BACKGROUND In adult mammalian organisms, multiple tissues—including the skin, blood, stomach, and intestines—are entrapped in a state of permanent regeneration; older cells are constantly shed, and the tissue is continuously being regenerated from resident stem cells. This phenomenon of “tissue renewal” was appreciated by Leblond in 1956, but the underlying mechanism has been unclear. It is now evident that a class of extracellular developmental signaling proteins, known as Wnt signals, animate the continued renewal of several mammalian tissues by fuelling stem cell activity. If the Wnt pathway is inhibited, tissue renewal is crippled. This signaling pathway is an ancient evolutionary program dating from when Wnt signals arose in the simplest multicellular organisms, in which Wnts acted as primordial symmetry-breaking signals crucial for the generation of patterned tissues during embryogenesis. In vertebrates, these signals also function in pattern maintenance: They sustain tissue renewal, enabling tissues to be continuously replenished and maintained over a lifetime. Multiple adult organs are in a state of continual regeneration. In tissues such as the skin, intestines, brain, and mammary glands, Wnt signaling proteins sustain this constant regeneration by inducing stem cells (green cells in the illustration) to grow. This leads to the robust supply of new cells (green) in order to replenish and maintain the tissue. [Image credits are available in the full article online.] ADVANCES In contrast to traditional “long-range” developmental signals, Wnts seem to act as short-range intercellular signals—acting mostly between adjacent cells. Lending credence to this notion, a membrane-tethered Wnt protein variant can fulfill most functions of a normal Wnt protein in Drosophila . Likely explaining the short-range nature of these signals, Wnt proteins are attached to a lipid and therefore are hydrophobic; they cannot freely traverse the extracellular space by themselves. This provides insight into how tissue renewal is regulated. It implies that Wnt signals emanating from the stem cell microenvironment (the “niche”) may influence adjacent stem cells without affecting a broad field of cells located farther away. The concept of an external niche, however, may have to be refined because it is clear that stem cells can sometimes act as their own niche and have unexpected developmental self-organizing capacities. Last, the widespread importance of Wnt signaling in driving tissue renewal has been revealed by the identification of Axin2 and Lgr5 , genes expressed in cells that are responding to Wnt signals. Genetically labeling Axin2 + or Lgr5 + cells in a variety of tissues has revealed that such cells fuel tissue renewal in the intestines, mammary gland, skin, and brain, among other organs. OUTLOOK The amazing continuous self-regeneration of various mammalian tissues over years and decades continues to be an enigmatic terra incognita in biology. For instance, visualization of stem cells in real-time in vivo (through intravital microscopy) has shown that when some stem cells are ablated, they are replaced by more differentiated cells that are recalled to the stem cell niche, whereupon they regain stem cell identity to effect tissue repair. Therefore, lineage barriers between stem cell and differentiated fates are not always stringent and can be traversed during times of tissue damage. Reactivated Wnt signals may be instrumental in this process, and perhaps such signals could be exploited in order to enkindle tissue regeneration after injury or disease. From a pragmatic perspective, Wnt signals have already found practical use in manipulating stem cells, enabling propagation of stem cells in vitro as self-renewing cell populations and as organoids.

An integral program for tissue renewal and regeneration: Wnt signaling and stem cell control

Familial hypercholesterolemia (FH) is an inherited disorder in humans that is caused by a deficiency of low density lipoprotein receptors (LDLRs). An animal model for FH, the Watanabe Heritable Hyperlipidemic rabbit, was used to develop an approach for liver-directed gene therapy based on transplantation of autologous hepatocytes that were genetically corrected ex vivo with recombinant retroviruses. Animals transplanted with LDLR-transduced autologous hepatocytes demonstrated a 30 to 50 percent decrease in total serum cholesterol that persisted for the duration of the experiment (122 days). Recombinant-derived LDLR RNA was harvested from tissues with no diminution for up to 6.5 months after transplantation.

http://science.sciencemag.org/content/sci/254/5039/1802.full.pdf

Long-term improvement of hypercholesterolemia after ex vivo gene therapy in LDLR-deficient rabbits.

长期以来许多学者认为独立的肝细胞移植是治疗严重肝脏疾病的有较大发展潜力的治疗方法.许多实验室已经进行了相关的动物试验.人体肝细胞移植曾在小范围、随机的个例中应用过.虽然这方面的统计数据很少,但已经体现了这种治疗方法的安全性、可行性.独立的肝细胞移植的潜力远未开发,除了尸肝外,其它可供移植的细胞资源也有待开发。

Hepatocyte transplantation

Objective. Transplantation of isolated hepatocytes in animal models has been shown to correct inborn errors of metabolism. Based on these studies and our experience with hepatocyte transplantation in a child with Crigler-Najjar syndrome, isolated hepatocyte trans- plantation was performed to attempt metabolic reconsti- tution in a male infant with severe ornithine transcar- bamylase (OTC) deficiency. Methods. An infant with an antenatal diagnosis of OTC deficiency was managed intensively to prevent hy- perammonemia. Isolated hepatocytes were obtained by collagenase perfusion of donated livers not used for transplantation. Hepatocytes were infused in batches over the first 4 weeks of life via an umbilical venous catheter positioned in the portal vein. Immunosuppres- sion consisted of tacrolimus and corticosteroids. Results. Over 4 billion viable hepatocytes were trans- planted during the first 3.5 weeks of life. A period of metabolic stability was achieved between days 20 and 31 during which normal protein intake was tolerated while phenylbutyrate was weaned. During this time, plasma ammonia and glutamine remained within normal limits. Hyperammonemia reappeared abruptly on day 31 of life. Protein tolerance diminished to baseline; metabolic sta- bility was subsequently reattained only following suc- cessful liver transplantation at 6 months of age. Conclusions. Isolated hepatocyte transplantation ap- peared to result in temporary relief of hyperammonemia and protein intolerance attributable to OTC deficiency. The metabolic stability achieved was lost after 11 days presumably because of rejection of the transplanted cells because of insufficient immunosuppression. Future at- tempts at isolated hepatocyte transplantation for inborn errors of metabolism in humans should include adequate immunosuppression and a liver biopsy as a means of proving hepatocyte engraftment and function. Pediatrics 2003;111:1262-1267; hepatocyte transplantation, infant, ornthine transcarbamylase deficiency, urea cycle disorder.

Isolated Hepatocyte Transplantation in an Infant With a Severe Urea Cycle Disorder

Pluripotent stem cells (PSCs) directed to various cell fates holds promise as source material for treating numerous disorders. The availability of precisely differentiated PSC-derived cells will dramatically affect blood component and hematopoietic stem cell therapies and should facilitate treatment of diabetes, some forms of liver disease and neurologic disorders, retinal diseases, and possibly heart disease. Although an unlimited supply of specific cell types is needed, other barriers must be overcome. This review of the state of cell therapies highlights important challenges. Successful cell transplantation will require optimizing the best cell type and site for engraftment, overcoming limitations to cell migration and tissue integration, and occasionally needing to control immunologic reactivity, as well as a number of other challenges. Collaboration among scientists, clinicians, and industry is critical for generating new stem cell-based therapies.

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Use of Differentiated Pluripotent Stem Cells in Replacement Therapy for Treating Disease

The Watanabe heritable hyperlipidemic (WHHL) rabbit reproduces human familial hypercholesterolemia due to a congenital low-density lipoprotein receptor deficiency and is characterized by elevated serum LDL cholesterol levels and early atherosclerosis. We attempted to transplant normal allogeneic hepatocytes into WHHL rabbits without chronic immunosuppression to cure the LDL receptor-deficient state. Livers from normal New Zealand White (NZW) rabbits were digested by intraportal perfusion of collagenase solution. Pure hepatocytes (PH) were obtained by Percoll gradient separation and nonparenchymal (NP) liver cells by pronase digestion. PH and NP were incubated with fluorescein isothiocyanate-monoclonal anti-rabbit class I, anti-class II, and anti-T cell antibodies and subjected to flow cytometry analysis. PH and NP were also used as stimulators in one way mixed lymphocyte-hepatocyte cultures (MLHC), before and after ultraviolet B light (UVB) exposure. Intraportal and intrasplenic injection of allogeneic PH were also performed in homozygous WHHL rabbits. PH were attached to collagen-coated dextran microcarriers (mc-PH) for intraperitoneal injection. Recipient control and transplanted WHHL rabbits received a single dose of cyclosporine subcutaneously (10 mg/kg/s.c.) at the time of transplantation. PH were mainly class I-positive (77.6%) and class II-negative (5.9%), while 31.5% of NP cells were class II-positive. In MLHC, PH did not stimulate proliferation, (stimulation index: 0.97 +/- 0.21), unlike NP (SI: 23.7). This latter response was abrogated by prior exposure of NP to UVB light. Intraportal injection of PH (n = 4) reduced serum LDL cholesterol to 60% of baseline, an effect lasting 2-3 weeks, and dose-dependent. Intraperitoneal mc-PH, 4 x 10(8) (n = 4), reduced serum LDL cholesterol levels to 45% of baseline more than 4 weeks posttransplant (P = 0.04). We conclude that transplantation of normal allogeneic NZW rabbit mc-PH reduces serum LDL cholesterol levels in homozygous WHHL rabbits without chronic immunosuppression. Longitudinal studies will establish if less atherosclerosis develops in mc-PH WHHL recipients than sham controls.

Hepatocyte transplantation for the low-density lipoprotein receptor-deficient state. A study in the Watanabe rabbit

Prostaglandins play an important role in cell-mediated immune responses. Their clinical use has been limited by poor oral bioavailability, short half-lives, and significant toxicity profiles. We studied the immunosuppressive properties of new, synthetic, prostaglandin E1 (PGE1) methyl ester analogs (misoprostol, enisoprost) with oral bioavailability using an allogeneic in vitro immunoassay. Our results show that the PGE1 analogs suppress alloproliferative responses and supplement the immunosuppressive activity of cyclosporine and methylprednisolone. Moreover, we demonstrate that addition of recombinant interleukin-2 to the PGE1 analogs restores alloimmune responsiveness and the expression of surface class II antigen and IL-2 receptors on responder lymphocytes. These studies, together with preliminary in vivo data in rodents and man, suggest that the new synthetic oral PGE1 analogs may provide therapeutic efficacy in clinical transplantation and a variety of immunologically mediated diseases.

The immunosuppressive properties of new oral prostaglandin E1 analogs.

Prostaglandins are reported to play an important regulatory role in cell-mediated immunity. The immunosuppressive properties of a new synthetic oral prostaglandin E1 analogue, misoprostol, were studied in vivo in a rat heterotopic cardiac allograft model, and in vitro in mixed lymphocyte reaction. The results show that parenteral misoprostol, alone or in combination with low dose cyclosporin immunosuppressive therapy, significantly prolonged cardiac allograft survival compared with appropriate controls. Oral misoprostol alone in this model did not allow for measurable cardiac allograft prolongation. In vitro misoprostol demonstrated significant dose-response inhibition of the mixed lymphocyte culture assay. It is concluded that new prostaglandin E1 analogues with oral bioavailability may have important applications to clinical transplantation in man, and may be cyclosporin sparing.

Julio C. Wiederkehr

Papers

Hepatocyte transplantation for the low-density lipoprotein receptor-deficient state. A study in the Watanabe rabbit

The immunosuppressive properties of new oral prostaglandin E1 analogs.

Immunosuppressive effect of misoprostol: a new synthetic prostaglandin E1 analogue.

A study of the time course of induction of class II and interleukin-2 receptors on human lymphocytes in response to alloantigen.