In the first reported instance of using stem cells to bioengineer a functional human organ, Paolo Macchiarini and his research group used a patient’s own stem cells to generate an airway, specifically a bronchus, and successfully grafted it into the patient to replace her damaged bronchus (See Figure 1). Macchiarini’s group bypassed the problem of immune rejection by using the patient’s own stem cells. Additionally, by combining a variety of bioengineering efforts, no synthetic parts were involved in the creation of the organ; it was made entirely of cadaveric and patient-derived tissues (Macchiarini et al., 2008; Hollander et al., 2009).

Figure 1. In order to create a patient-compatible replacement bronchus, Macchiarini’s group removed and decellularized a trachea from a cadaveric donor, grew cells removed from the patient on the trachea in a bioreactor, and then transplanted the bioengineered airway into the patient, successfully replacing their defective bronchus (Macchiarini et al., 2008).

The relatively unique and tragic situation of the patient led Macchiarini’s group to test this novel organ transplant on her, which had previously been tried in mouse and pig models. Due to a severe tuberculosis infection, the 30-year-old female patient’s left bronchus had become near-collapse; breathing was so impaired that the patient could no longer carry out simple domestic chores. After several other approaches did not succeed in fixing the bronchus, it was decided that the best option was to remove and replace the bronchus. Normally replacement of large airway pieces and other organs is a significant problem because the patient must be on immunosuppressant medications for life to prevent rejection of the new tissue, and this can shorten the patient’s lifespan by 10 years on average; using the patient’s own stem cells got around rejection (Macchiarini et al., 2008; Hollander et al., 2009).

To create the replacement bronchus, a cadaveric donor airway was obtained and decellularized, or treated so that all donor cells would be removed. A segment of trachea was removed from a cadaveric donor and all connected tissues carefully detached. To prevent immune rejection by the patient, which can be caused by the presence of foreign cells and different major histocompatibility complexes (MHC), all cells and parts of cells had to be removed from the donor trachea. To ensure complete removal of all donor cellular components, the trachea underwent an extensive, previously established decellularization procedure over a period of 6 weeks, which involved the trachea being incubated with detergents and deoxyribonucleases (enzymes that degrade DNA) for 25 cycles (Macchiarini et al., 2008; Conconi et al., 2005). The researchers confirmed that donor cells, including MHC-positive cells, were absent, leaving only the cartilage of the trachea intact (Macchiarini et al., 2008).

The decellularized trachea acted as a scaffold for the patient’s cells to be grown on; the stripped airway was incubated in a novel bioreactor with two different kinds of cells from the patient. Epithelial cells were removed from the mucosa, or moist tissue lining, of the patient’s right bronchus. These cells were taken and cultured, or grown, inside the donor trachea. The second type of cell used was chondrocytes. To create chondrocytes the researchers removed bone marrow from the patient and isolated out a population of mesenchymal stem cells (MSCs). The MSCs were induced to differentiate into, or become, chondrocytes using a standard protocol (i.e. specific factors were added to the growth media) for three days. These chondrocytes were seeded on the outside of the trachea. The cells were grown in different media used inside and outside of the bioreactor, media specific to the epithelial cells or chondrocytes. The cells were cultured on the trachea in the bioreactor for four days, at which point the researchers had bioengineered a human airway lacking any synthetic parts (Macchiarini et al., 2008).

The portion of the patient’s left bronchus that was near-collapse was removed and successfully replaced by the bioengineered trachea, now acting as a segment of bronchus. After a month in the patient, the transplanted trachea was indistinguishable from a normal bronchus, as compared to the patient’s unaffected right bronchus and the surrounding bronchus tissue. The transplanted airway quickly also displayed completely normal function (Macchiarini et al., 2008). One year later, the graft and patient are still doing fine (Asnaghi et al., 2009).

While the case of this successfully bioengineered and transplanted organ is a breakthrough, improvements are needed to make such transplants feasible. Because Macchiarini’s group used a donor graft, the original cadaveric trachea segment, these transplants are limited by available donors. It is hoped that research efforts will lead to fully-tissue engineered organ transplants without the need of such donor grafts. If this is possible, the current shortage of donor tissue and organs can be dealt with and a large aging population can be much more effectively treated (Hollander et al., 2009).

Aside from Macchiarini’s report, several other research groups have made breakthroughs in bioengineering organs and tissues recently. One group reported creating skeletal muscle segments using a synthetic scaffold to shape and grow cells on (Bian and Bursac, 2009). Specifically, these researchers used a silicon-based polymer (polydimethylsiloxane, or PDMS) to create micromolds with pegs, or elongated posts, sticking up from the molds. Muscle cells in a gel solution were poured onto the mold and polymerized together. This created a porous skeletal muscle network that was densely packed, with uniformly aligned muscle fibers that spontaneous contracted at the macroscopic level. In the future this approach could create customized, functional skeletal muscle tissue for reconstructing damaged muscle (Bian and Bursac, 2009). Similarly, another group discusses potential in using stem cells to rescue damaged heart muscles (Shimizu et al., 2009). Researchers are also investigating the feasibility of using epithelial stem cells in bioengineered intestines, based on polymer scaffold experiments performed in rats (Day, 2006). Intestinal transplantation, often needed for short bowel syndrome caused by a variety of reasons, is a significant problem because of the extremely active immune system of the intestines (Day, 2006). Other researchers are focusing on the great potential of mesenchymal stem cells (such as were used in Macchiarini’s report) in general wound healing; these cells can differentiate into many different kinds of cells, be isolated in significant numbers, potentially migrate to areas they are needed in, and may be immunosuppressive (Fu and Li, 2009). The use of nanomaterials, which can mimic proteins on the surface of cells and tissues, also hold much potential for future scaffold designs in regenerative medicine (Zhang and Webster, 2008).

While Macchiarini’s patient represents a significant breakthrough, it is still a single success that must be repeated to be proven. The transition to the clinic of other stem cell-based regenerative therapies will also require extremely careful characterization of each individual procedure. There are still many obstacles to overcome before such therapies can become common practice. Those interested in receiving stem cell therapies should be aware of the possible risks involved; the Department of Health’s Gene Therapy Advisory Committee lists such potential hazards associated with undergoing stem cell therapies.

References

Asnaghi, M. A., Jungebluth, P., Raimondi, M. T., Dickinson, S. C., Rees, L. E. N., Go, T., Cogan, T. A., Dodson, A., Parnigotto, P. P., Hollander, A. P., Birchall, M. A., Conconi, M. T., Macchiarini, P., and Mantero, S. A double-chamber rotating bioreactor for the development of tissue-engineered hollow organs: From concept to clinical trials. Biomaterials. 2009. 30(29): 5260-5269.
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Bian, W. and Bursac, N. Engineered skeletal muscle tissue networks with controllable architecture. Biomaterials. 2009. 30(7): 1401-1412.
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Conconi , M. T., De Coppi, P., Di Liddo, R., Vigolo, S., Zanon, G. F., Parnigotto, P. P., and Nussdorfer, G. G. Tracheal matrices, obtained by a detergent-enzymatic method, support in vitro the adhesion of chondrocytes and tracheal epithelial cells. Transpl. Internat. 2005. 18(6): 727-734.
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Day, R. M. Epithelial stem cells and tissue engineered intestine. Curr. Stem Cell Res. Ther. 2006. 1(1): 113-120.
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Fu, X. and Li, H. Mesenchymal stem cells and skin wound repair and regeneration: possibilities and questions. Cell and Tiss. Res. 2009. 335(2): 317-321.
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Hollander, A., Macchiarini, P., Gordijn, B., and Birchall, M. The first stem cell-based tissue-engineered organ replacement: implications for regenerative medicine and society. Regen. Med. 2009. 4(2): 147-148.
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Macchiarini, P., Jungebluth, P., Go, T., Asnaghi, M. A., Rees, L. E., Cogan, T. A., Ddson, A., Martorell, J., Bellini, S., Parnigotto, P. P., Dickinson, S. C., Hollander, A. P., Mantero, S., Conconi, M. R., Birchall, M. A. Clinical transplantation of a tissue-engineered airway. The Lancent. 2008. 372(9655): 2023-2030.
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Shimizu,T., Sekine, H., Yamato, M., Okano, T. Cell Sheet-Based Myocardial Tissue Engineering: New Hope for Damaged Heart Rescue. Curr. Pharm. Design. 2009. 15(24): 2807-2814.
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Zhang, L., and Webster, T. J. Nanotechnology and nanomaterials: Promises for improved tissue regeneration. Nanotoday. 2009. 4(1): 66-80.
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Image of “Macchiarini’s Bioengineered Bronchus Replacement” was modified from Wikipedia and redistributed freely as it is in the public domain.

In salamanders, when a limb is severed the resultant limb bud undergoes a distinct process to regenerate the lost limb. The epithelial layer quickly spreads across the amputation site, closing the wound within 24 hours (Mescher, 1996). This epithelial layer thickens and becomes what is referred to as the wound epithelium (WE). As the immune system responds to the injury, macrophages and neutrophils arrive to clean up the wound site beneath the WE. The existing injured tissues and cells are broken down as well as the extracellular matrix, which is made up of proteins that surround cells to hold them together and stimulate normal cellular functions. It was thought that at this time in the regenerative process other resident cells below the WE become multipotent mesenchymal stem cells (MSCs) (see Figure). These eventually form a mass of MSCs called a blastema (Mescher, 1996; Brockes and Kumar, 2005). The blastema was thought to contain a homogenous group of pluripotent stem cells that had “dedifferentiated” or “redifferentiated,” meaning they had reverted back from their committed fates to function as very potent stem cells in order to recreate the limb. The WE stimulates the cells in the blastema to proliferate, making new cells and extracellular matrix, though more than is required for simple repair; the WE signals the blastema cells to regenerate the entire lost limb (Mescher, 1996; Kragl et al., 2009).

Limb regeneration in the salamander after limb amputation (time course going from the top down). Shortly after the limb is amputated, the epithelium layer covers the exposed limb bud, forming the wound epithelium (WE). A group of stem cells collects below this layer, forming the blastema (at the tip of the bud). The WE signals the stem cells below it to rebuild the limb, recreating the limb from the point of injury out towards the hand. The final regenerated limb is indistinguishable from the original.

Recently, Elly Tanaka’s group showed that multiple different groups of stem cells with relatively limited fates, being only multipotent or unipotent, may actually regenerate the salamander limb, in contrast to the previously held belief that one homogenous group of pluripotent stem cells was responsible (Kragl et al., 2009). The group labeled the major tissue types in the limb (using green fluorescent protein [GFP]) to track the tissue types during regeneration. While the blastema does appear histologically homogenous, they found it may be made up of all the different tissue types found in the complete limb; all the cells in the blastema may be types of progenitor cells that can become only one or two specific adult tissue types. In short, many different cell types may coordinate to recreate the limb (Kragl et al., 2009).

Only one tissue cell type in the blastema was able to differentiate into a cell of a different tissue layer in Tanaka’s experiments. Specifically, Tanaka’s group reported that precursor cells for muscle, epidermis, cartilage, or Schwann cells (neural cells) could only create muscle, epidermis, cartilage, or Schwann cells, respectively; each of these cell types was limited to become its own cell type. The dermis tissue layer was the only cell type found to be able to become more than one fate; the dermis could become both dermis and skeleton/cartilage (but not muscle or Schwann cells). Dermis and cartilage have a common developmental origin in the mesoderm, which helps explain why the dermis layer cells could become both of these cell types. Additionally, the group investigated whether these progenitors know their proper final position in the limb along the proximal-distal (i.e. shoulder-hand) axis. The researchers found that cartilage precursors do not have such positioning abilities, while the Schwann cells do. This indicates that the positional identity is tissue specific (Kragl et al., 2009).

While this research was conducted using salamanders, it may be quite relevant for future regenerative medicine research in humans; it shows that we may not need a pluripotent state for complex tissue regeneration, but instead could use multiple different stem cells with much more restricted fates. However, this does not necessarily make the process more feasible technically, but it may give researchers a more focused direction for future studies. To read other coverage of this ground-breaking work by Tanaka’s group, reported just last month, take a look at (Baker, 2009) or (Johnson, 2009).

References

Baker, M. Regenerating limb tissue may not dedifferentiate. Nat. Rep. Stem Cells. 2009.
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Brockes, J. P. and Kumar, A. Appendage Regeneration in Adult Vertebrates and Implications for Regenerative Medicine. Science. 2005. 310(5756): 1919-1923.
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Johnson, S. L. Memory of Fate and Position, Colorized. Dev. Cell. 2009. 17(1): 5-6.
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Kragl, M., Knapp, D., Nacu, E., Khattak, S., Maden, M., Epperlein, H. H., Tanaka, E. M. Cells keep a memory of their tissue of origin during axolotl limb regeneration. Nature. 2009. 460: 60–65.
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Mescher, A. L. The cellular basis of limb regeneration in urodeles. Int. J. Dev. Biol. 1996. 40: 785-795.
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Original “Salamander Limb Regeneration” image modified from the Wikimedia Commons and redistributed freely as it is under GNU Free Documentation License.

The endoderm layer later becomes skin (epidermis) and the nervous system, the ectoderm becomes the digestive tract and respiratory system, and the mesoderm becomes bone, blood, muscles, connective tissue, and several organs (heart, kidney, and gonads).

However, calling MSCs “mesenchymal” can be misleading. Because this term refers to a precursor of the large MSC family, it is referring to an embryonic tissue, though the descendant MSCs can be found in both fetal and adult tissues. MSCs have been isolated from adult muscle, bone marrow, adipose tissue, cartilage, bone, potentially teeth (Caplan, 2005) as well as some fetal tissues (fetal liver, lung, amniotic fluid, and umbilical cord) (Phinney and Prockop, 2007). The MSCs isolated from any one of these tissues are multipotent and are usually shown to be MSCs by being able to differentiate into at least three different, standard mesenchymal cell types: osteocytes (bone), chondrocytes (cartilage), and adipocytes (fat) (Baksh et al., 2004). There is much evidence, though somewhat inconsistent, showing that MSCs can also differentiate into neuronal cells, which may be from mesenchyme derived from the endoderm instead of the mesoderm (Gilbert, 2003; Phinney and Prockop, 2007). Overall, MSC differentiation potentials can vary depending on what mesenchyme-derived tissue the MSCs were harvested from (Phinney and Prockop, 2007). However, MSCs cannot become hematopoietic cells (which are derived from hematopoietic stem cells), even though these cells are derived from the mesenchyme, making the label “mesenchymal” more deceptive (Gilbert, 2003; Caplan, 2005).

Though MSCs have a relatively long history, only recently have they been fully recognized as a valid stem cell family and their presence in a large array of tissue types discovered. From the 1960s to 1970s, MSCs were mainly studied from bone marrow and cartilage and mostly characterized in model organisms (Friedenstein et al., 1974; Caplan, 2005; Bianco et al., 2008). Specifically, in the 1970s Friedenstein’s group discovered that certain cells isolated from bone marrow can create clonal colonies all descendent from one original cell, and, furthermore, the colonies derived from the single cell precursor can become multiple different cell types (Friedenstein et al., 1974; Bianco et al., 2008). However, because hematopoietic stem cells were already a known stem cell population residing in bone marrow, it was many years before MSCs were widely accepted as a second stem cell population within the bone marrow. The term “mesenchymal stem cell” was later coined in 1991 by Arnold Caplan, but not widely used until 1999, though, as discussed above, its appropriateness is still in question (Bianco et al., 2008). In the 1990s progress was made using human MSCs in optimizing preservation and isolation of these cells, as well as trials in regenerative medicine. Just in the last decade, it has been found that MSCs can be isolated from skeletal muscle, adipose tissue, umbilical chords, the circulatory system, potentially dental pulp, amniotic fluids, and fetal tissues (Phinney and Prockop, 2007).

To improve future MSC applications, standardization of practices is important, and a full understanding of their potential uses in regenerative medicine is essential. Though MSCs are made up of a wide variety of cell types, most share some common proteins expressed on their cell surface that can classify them as a MSC in an assay, to some degree. However, there are no standard isolation methods for MSCs and the harvested populations, which are themselves quite heterogeneous, vary depending on the donor (Phinney and Prockop, 2007). Despite the apparent need for standardization, MSCs are becoming increasingly important in the field of regenerative medicine, having great potential for tissue repair. Specifically, MSCs have properties that inhibit inflammation and immune responses, making them ideal for this field (Phinney and Prockop, 2007). As set protocols are put into place and the large family of MSCs continues to be better understood, MSCs hold the promise of being major players in the future world of regenerative medicine.

References

Baksh, D., Song, L., Tuan, R. S. Adult mesenchymal stem cells: characterization, differentiation, and application in cell and gene therapy. J. Cell. Mol. Med. 2004. 8(3): 301-16.
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Bianco, P., Robey, P. G., Simmons, P. J. Mesenchymal Stem Cells: Revisiting History, Concepts, and Assays. Cell Stem Cell. 2008. 2(4): 313-9.
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Caplan, A. I. Review: Mesenchymal Stem Cells: Cell–Based Reconstructive Therapy in Orthopedics. Tissue Eng. 2005. 11(7-8): 1198-211.
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Conrad, C., Niess, H., Huss, R., Huber, S., von Luettichau, I., Nelson, P. J., Ott, H. C., Jauch, K., Bruns, C. J. Multipotent Mesenchymal Stem Cells Acquire a Lymphendothelial Phenotype and Enhance Lymphatic Regeneration In Vivo. Circulation. 2009. 119: 281-9.
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Friedenstein, A. J., Deriglasova, U. F., Kulagina, N. N., Panasuk, A. F., Rudakowa, S. F., Luria, E. A., Ruadkow, I. A. Precursors for fibroblasts in different populations of hematopoietic cells as detected by the in vitro colony assay method. Exp. Hematol. 1974. 2(2): 83–92.
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Gilbert, Scott F. (2003). Developmental Biology, Seventh Edition. Sunderland: Sinauer Associates Inc.

Phinney, D. G. and Prockop, D. J. Concise Review: Mesenchymal Stem/Multipotent Stromal Cells: The State of Transdifferentiation and Modes of Tissue Repair – Current Views. Stem Cells. 2007. 25:2896-902.
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Original “The Three Germ Layers” image from the Wikimedia Commons and redistributed freely as it is in the public domain.