 1). Diastolic function parameters, dP/dTmin, relaxation constant (Tau), EDV, and EDP were increased in the MI rats and returned towards sham control parameters after sheet treatment (Table 1). However, load-independent systolic function, Emax, was decreased after MI. Treatment with human sheet improved Emax, while treatment with rat sheet had no effect (Table 1). Treatment with either rat or human sheets retarded LV remodelling; as such that it increased the ratio of anteriolateral wall thickness/LV inner diameter (t/Di) and wall thickness/LV outer diameter (t/Do) (see Supplementary material online, Table S3). However, human sheets appear to further improve LV remodelling compared with rat sheets as indicated by increased ratio of wall thickness to ventricular diameter and decreased both EDV and EDP (Table 1 and see Supplementary material online, Table S3).
Table 1 Hemodynamic parameters
Table 1. hemodynamic parameters

Discussion

The majority of the cardiac progenitor cells delivered using our scaffold-free cell sheet survived after transplantation onto the infarcted heart. A significant percentage of transplanted cells migrated from the cell sheet to the site of infarction and differentiated into car-diomyocytes and vasculature leading to improving cardiac contractile function and retarding LV remodelling. Thus, delivery of cardiac progenitor cells together with cardiac mesenchymal cells in a form of scaffold-free cell sheet is an effective approach for cardiac regeneration after MI.
Consistent with previous studies,5,11 here we showed that cardio-spheres are composed of multipotent precursors, which have the capacity to differentiate to cardiomyocytes and other cardiac cell types. When we fractioned cardiospheres based on c-Kit expression, we identified two subsets: Kitþ /KDR2/low/Nkx2.5þ and Kit2/KDRþ/ Nkx2.52(Supplementary material online, Figure S3d), which are likely reflecting cardiac and vascular progenitors.20
In the present study, delivery of cardiac progenitor cells as a cell sheet facilitates cell survival after transplantation. Necrotic cores, commonly observed in tissue engineered patches,23,24 are absent in cardiosphere sheets prior to transplantation (Figure 1B and C). Poor cell survival is caused by multiple processes such as: ischemia from the lack of vasculature and anoikis due to cell detachment from sub-strate.25 A possible mechanism of cell survival within the sheet is the induction of neo-vessels soon after transplantation due to the presence of endothelial cells within the sheet before transplantation (Figure 10). The cell sheet continued to grow in vivo (Figure 2B and C), suppressed cardiac wall thinning, and prevented LV remodelling at 21 days after transplantation (see Supplementary material online, Table S3). This maybe due to the induction of neovascularization (Figure 3), which may prevents ischemia-induced cell death (Figure 2G). Another likely mechanism of cell survival is that the cells within the scaffold-free sheet maintained cell-to-cell adhesion16 as shown by ICAM expression (Figure 1F). The cells also exhibit C43-positive junctions (Figure 10, see Supplementary material online, Figure S6), which may facilitate electromechanical coupling between the transplanted cells and the native myocardium.
We observed cell migration from the sheet to the infarcted myocardium (Figure 4A and B, E and F), which may be facilitated by the strong expression of MMP2 in the cell sheet (Figure 1F). Although, the mechanism of cardiac progenitor cell migration remains unclear, previous observations showed that SDF-1 is upregulated after MI and plays a role in bone-marrow and cardiac stem cell migration.26,27 Our data suggest that SDF-1-CXCR4 axis plays, at least in part, a role in cardiac progenitor cell migration from cell sheet to the infarcted myocardium. This conclusion is based on the following observations: (1) cell sheet expresses CXCR4 prior to transplantation (Figure 1F), (2) migrated cells are located in the vicinity of SDF-1 release (Figure 5A and B), and (3) about 20% of migrated cells expressed CXCR4. Note, not all the migrated cells expressed CXCR4 suggesting other mechanisms are involved in cell migration (Figure 5C).
Here we report that implanting cardiosphere-generated cell sheet onto infarcted myocardium not only improved vascularization but also promoted cardiogenesis within the infarcted area (Figure 6). A larger number of newly formed cardiomyocytes were found deep within the infarct compared with the cell sheet periphery. Notably the transplantation of the cell sheet resulted in a significant improvement of the cardiac contractile function after MI, as was shown by an increase of EF and decrease of LV end diastolic pressure (Table 1).
The beneficial effect of cell sheet is, in part, due to the presence of a large number of activated cardiac mesenchymal stromal cells (myofibroblasts) within the sheet. Myofibroblasts are known to provide a mechanical support for grafted cells, facilitating contraction28 and to induce neovascularization through the release of cytokines.17 In addition, mesenchymal cells are uniquely immunotolerant. In xenograft models unmatched mesenchymal cells transplanted to the heart of immunocompetent rats were shown to suppress host immune response29 presumably due to inhibition of T-cell activation.30 Consistently with previous study from our laboratory,31 here, we demonstrated host tolerance to the cell sheet 21 days after MI. Finally, phase II and III clinical trials are currently undergoing in which allogeneic MSCs are used to treat MI in patients (Osiris Therapeutic, Inc.).
In summary, our results show that cardiac progenitor cells can be delivered as a cell sheet, composed of a layer of cardiac stromal cells impregnated with cardiospheres. After transplantation, cells from the cell sheet migrated to the infarct, partially driven by SDF-1 gradient, and differentiated into cardiomyocytes and vasculature. Transplantation of cell sheet was associated with prevention of LV remodelling, reconstitution of cardiac mass, reversal of wall thinning, and significant improvement in cardiac contractile function after MI. Our data also suggest that strategies, which utilize undigested cells, intact cell–cell interactions, and combined cell types such as our scaffold-free cell sheet should be considered in designing effective cell therapy.

References

Fuchs JR, Nasseri BA, Vacanti JP, Fauza DO. Postnatal myocardial augmentation with skeletal myoblast-based fetal tissue engineering. Surgery 2006;140:100–107.
Orlic D, Kajstura J, Chimenti S, Bodine DM, Leri A, Anversa P. Bone marrow stem cells regenerate infarcted myocardium. Pediatr Transplant 2003;7(Suppl. 3):86–88.
Kawamoto A, Tkebuchava T, Yamaguchi J, Nishimura H, Yoon YS, Milliken C et al. Intramyocardial transplantation of autologous endothelial progenitor cells for therapeutic neovascularization of myocardial ischemia. Circulation 2003;107:461–468.
Iwasaki H, Kawamoto A, Ishikawa M, Oyamada A, Nakamori S, Nishimura H et al. Dose-dependent contribution of CD34-positive cell transplantation to concurrent vasculogenesis and cardiomyogenesis for functional regenerative recovery after myocardial infarction. Circulation 2006;113:1311–1325.
Beltrami AP, Barlucchi L, Torella D, Baker M, Limana F, Chimenti S et al. Adult cardiac stem cells are multipotent and support myocardial regeneration. Cell 2003;114: 763–776.
Oh H, Bradfute SB, Gallardo TD, Nakamura T, Gaussin V, Mishina Y et al. Cardiac progenitor cells from adult myocardium: homing, differentiation, and fusion after infarction. Proc Natl Acad Sci USA 2003;100:12313–12318.
Laugwitz KL, Moretti A, Lam J, Gruber P, Chen Y, Woodard S et al. Postnatal isl1+ cardioblasts enter fully differentiated cardiomyocyte lineages. Nature 2005;433: 647–653.
Pfister O, Mouquet F, Jain M, Summer R, Helmes M, Fine A et al. CD31- but Not CD31+ cardiac side population cells exhibit functional cardiomyogenic differentiation. Circ Res 2005;97:52–61.
Dawn B, Stein AB, Urbanek K, Rota M, Whang B, Rastaldo R et al. Cardiac stem cells delivered intravascularly traverse the vessel barrier, regenerate infarcted myocardium, and improve cardiac function. Proc Natl Acad Sci USA 2005;102:3766–3771.

 

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Image

Author and Reporter: Ritu Saxena, Ph.D.

Introduction

Blood vessels arise from endothelial precursors that are thin, flat cells lining the inside of blood vessels forming a monolayer throughout the circulatory system. ECs are defined by specific cell surface markers including CD31, CD34, CD105, VE-cadherin, vascular endothelial growth factor receptor 1 [VEGFR-1], VEGFR-2, Tie-1, Tie-2) that characterize their phenotype. Angiogenesis is the growth of new blood vessels from preexisting ones and is required for growth and repair. Malignancy is a pathological scenario that requires angiogenesis. The definite cellular origin of adult blood vessel-forming cells necessary for neoangiogenesis has been unknown. Weissman and fellow coworkers in their previous work indicated that the address of these cells might be local, residing in non-circulating tissue. Also, very low numbers of cells with endothelial characteristics and high proliferative potential have been reported in umbilical cord blood or in peripheral blood. The function of circulating endothelial progenitor cells and pharmacotherapy targeted at the      endogenous augmentation of these cells for their use in cardiovascular repair has been discussed in detail in a post authored by Aviva Lev-Ari on August 28, 2012.

Research

Scientists at the University of Helsinki, Finland, wanted to find out if there exists a rare vascular endothelial stem cell (VESC) population that is capable of producing very high numbers of endothelial daughter cells, and can lead to neovascular growth in adults.  They were not only able to define the characteristic cells responsible for giving rise of blood vessels in adults, but took a leap forward by generating blood vessels from a single cells from the VESC population. (Figure:  VESCs discovered that reside at the blood vessel wall endothelium. These are a small population of CD117+ ECs capable of self-renewal.  Image Courtesy: Fang et al, 2012).

The VESCs, as explained by the Fang and coworkers, reside in the blood vessel wall endothelium and constitute a small subpopulation within CD117+ (c-kit+) endothelial cells (ECs). These cells are capable of undergoing clonal expansion unlike the surrounding ECs that bear limited proliferating potential. VESC discovered in this study were found to a have a certain characteristic phenotype defined by the presence of a few surface proteins. The authors utilized the technique of FACS (Fluorescence Activated Cell Sorting) to isolate the cells capable of undergoing clonal expansion. The sorting was performed against endothelial-specific protein markers CD31 and CD15, and against CD117 and Sca-1 molecules that are expressed by many adult stem cell types including hematopoietic stem cells (HSCs) and prostate and mammary gland stem cells. The experimental results defined the surface characteristics or the phenotype of the isolated cells to be lin2CD31+CD105+Sca1+CD117+A.  A single VESC cell isolated from the endothelial population was able to generate functional blood vessels that connected to host circulation after transplantation in mouse. In cell culture, these cells were shown to generate tens of millions of daughter endothelial cells. Also, within cell culture, the isolated VESCs showed long-term self-renewal properties, bearing similarity to adult stem cells. The self-renewal capacity of VESCs was evident even in vivo, when the ‘isolated’ ECs containing VESCs retained the capacity to generate functional blood vessels during serial transplantations. The transplanted ECs were monitored with the help of Green Fluorescent protein (GFP). Fluorescent blood vessels were observed in secondary, tertiary, and quaternary transplants providing direct evidence that the GFP-tagged ECs contained VESCs with self-renewal capacity.

Furthermore, the cell culture and animal experiment results were supported by the observation that abundant CD117+ ECs were discovered in human malignant melanomas and invasive breast cancer samples.

Research relevance

The discovery of VESCs is seminal and could be of tremendous therapeutic potential. It could be useful in the following ways leading way for related research endeavors including-

  • Cell-based therapies: VESCs could be used in cell-based therapies for cardiovascular repair to restore tissue vascularization i.e., the daughter cells arising from VESCs at the target site could assist in repair by generation of  neoangiogenic ECs required for the formation of blood vessels.
  • Therapeutic target: VESCs could serve as a possible cellular and molecular target to restrain angiogenesis by inhibiting endothelial-cell proliferation thereby blocking cancer progression.

Sources:

Fang S et al, Generation of Functional Blood Vessels from a Single c- kit + Adult Vascular Endothelial Stem Cell. PLoS Biol. 2012;10(10):e1001407. http://www.ncbi.nlm.nih.gov/pubmed/23091420

News Brief: http://www.business-standard.com/generalnews/news/scientists-discover-new-blood-vessel-generating-cells/69329/

Related reading:

Cardiovascular and endothelial cells

Statins’ Nonlipid Effects on Vascular Endothelium through eNOS Activation Curator, Author,Writer, Reporter: Larry Bernstein, MD, FCAP

Cardiovascular Outcomes: Function of circulating Endothelial Progenitor Cells (cEPCs): Exploring Pharmaco-therapy targeted at Endogenous Augmentation of cEPCs Author and Curator: Aviva Lev-Ari, PhD, RN

Vascular Medicine and Biology: Macrovascular Disease – Therapeutic Potential of cEPCs Curator and Author: Aviva Lev-Ari, PhD, RN

Repair damaged blood vessels in heart disease, stroke, diabetes and trauma: Cellular Reprogramming amniotic fluid-derived cells into Endothelial Cells Reporter: Aviva Lev-Ari, PhD, RN

Stem cells in therapy

A possible light by Stem cell therapy in painful dark of Osteoarthritis” – Kartogenin, a small molecule, differentiates stem cells to chondrocyte, healthy cartilage cells Author and Reporter: Anamika Sarkar, Ph.D and Ritu Saxena, Ph.D.

Human embryonic pluripotent stem cells and healing post-myocardial infarction Author: Larry H. Bernstein, MD

Stem cells create new heart cells in baby mice, but not in adults, study shows Reporter: Aviva Lev-Ari, PhD, RN

Stem cells for the rescue of mitochondrial dysfunction in Parkinson’s disease Reporter: Ritu Saxena, Ph.D.

Stem Cell Research — The Frontier is at the Technion in Israel Reporter: Aviva Lev-Ari, PhD, RN

Research articles by MA Gaballa, PhD

Harris DT, Badowski M, Nafees A, Gaballa MA. The potential of Cord Blood Stem Cells for Use in Regenerative Medicine. Expert Opinion in Biological Therapy 2007. Sept 7(9): 1131-22.

Furfaro E, Gaballa MA. Do adult stem cells ameliorate the damaged myocardium?. Human cord blood as a potential source of stem cells. Current Vascular Pharmacology 2007, 5; 27-44.

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