Promises and Limitations in the Application of Cell Therapy for Tissue Regeneration

RAPHAEL GORODETSKY

Laboratory of Biotechnology and Radiobiology, Sharett Institute of Oncology, Hadassah Hebrew University Medical Center, Jerusalem, Israel

1.1 Factors Affecting Morphogenesis and Normal Cellular Organization in Tissues and Organs

Differentiated tissues are composed of different types of specialized cells organized into complex structures that form functional organs. Mammals higher in the phylogenetic tree are estimated to have at least 250 different cell types, which develop from early common embryonic stem cells and get organized in the different organs by a complex homing process which is so far only partially understood. In spite of the vast interest in this issue, very limited information is available with regard to the full cascades which regulate the migration of progenitor cells and integration in damaged tissues.

From early embryogenesis onward, the different cell types in complex organs get organized in a manner dictated by the fine balance between their rate of proliferation and self renewal on one hand, and cell death or apoptosis on the other hand. Signaling by a large number of membrane cell receptors is a crucial factor in the organization of tissues and organs from single cells, but the exact role of only a small portion of such signals has so far been investigated and deciphered. With current knowledge it is clear that the behavior of cells in normal tissues and tumors and their targeting and homing is dictated by interactions with the extracellular matrix (ECM) and chemokines. There are also numerous direct messages transmitted by physical contact and various physical interactions, membrane potential, chemical messages, signaling agents that diffuse out from adjacent cells via cell–cell junctions, signals that are delivered systemically by the circulation and lymph pathways, or controlled stimulation by nerve ends through synapses. Not only the cells, but the whole organs do not operate autonomously and the function of one organ may be immediately affected by malfunctioning of others — even in cases where this interaction is not straightforward. For instance, lack of nerve input to tissues such as muscle will result in the degeneration of the muscles and massive cell loss in this tissue though it may be otherwise intact.

1.2 Endogenous Cell-based Repair of Damaged Tissues

Since in many disorders and injuries the repair of the damage and tissue regeneration is attributed to cells that reside in the damaged tissues, it is tempting to try to seek external delivery of reinforcement in the form of implanted differentiated or multipotent cells to help the organs overcome the complex healing process. The different options of cell-based therapies with different cell sources, matrix scaffolds and growth factors are summarized in Figure 1.1.

It should be noted that, in general, though the adult animals higher in the phylogenetic tree have the ability to somehow overcome tissue damage and repair trauma, this ability is limited to moderate injuries and in most cases such repair is also associated with the formation of non-functional scar tissue that replaces severely damaged tissue and may even interfere with the function of the healed organ. With a few exceptions, the ability to replace damaged organs is lost from the early stage of prenatal development and after birth, with a gradual decrease in the natural ability to fully repair severe damage and regenerate tissues in damaged organs. Therefore, it was suggested that organs can maintain functionality and repair spontaneously limited damage using a reservoir of an adequate stock of progenitor cells within the tissue. Such cells should exist as progenitors in soft and hard tissues with fast cell turnover. For instance in the gut, where the crypt cells divide regularly to repair and replace worn-out damaged gut tissue, and in bones, where the osteoblasts are constantly going through natural turnover which is regulated, among other factors, by physical load and growth hormones. In some cases it has been shown that the adult progenitor cells may have also limited trans-differentiation potential, which renders them partially multipotent stem cells. In most cases, the healing and cell replacement in damaged tissues is not performed by the differentiated functional cells, which have lost their proliferative potential along with their specialization. It is disputed as to what extent progenitor cells within tissues (often referred as pericytes) are involved in the repair of the damaged tissue by providing an adequate cell reservoir to replace functional cells in the tissues where they reside.

1.3 Cellular Implants for Regenerative Therapies of Damaged Organs and Tissues

The discovery of progenitors and stem cells of all kinds raised the expectation that cell-based therapy will be able to solve major problems associated with tissue regeneration, especially where autologous cell implants are involved (Figure 1.2). With these high expectations, numerous studies have been published in this field of which only a few showed significant prospects of feasibility.

The simple approach of progenitor cells delivery in suspension for the induction of organ regeneration seems highly appealing. Nevertheless, in most cases where this approach has been investigated, it was not found to provide an easy and straightforward solution. Even allogeneic cells were examined, though it is expected that such cells will not survive long in the regenerated tissue due to rejection. As to autologous cells, though they are not expected to be rejected by the immune system, both experimental and early preliminary clinical works showed that in the best case only a small fraction of cells could become incorporated in the damaged tissues after local or systemic delivery as a cell suspension; this was also the case when progenitors that had been induced to differentiate specifically to the cells of the target organs were implanted. The fact that similar results were obtained in many cases with the implant of cells from syngeneic or allogeneic sources hints that the cells were probably not incorporated in the repaired tissue and that their claimed fringe effect on the regeneration of the damaged tissues was indirect.

1.4 Potential Practical Application of Stem Cells for Tissue Regeneration

1.4.1 Definition of Stem Cells

Stem cells have no clear-cut definition. In general this terminology refers to cells that are less differentiated or are part of a reservoir of unspecialized precursor which can divide and differentiate to form adult specialized cells. All these qualities can be summarized in two major properties — self-renewal by multiple cell division and the ability to differentiate into the target specialized cells. The highest degree of 'stemness' is associated with the ability of such cells to turn into a wide range of phenotypes which are found in the three main germ layers. This quality, which is also termed pluripotency, is normally associated with the early embryonic stage. With the progress in embryonic development, most cells irreversibly lose their plasticity to trans-differentiate while irreversibly shutting down the expression of most genes by an epigenetic process of non-reversible gene inactivation. Nevertheless, some small populations of cells with the ability to form a few cell types, mostly of the same germ layer, still remain in adults and may contribute to the maintenance of the adult organs and their ability to overcome damage. Those are called multipotent stem cells. The other feature of stem cells is associated with their ability to proliferate and have multiple divisions. This ability is believed to be mainly associated with the ability of stem cells to maintain and regenerate their longer chromosomal telomeres, which are normally shortened in specialized adult cells along cell divisions and with aging.

1.4.2 Somatic ('Adult') Multipotent Stem Cells

The first clonogenic stem cells were first identified in bone marrow, where they could replace radiation-irradiated bone marrow. This is also the organ from which hematopoietic stem cells have been routinely and successfully used for the last few decades in bone marrow transplantation (termed bone marrow hematopoietic stem cell transplantation — HSCT) to treat many disorders associated with a malfunctioning or malignant hematopoietic system. Such stem cells may derive from different sources besides bone marrow, including mobilized blood (growth factors induced acceleration of hematopoietic cell generation) or cord blood. But in addition to the vast number of hematopoietic stem cells, a fraction of mesodermal cells can be isolated from the stroma of bone marrow. They were initially termed stromal cells which can be induced to differentiate to various cell phenotype of the mesenchymal germ layer. There- fore, these cells have been referred to in the last decade as mesenchymal stem cells. Among other major sources that could provide such multipotent cells are the adipose derived progenitor, which can be available in high numbers from simple liposuction procedures. The easily accessible raw material renders it a major candidate for future regenerative medicine.

A claim for the existence of a naturally occurring subpopulation within adult bone marrow derived stem cell progenitors that are multipotent (multipotent adult progenitor cells — MAPC), which can produce cell types of the three germ layers was proposed and gained a lot of attention. Some of the data behind these findings were eventually found to be problematic and a relevant publication was later withdrawn due to a claim that part of the data were ?awed. This could serve as a good example as how too high expectations in this field can distort the scientific integrity of the bench researchers who are keen to deliver in spite of objective unsolved difficulties.

1.4.3 'Embryonic' Pluripotent Stem Cells

Researchers discovered ways to derive embryonic stem cells from early mouse embryos in 1981. The detailed study of the biology of mouse stem cells led to the discovery of a method to derive stem cells from human embryos and grow them in the laboratory. The embryos used in these studies were created for reproductive purposes through in vitro fertilization procedures. The isolated cells could be grown with feeder cells in special medium conditions. They produced non-differentiated cell lines that could be induced to differentiate to cells from different germlines. Their other main feature is their tendency to form spontaneously teratomas — tumors of a mixture of different partially differentiated embryonic tissues. This turned out to be the main obstacle to their use apart from the difficulties in their derivation and expansion. Their tendency to regain the donor human leukocyte antigen (HLA) phenotype after differentiation and thereby to be rejected post-implantation is another major concern in the attempt to use such cells for tissue regeneration.

1.4.4 'Induced' Pluripotent Stem Cells

In 2006 a breakthrough was made with the identification of conditions that would allow some specialized adult cells to be 'reprogrammed' genetically to assume a stem cell-like state and reverse their epigenetic barriers. This new type of stem cells was termed 'induced pluripotent stem cells' (iPS cells). By practicing this approach any adult cell could be 'reversed' to embryonic-like pluripotent cells by the introduction of a selected set of regulatory genes, either by viral vectors or other non-viral transfection. The advantage of this approach is that it could provide individualized therapy with no need to develop numerous cell lines and without the risk of rejection due to the self origin of the donor cell. It was suggested that iPS cells have a lower proliferative potential than embryonic stem cells and in that perspective they may behave more like other adult progenitors. It was also proposed that they can form teratomas or be hazardous due to the use of viral vectors. These issues pose a major difficulty in their future application.

1.5 Regenerative Medicine

1.5.1 The Combination of Artificial Scaffolds and Cells

Regenerative medicine was initiated a few decades ago under the title of 'tissue engineering'. This field has been led by researchers many of whom originated from engineering, technical and material science.

The basis of tissue engineering is to try to replace non-functioning tissues and organs by their reconstruction, or to repair damaged organs by artificial cellular implants. The rational is that the cells that handle normal wear may have only limited ability to handle major damage. Therefore, it was proposed to aid natural spontaneously occurring cellular maintenance–repair by external intervention with major biological cellular components that can replace missing functional cells in the damaged tissues. Essentially, this approach is based on the introduction of the tissue derived cells or stem cells, which could be delivered either directly into damaged organs or through the circulation, counting on the possible homing of the cells to replace the nonfunctional cells and thereby help repair or even reconstruct failing organs (Figure 1.1).

This approach is based on the expectation that complex biological systems could operate according to the principles behind sophisticated mechanics and material science. It was anticipated that components such as cells and scaffolds (Figure 1.1) could efficiently replace the naturally organized cells in the tissues and organs.

Indeed, tissue engineering has captured the imagination of professionals, as well as non-professionals, with a logarithmic increase of works on this direction. Nevertheless, even the early works in this field indicated that there are enormous drawbacks in trying to use cells from different organs to help repair and enhance the healing of organs, not mentioning their reengineering. Most techniques that have been proposed are based on in vitro prepared cell-bearing scaffolds, synthetic or biological polymer materials — either with the differentiated cells of the damaged tissue or pluripotent stem cells with the addition of a cocktail of growth factors. Nevertheless, long experimental records over the years indicate that tissues cannot so easily be engineered by this simplified approach as implantable functional tissue replacement.

1.5.2 Main Biological Di?culties Associated with Tissue Engineering

Matrices for tissue engineering need to be cell friendly to allow cells to load and attach, to secrete extracellular matrices, to proliferate and to gain functionality. For in vitro studies this may be sufficient, but for in vivo applications, the problem is that most artificial matrices are conceived by the body as foreign bodies and are prone to induce inflammation and rejection. In this process the cells are also prone to be lost within the inflamed area. A matrix containing foreign proteins may also be immunogenic. Among the more practical approaches to overcome this problem is the attempt to reduce rejection by implants coated with relatively inert polymers or made of human derived or modified proteins and macromolecules that are expected to be accepted by the recipient's immune system.