Philippe A. Lysy

Native umbilical cord matrix stem cells express hepatic markers and differentiate into hepatocyte-like cells.

BACKGROUND & AIMSnUmbilical cord matrix stem cells (UCMSCs) are able to differentiate into mesodermal and ectodermal lineages. The present study investigates the differentiation potential of human UCMSCs into hepatic lineage.nnnMETHODSnWe isolated human UCMSCs and characterized them in vitro by measuring their expansion potential, by assessing expression of mesenchymal stem cell (MSC) markers, and by evaluating their ability to differentiate into adipocytes and osteocytes. UCMSCs were thereafter subjected to a hepatogenic differentiation protocol. Expression of hepatic and MSC markers in differentiated cells was analyzed by reverse-transcription polymerase chain reaction, flow cytometry, and immunocytochemical assays and compared with undifferentiated UCMSCs and freshly isolated liver cells. UCMSCs were transplanted into livers of hepatectomized-SCID mice, and engraftment capacity was investigated by detection of human nucleus and mitochondria and human hepatic-specific proteins.nnnRESULTSnIn vitro expanded UCMSCs constitutively expressed markers of hepatic lineage, including albumin, alpha-fetoprotein, cytokeratin-19, connexin-32, and dipeptidyl peptidase IV. In vitro-differentiated UCMSCs exhibited hepatocyte-like morphology, up-regulated several hepatic markers, stored glycogen, produced urea, and exhibited an inducible CYP 3A4 activity. However, absence of some hepatic markers in differentiated UCMSCs, as HepPar1 or hepatocyte nuclear factor 4 (HNF-4), implied that their differentiation did not reach the level of mature hepatocytes. We also noticed that differentiated UCMSCs partially preserved MSC markers. Engraftment capacity of UCMSCs was observed, and expression of human albumin and alpha-fetoprotein was detected 2, 4, and 6 weeks after transplantation in mice livers, while cytokeratin 19 was completely down-regulated.nnnCONCLUSIONSnWe conclude that UCMSCs, with a newly demonstrated endodermic differentiation potential, might be an alternative source for liver-directed cell therapies.

Gigantism and Acromegaly Due to Xq26 Microduplications and GPR101 Mutation

BACKGROUNDnIncreased secretion of growth hormone leads to gigantism in children and acromegaly in adults; the genetic causes of gigantism and acromegaly are poorly understood.nnnMETHODSnWe performed clinical and genetic studies of samples obtained from 43 patients with gigantism and then sequenced an implicated gene in samples from 248 patients with acromegaly.nnnRESULTSnWe observed microduplication on chromosome Xq26.3 in samples from 13 patients with gigantism; of these samples, 4 were obtained from members of two unrelated kindreds, and 9 were from patients with sporadic cases. All the patients had disease onset during early childhood. Of the patients with gigantism who did not carry an Xq26.3 microduplication, none presented before the age of 5 years. Genomic characterization of the Xq26.3 region suggests that the microduplications are generated during chromosome replication and that they contain four protein-coding genes. Only one of these genes, GPR101, which encodes a G-protein-coupled receptor, was overexpressed in patients pituitary lesions. We identified a recurrent GPR101 mutation (p.E308D) in 11 of 248 patients with acromegaly, with the mutation found mostly in tumors. When the mutation was transfected into rat GH3 cells, it led to increased release of growth hormone and proliferation of growth hormone-producing cells.nnnCONCLUSIONSnWe describe a pediatric disorder (which we have termed X-linked acrogigantism [X-LAG]) that is caused by an Xq26.3 genomic duplication and is characterized by early-onset gigantism resulting from an excess of growth hormone. Duplication of GPR101 probably causes X-LAG. We also found a recurrent mutation in GPR101 in some adults with acromegaly. (Funded by the Eunice Kennedy Shriver National Institute of Child Health and Human Development and others.).

Adult-derived human liver mesenchymal-like cells as a potential progenitor reservoir of hepatocytes?

It is currently accepted that adult tissues may develop and maintain their own stem cell pools. Because of their higher safety profile, adult stem cells may represent an ideal candidate cell source to be used for liver cell therapies. We therefore evaluated the differentiation potential of mesenchymal-like cells isolated from adult human livers. Mesenchymal-like cells were isolated from enzymatically digested adult human liver and expanded in vitro. Cell characterization was performed using flow cytometry, RT-PCR, and immunofluorescence, whereas the differentiation potential was evaluated both in vitro after incubation with specific media and in vivo after intrasplenic transplantation of uPA+/+-SCID and SCID mice. Adult-derived human liver mesenchymal-like cells expressed both hepatic and mesenchymal markers among which albumin, CYP3A4, vimentin, and α-smooth muscle actin. In vitro differentiation studies demonstrated that these mesenchymal-like cells are preferentially determined to differentiate into hepatocyte-like cells. Ten weeks following intrasplenic transplantation into uPA+/+-SCID mice, recipient livers showed the presence of human hepatocytic cell nodules positive for human albumin, prealbumin, and α-fetoprotein. In SCID transplanted liver mice, human hepatocyte-like cells were mostly found near vascular structures 56 days posttransplantation. In conclusion, the ability of isolated adult-derived liver mesenchymal stem-like cells to proliferate and differentiate into hepatocyte-like cells both in vitro and in vivo leads to propose them as an attractive expandable cell source for stem cell therapy in human liver diseases.

Human skin fibroblasts: From mesodermal to hepatocyte-like differentiation†

The phenotypic homology of fibroblasts and mesenchymal stem cells (MSCs) has been recently described. Our study investigated the in vitro potential of human skin fibroblasts to differentiate into mesodermal (osteocyte and adipocyte) and endodermal (hepatocyte) cell lineages by comparison with human bone marrow (hBM) MSCs. The endodermal potential of fibroblasts was then explored in vivo in a mouse model of liver injury. Fibroblasts were able to acquire osteocyte and adipocyte phenotypes as assessed by cytochemistry and gene expression analyses. After exposure to a specific differentiation cocktail, these cells presented hepatocyte‐like morphology and acquired liver‐specific markers on protein and gene expression levels. Furthermore, these fibroblast‐derived hepatocyte‐like cells (FDHLCs) displayed the ability to store glycogen and synthesize small amounts of urea. By gene expression analysis, we observed that fibroblasts remained in a mesenchymal‐epithelial transition state after hepatocyte differentiation. Moreover, FDHLCs lost their hepatocyte‐like phenotype after dedifferentiation. In vivo, human fibroblasts infused directly into the liver of hepatectomized severe combined immunodeficient (SCID) mice engrafted in situ and expressed hepatocyte markers (albumin, alpha‐fetoprotein, and cytokeratin 18) together with the mesodermal marker fibronectin. Despite lower liver‐specific marker expression, the in vitro and in vivo differentiation profile of fibroblasts was comparable to that of mesenchymal‐derived hepatocyte‐like cells (MDHLCs). In conclusion, our work demonstrates that human skin fibroblasts are able to display mesodermal and endodermal differentiation capacities and provides arguments that these cells share MSCs features both on the phenotypic and functional levels. (HEPATOLOGY 2007;46:1574–1585.)

X-linked acrogigantism syndrome: clinical profile and therapeutic responses.

X-linked acrogigantism (X-LAG) is a new syndrome of pituitary gigantism, caused by microduplications on chromosome Xq26.3, encompassing the gene GPR101, which is highly upregulated in pituitary tumors. We conducted this study to explore the clinical, radiological, and hormonal phenotype and responses to therapy in patients with X-LAG syndrome. The study included 18 patients (13 sporadic) with X-LAG and microduplication of chromosome Xq26.3. All sporadic cases had unique duplications and the inheritance pattern in two families was dominant, with all Xq26.3 duplication carriers being affected. Patients began to grow rapidly as early as 2-3 months of age (median 12 months). At diagnosis (median delay 27 months), patients had a median height and weight standard deviation scores (SDS) of >+3.9 SDS. Apart from the increased overall body size, the children had acromegalic symptoms including acral enlargement and facial coarsening. More than a third of cases had increased appetite. Patients had marked hypersecretion of GH/IGF1 and usually prolactin, due to a pituitary macroadenoma or hyperplasia. Primary neurosurgical control was achieved with extensive anterior pituitary resection, but postoperative hypopituitarism was frequent. Control with somatostatin analogs was not readily achieved despite moderate to high levels of expression of somatostatin receptor subtype-2 in tumor tissue. Postoperative use of adjuvant pegvisomant resulted in control of IGF1 in all five cases where it was employed. X-LAG is a new infant-onset gigantism syndrome that has a severe clinical phenotype leading to challenging disease management.

Concise Review: Pancreas Regeneration: Recent Advances and Perspectives

The replacement of functional pancreatic β‐cells is seen as an attractive potential therapy for diabetes, because diabetes results from an inadequate β‐cell mass. Inducing replication of the remaining β‐cells and new islet formation from progenitors within the pancreas (neogenesis) are the most direct ways to increase the β‐cell mass. Stimulation of both replication and neogenesis have been reported in rodents, but their clinical significance must still be shown. Because human islet transplantation is limited by the scarcity of donors and graft failure within a few years, efforts have recently concentrated on the use of stem cells to replace the deficient β‐cells. Currently, embryonic stem cells and induced pluripotent stem cells achieve high levels of β‐cell differentiation, but their clinical use is still hampered by ethical issues and/or the risk of developing tumors after transplantation. Pancreatic epithelial cells (duct, acinar, or α‐cells) represent an appealing alternative to stem cells because they demonstrate β‐cell differentiation capacities. Yet translation of such capacity to human cells after significant in vitro expansion has yet to be achieved. Besides providing new β‐cells, cell therapy also has to address the question on how to protect the transplanted cells from destruction by the immune system via either allo‐ or autoimmunity. Encouraging developments have been made in encapsulation and immunomodulation techniques, but many challenges still remain. Herein, we discuss recent advances in the search for β‐cell replacement therapies, current strategies for circumventing the immune system, and mandatory steps for new techniques to be translated from bench to clinics.

Clinical and genetic characterization of pituitary gigantism: an international collaborative study in 208 patients

Despite being a classical growth disorder, pituitary gigantism has not been studied previously in a standardized way. We performed a retrospective, multicenter, international study to characterize a large series of pituitary gigantism patients. We included 208 patients (163 males; 78.4%) with growth hormone excess and a current/previous abnormal growth velocity for age or final height >2 s.d. above country normal means. The median onset of rapid growth was 13 years and occurred significantly earlier in females than in males; pituitary adenomas were diagnosed earlier in females than males (15.8 vs 21.5 years respectively). Adenomas were ≥10u200amm (i.e., macroadenomas) in 84%, of which extrasellar extension occurred in 77% and invasion in 54%. GH/IGF1 control was achieved in 39% during long-term follow-up. Final height was greater in younger onset patients, with larger tumors and higher GH levels. Later disease control was associated with a greater difference from mid-parental height (r=0.23, P=0.02). AIP mutations occurred in 29%; microduplication at Xq26.3 - X-linked acrogigantism (X-LAG) - occurred in two familial isolated pituitary adenoma kindreds and in ten sporadic patients. Tumor size was not different in X-LAG, AIP mutated and genetically negative patient groups. AIP-mutated and X-LAG patients were significantly younger at onset and diagnosis, but disease control was worse in genetically negative cases. Pituitary gigantism patients are characterized by male predominance and large tumors that are difficult to control. Treatment delay increases final height and symptom burden. AIP mutations and X-LAG explain many cases, but no genetic etiology is seen in >50% of cases.

Persistence of a chimerical phenotype after hepatocyte differentiation of human bone marrow mesenchymal stem cells

Abstract.u2002 Objectives: Recent studies have suggested the potential of mesenchymal stem cells (MSCs) to differentiate into a hepatocyte‐like lineage. Here, we evaluate the efficacy of hepatocyte differentiation of MSCs by studying acquisition of hepatocyte‐like features together with alteration of the native mesenchymal phenotype. Material and methods: In vitro, we have investigated protein and mRNA level expression of hepatocyte and mesenchymal markers of mesenchymal‐derived hepatocyte‐like cells (MDHLCs) and we have evaluated their functionality using metabolic assays. In vivo, we investigated co‐expression of hepatocyte (albumin, α‐foetoprotein, cytokeratin 18) and mesenchymal (fibronectin, vimentin) markers after transplantation of MSCs or MDHLCs into severe combined immune deficiency mice. Results: We observed that while in vitro these cells acquired some phenotypic and functional features of mature hepatocytes, they partially preserved their mesenchymal phenotype. After intrasplenic transplantation, engrafted MSCs with isolated expression of fibronectin and α‐foetoprotein were observed. When these cells were injected into the liver, they expressed all analysed markers, confirming the chimaeric co‐expression observed in vitro. Conversely, liver‐engrafted MDHLCs conserved their hepatocyte‐lineage markers but lost their chimaeric phenotype. Conclusions: Hepatocyte differentiation of MSCs predominantly allows the acquisition of phenotypic hallmarks and provides chimaeric cells that maintain expression of initial lineage markers. However, advanced maturation to the hepatocyte‐like phenotype could be obtained in vivo by conditioning MSCs prior to transplantation or by infusing cells into the liver micro‐environment.

Making β cells from adult cells within the pancreas

Cell therapy is currently considered as a potential therapeutic alternative to traditional treatments of diabetes. Islet and whole pancreas transplantations provided the proof-of-concept of glucose homeostasis restoration after replenishment of the deficiency of β cells responsible for the disease. Current limitations of these procedures have led to the search for strategies targeting replication of pre-existing β cells or transdifferentiation of progenitors and adult cells. These investigations revealed an unexpected plasticity towards β cells of adult cells residing in pancreatic epithelium (eg, acinar, duct, and α cells). Here we discuss recent developments in β-cell replication and β-cell transdifferentiation of adult epithelial pancreatic cells, with an emphasis on techniques with a potential for clinical translation.

GHRH excess and blockade in X-LAG syndrome

X-linked acrogigantism (X-LAG) syndrome is a newly described form of inheritable pituitary gigantism that begins in early childhood and is usually associated with markedly elevated GH and prolactin secretion by mixed pituitary adenomas/hyperplasia. Microduplications on chromosome Xq26.3 including the GPR101 gene cause X-LAG syndrome. In individual cases random GHRH levels have been elevated. We performed a series of hormonal profiles in a young female sporadic X-LAG syndrome patient and subsequently undertook in vitro studies of primary pituitary tumor culture following neurosurgical resection. The patient demonstrated consistently elevated circulating GHRH levels throughout preoperative testing, which was accompanied by marked GH and prolactin hypersecretion; GH demonstrated a paradoxical increase following TRH administration. In vitro, the pituitary cells showed baseline GH and prolactin release that was further stimulated by GHRH administration. Co-incubation with GHRH and the GHRH receptor antagonist, acetyl-(d-Arg(2))-GHRH (1-29) amide, blocked the GHRH-induced GH stimulation; the GHRH receptor antagonist alone significantly reduced GH release. Pasireotide, but not octreotide, inhibited GH secretion. A ghrelin receptor agonist and an inverse agonist led to modest, statistically significant increases and decreases in GH secretion, respectively. GHRH hypersecretion can accompany the pituitary abnormalities seen in X-LAG syndrome. These data suggest that the pathology of X-LAG syndrome may include hypothalamic dysregulation of GHRH secretion, which is in keeping with localization of GPR101 in the hypothalamus. Therapeutic blockade of GHRH secretion could represent a way to target the marked hormonal hypersecretion and overgrowth that characterizes X-LAG syndrome.