Stem Cells in Tissue Repair and Regeneration

Summary

The field of tissue repair and wound healing has blossomed in the last 30 years. We have gone from recombinant growth factors, to living tissue engineering constructs, to stem cells. The task now is to pursue true regeneration, thus achieving full restoration of structures and their function.

For most of this discussion, we will actually mean tissue repair, with its attendant compromise of achieving rapid wound closure at the expense of regeneration. Technically, the term “wound healing” implies complete restoration of tissue (i.e.,regeneration) as the outcome. However, we believe that over time wound healing has also come to signify the overall processes taking place after injury, and more so from a biological standpoint. We should remain hopeful that true regeneration is possible in adult human tissues. Thus far, the evidence has not been very encouraging, except for fetal wound healing. Even the liver, which many erroneously cite as an example of regeneration in adults, simply gets larger after lobe resection or injury; no true regeneration takes place. We show Figure 1 to indicate the immense difficulties underlying chronic wounds. Regeneration of the tissue shown in the Figure would be extremely difficult, and would most likely first require complete removal of the non-healing and affected skin. Therefore, as we approach the topic of stem cells for wound healing/tissue repair, we must keep in mind the clinical realities and what we can achieve with the methods and agents presently available. This is not meant to be a pessimistic view, but rather to stimulate us towards our ultimate goal of achieving true regeneration.

Figure 1.

The left side of the figure shows a non-healing chronic wound due to a combination of venous and lymphatic disease. Both arrows point to the non-migrating epidermis. The solid arrow shows the steep edges at the periphery of the wound, while the dashed arrow is directed at an island of fully epithelialized tissue within the wound bed, but with continued failure of its epidermis to migrate. The right side of the figure is a diagrammatic representation of the different phases of wound healing which, in the case of chronic wounds, are not linear or predictable in their sequence. Also, different areas of the wound are likely to be in a different phase of wound healing. The dashed lines in the diagram point to the fact that this is a dynamic situation and, thus, not easily predictable.

With a focus on wound healing and tissue repair, and particularly with the immense need represented by chronic non-healing human wounds, in the 1980's the excitement was all centered on growth factors. (Falanga, 2005) However, in spite of the great potential of recombinant growth factors, multiple clinical trials involving thousands of patients failed for the most part to show any or more than modest results. This failure was surprising to most investigators and pharmaceutical companies, because growth factors were so very active on multiple cell types in vitro (including fibroblasts, endothelial cells, keratinocytes, etc.), and had been shown to accelerate healing in experimental animal models. The scientific problem is that animals do not get chronic wounds, and even innovative models could not reproduce the failure to heal that characterizes most human chronic wounds. Some investigators were not to be deterred. They hypothesized that growth factors either could not penetrate the wound bed in sufficient concentrations, or are easily broken down in the wound, or require the administration of multiple peptides in a specific sequence and at certain concentrations. We even hypothesized that growth factors are “trapped” or sequestered within the wound, for example by albumin or α₂-macroglobulin, the latter being a known scavenger molecule for PDGF. (Falanga, 2005)That hypothesis, like the others just mentioned, remains viable. Still, the problem with complex hypotheses is that, from a regulatory standpoint, it would be very difficult to move from the bench to the bedside, i.e., like using multiple growth factors in humans.

However, the field of wound healing is very important and kept moving. Tissue engineering and bioengineered skin constructs, first developed many years earlier, began to dominate that field in the 1990's. Indeed, the scientific and clinical rationale for certain constructs, especially those comprising living cells like fibroblasts, keratinocytes, or both, was that a tissue-like product could overcome the deficiencies potentially uncovered with the use of growth factors. In engineering terms, a living construct could act in a “smart” way, meaning that it would adapt to the wound microenvironment, release just enough peptide growth factors and in the right sequence, and possibly provide additional and much needed extracellular matrix proteins (ECM). In a way, this approach and hypothesis was a way to admit our own ignorance about the underpinning of failure to heal, and therefore relying on cells and constructs to do the work. It should be noted that the cells in allogeneic constructs are not detectable after 4-6 weeks, further suggesting that the constructs act in a pharmacological mode, rather than replacement therapy.(Phillips et al., 2002) There have been notable successes with the use of bioengineered skin, and indeed some are used in major burns and some are approved by the Food and Drug Administration (FDA) for use in chronic wounds. (Falanga and Sabolinski, 1999) (Marston et al., 2003) Why the use of these constructs is not even greater is interesting but remains speculative. A very dramatic turnaround in the outcome of a difficult to heal wound is sometimes achieved but is not the norm. Careful use and clinical time are needed to achieve optimal results. Indeed, the experience with growth factors and tissue engineering led us to develop the concept of wound bed preparation (WBP), whereby all aspects of the wound (and not just the need for debridement and for removing the bacterial burden) are optimized before these therapeutic agents are used in a more successful manner. (Falanga, 2000; Panuncialman and Falanga, 2009) In fact, it is probably a two-way street, in that a complex therapeutic agent (i.e., a tissue engineering construct) can also improve WBP, while the latter can in turn improve the outcome of the construct. We thought that a didactic paradigm (see below with stem cells) may be operative in difficult chronic or complex traumatic wounds, whereby the cellular and extracellular components we introduce in vivo has a beneficial effect on the resident cells of the wound bed, on the ability of keratinocytes to migrate, and even on the remodeling process. It should be added that surgeons became more interested in difficult and chronic wound around this time, and have since introduced more procedures, devices, and clinical evidence in wound management. Moreover, a proper surgical approach was always an important component of WBP.

It was not long before interest in stem cells would emerge in the field of wound healing and offer fresh hope. This interest is now dominating our time and is obscuring, perhaps unwisely, some of the useful approaches achieved with growth factors and bioengineered constructs. At first and in principle, turning to human embryonic stem cells seemed attractive, were one to overcome the ethical issues regarding their use and which may also compromise the “quality” or reproducible behavior of cells kept long-term in culture. After all, although not totipotent (i.e., able to also form placental tissues), embryonic stem cells are pluripotent and can thus give rise to all cells by using appropriate culture conditions. The ethical issues seemed to diminish overnight when in 2007 two separate publications described the development of induced pluripotent stem cells (iPS cells) from fibroblast cultures. A cocktail of genes was used for this development. One investigative team used Oct3/4, Sox2, Klf4, and c-Myc by retroviral delivery, (Takahashi et al., 2007) while the other research group used Oct4, Sox2, Nanog, Lin28 by lentiviral vectors. (Yu et al., 2007) The yield of producing a single iPS cell from fibroblast cultures was relatively low (requiring 5-10 thousand cells), but a major coup had taken place. Suddenly, human embryonic stem cells and their pluripotential capacity lost some of their attractiveness, because we were now able to develop pluripotent cells from skin fibroblasts. Over time, other methods of delivery (i.e., naked DNA, proteins, etc.) of these and other critical reprogramming genes have become possible. Questions linger about whether iPS cells are truly embryonic-like, but nobody can deny the importance of this major discovery of taking a differentiated cell back to its possible origin, like in a time machine.

For wound healing and tissue repair, however, the pluripotent stem cells remain a problem. We may not need all that pluripotentiality to treat wounds safely, and the teratoma concern and regulatory and safety hurdles remain. Immunological rejection of these cells, recently identified as an unexpected finding, is probably not so important in wound healing but that remains to be seen. However, alternatives have emerged or at least are enjoying greater attention. Among these alternatives are bone marrow mobilization and peripheral blood harvesting of stem cells with apheresis after the systemic administration of GCSF, the use of stem/progenitor cells from umbilical cord blood, the actual culturing of multipotent (giving rise to one tissue type) stem cells from bone marrow, epidermis, hair, and now sweat glands, among others. From a scientific and practical standpoint, which has ramifications for safety and regulatory oversight, an advantage of multipotent stem cells is that we can more reliably depend on certain surface markers and appearance/behavior in culture to grow the same type of cells. For example, when our group first decided to use autologous cultured cells from bone marrow in wounds, we focused on mesenchymal stem cells. Although the mesenchymal cell surface markers are not specific, the combination of such markers (i.e., lineage negative and CD34-, positivity for CD29, CD44, CD105, CD166), adherence to tissue culture plastic and cell culture appearance, and functional assays showing differentiation to bone, cartilage, and adipose tissue, taken together provided a reproducible system. (Falanga et al., 2007) We could have chosen a hematopoietic stem/progenitor cell type, which could have possibly been even more effective, but then we would not necessarily know the attributes of that cell and could not ensure consistent and reproducible cultures in our GMP facilities. The mesenchymal stem cells we have used in human wounds appear to differentiate along fibroblastic pathways and, in an animal model, do not seem to persist for more than 30 days, in spite of them being autologous. Still, they appear to be promising in difficult wounds and, as suggested by published reports, may actually decrease scarring. That would be a major advantage in burns. We are intrigued by the report that stem cells isolated from sweat glands have features highly suggestive of endothelial cells. (Danner, In Press) That brings up the issue of whether stem cell transdifferentiation or plasticity, a process that some regard as controversial, is playing a role. Some have suggested that plasticity is the result of co-purification with dormant pluripotent very small embryonic-like (VSEL) stem cells in various adult organs and tissues. (Kucia et al., 2006) VSEL stem cells might be of interest in wound healing, provided that an adequate yield from apheresis can be achieved and that the potential for tumor formation, which is associated with embryonic stem cells, is not as great.

The use of multipotent, rather than pluripotent stem cells, in wound healing appears to be beneficial and probably more practical at this point, also given the regulatory and safety concerns. Regulatory requirements keep increasing, even as (or because) we are placing more emphasis on translational research. Therefore, investigators interested in applied approaches for stem cells have to take into account whether the stem cells they choose to study will be deemed to be a relatively low risk in the field of tissue repair. Moreover, the lessons from tissue engineering constructs and from WBP have taught us that true replacement of cells may not be necessary for most wounds. The didactic paradigm we were alluding to earlier may be operative. It is indeed possible that certain subpopulations of multipotent stem cells may “teach” the wound microenvironment how to rectify the cellular and molecular phenotypic flaws that lead to impaired or failed healing. It is also plausible that recruitment of circulating stem cells or neighboring tissue differentiated cells may take place with the topical administration of multipotent stem cells. The number of delivered stem cells seems critical. In our mesenchymal stem cell studies in humans, we found that at least one million autologous cultured stem cells per cm² of wound surface were required to achieve a beneficial outcome.

In conclusion, progress continues to take place in our abilities to isolate stem cells, to characterize them, and to think of ways we can deliver them and use them in translational research. For now, multipotent stem cells have a definite advantage in being used for the acceleration of healing. This has to do with their generally favorable risks/benefits ratio. Still, we do not reject the notion that pluripotent stem cells may find an extraordinary role in wound healing. When properly handled and controlled, such cells could be directed towards diverse differentiation pathways that might bring the field of tissue repair closer to regeneration, or true wound healing.