Topical Application of Mesenchymal Stem Cells to Somatic Organs—A Preliminary Report (2011)

Human mesenchymal stem cells (MSCs) are increasingly being advocated in cell-based therapeutic strategies for the treatment of traumatic and degenerative diseases (1). Systemic delivery of MSCs through arterial or venous system is an appealing strategy because by selective delivery through cannulation of specific blood vessels, large amount of cells can be transplanted by infusion. However, the infused cells are mostly trapped by the lung (2). Moreover, there is a threat of arterial embolism or occlusion during intraarterial administration of MSCs. Another alternate approach is by the direct implantation of cells into target organs using Hamilton syringes. Unfortunately, this approach may pose certain risk of bleeding, and also, the trituration of cell during the preparation of cell suspension may lead to inevitably damage of the cell membranes and subsequently may adversely affect the viability of the MSCs. Therefore, a better cell delivery approach is needed to overcome these obstacles. We would like to share our recent preliminary study of the direct topical application of MSC to visceral organs. In our series of experiments, MSCs were first derived from the subcutaneous adipose tissue of transgenic Sprague-Dawley rats expressing green fluorescent protein (GFP). The MSCs expressed CD90 (79%), but not CD45, and were capable of in vitro adipogenic, chondrogenic, and osteogenic differentiation under selective culture conditions. To stimulate the mobilization of the topically applied MSCs, experimental models of severe ischemia-reperfusion injury (IRI) of kidney, liver, and small intestine were induced in test wild-type Sprague-Dawley rats (n=19) by occlusion of kidney pedicle for 40 min (3); clamping portal vein and hepatic artery and bile duct for 30 min (4); and occlusion of main vascular pedicle, together with ligation of collateral vessels to the bowel segment for 90 min (5). The IRI caused severe acute inflammation in the organs and high mortality to the test animals. Four and two test animals died immediately after releasing the clamps from liver and kidney, respectively. Two days after the IRI, 7×106 GFP-MSCs at passage 2 to 3 suspended in 200 μL phosphate buffer saline were applied directly to the organ surfaces of the ischemic kidney, liver, and small intestine of the test animals (n=9). A thin layer of fibrin glue (Tisseel, Baxter Healthcare S.A., Wallisellen, Switzerland) was then applied to the recipient surfaces. In the control groups, MSCs alone were applied to the ischemic organs (n=6) or MSCs together with fibrin glue were applied to the nonischemic organs (n=2). The trafficking of GFP-MSCs was examined by immunohistochemistry staining on serial paraffin sections with rabbit polyclonal anti-GFP (1:4000; Abcam Inc., Cambridge, UK). In five of the nine test animals, some topically applied GFP-MSCs were found histologically in the renal cortex, liver parenchyma, and small intestinal mucosa few days after cell transplant (Fig. 1). In both control groups, GFP-MSCs were not found within the liver, kidney, and small intestine. Only few topically applied GFP-MSCs resided on the organ surfaces if fibrin glue was not added.FIGURE 1.: Homing of adipose tissue–derived mesenchymal stem cells (MSCs) into the kidney, liver, and small intestine. (a) Significant inflammatory cells (arrows) are seen infiltrating into the renal cortex as a subcapsular wedge-shaped scar, characterized by tubular atrophy with interstitial fibrosis. (b) MSCs as indicated by anti-green fluorescent protein (GFP) antibody (anti-GFP, Abcam Inc., Cambridge, UK) are shown to migrate into injured renal cortical parenchyma 3 days after cell transplant. Magnification, ×100. (c) Injured liver parenchyma is replaced by fibrovascular tissue with inflammatory cells infiltrate and extravasated red blood cells. Residual hepatic lobules are indicated by star. (d) MSCs as indicated by anti-GFP antibody (anti-GFP, Abcam Inc.) are seen to migrate into the injured site of the liver 5 days after cell transplant (arrows). Magnification, ×200. (e) Fibroblastic proliferation, associated with moderate inflammation, is noted on the serosal surface of the injured site of the small intestine (arrows), where MSCs were topically applied. Sloughing of the epithelium at the tips of villous (arrow-head) and the uneven muscularis propria marked the ischemic damaged site of the small intestine. (f) Viable GFP-labeled MSCs (anti-GFP, Abcam Inc.) are seen among the fibroblastic proliferation on the serosal surface (arrows), and some MSCs have migrated into lamina propria of the injured small intestine 7 days after cell transplant (arrow head). Magnification, ×200.Although the underlying mechanism of migration of systemically infused or topically applied MSCs is unclear, the stimulation cue to mobilize MSCs depends on the physiologic and pathologic conditions of the microenvironment of the injured sites. It is believed that mobilization of MSCs and their subsequent homing to injured tissues might be mediated by various chemokines and their receptors (6). As the MSCs were topically applied to the organs 2 days after the IRI, the homed MSCs were exposed to a hostile microenvironment with hypoxia and the infiltrative activated neutrophils. We postulated that some of the migrating MSCs might have died or degenerated. This can explain that in four of the nine test animals, no viable GFP-MSCs were detected in the IRI organs. Because of its hemostatic and sealing properties, fibrin glue is used in many areas of surgery. It also acts as a delivery carrier for cultured keratinocytes (7). To the best of our knowledge, we are the first group to apply MSCs topically on the surface of somatic organs and use fibrin as an exogenous matrix to keep the MSCs in place on the recipient surfaces. Hence, the cells are held sandwiched between the organ surface and fibrin glue. Before the fibrin glue is completely absorbed, the extracellular matrix provides a three-dimensional template for the MSCs to proliferate. In vitro studies have demonstrated that a broad range of antiinflammatory, neurotrophic, immunomodulatory, and antifibrotic factors are secreted by the MSCs. More recent studies clearly showed that MSCs exerted a therapeutic effect primarily through the release of these soluble proregenerative factors that acted on local and possibly distant target tissues, instead of or in addition to differentiation and integration into impaired organs (8). Topical application offers an efficient delivery of most of the transplanted MSCs to the target organs. Moreover, the concern of MSCs retention by other normal organs is minimized. A sustained therapeutic effect of cell therapy has to be maintained by repeated administrations of cells. Topical application allows multiple MSCs transplantation without causing major trauma to the target organs and related complications. Hundreds of millions of MSCs can be directly applied to the injured organs in a single topical transplantation. Consequently, a large dosage of paracrine mediators released from the MSCs can be continuously given to the recipient organs. We aim to introduce the idea of topical application to the readers. Further studies will be conducted to compare the integration and differentiation of MSCs delivered by topical application and conventional routes of administration. If the concept of topical application of MSCs works, it will have a great impact on future development of stem-cell therapy because the therapeutic potential will be greatly enhanced. Ping Kuen Lam1 Chi Fai Ng1 Ka Fai To2 Simon Siu Man Ng1 Tony Wing Chung Mak1 Eddie Shu Yin Chan1 Anthony Wing Ip Lo2 Fernand Mac Moune Lai2 Wai San Poon1 Paul Bo San Lai1,3 1 Department of Surgery, The Chinese University of Hong Kong Prince of Wales Hospital Shatin, Hong Kong SAR, China 2 Department of Anatomical & Cellular Pathology The Chinese University of Hong Kong Prince of Wales Hospital Shatin, Hong Kong SAR, China