[Canadian founders of haematopoietic stem cell research receive the Lasker Award].
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Animals↗
Biomedical subjects
Danielle Larouche
Publications and source records attributed to Danielle Larouche.
Keratin 19 as a stem cell marker in vivo and in vitro.
The skin is a dynamic tissue in which terminally differentiated keratinocytes are replaced by the proliferation of new epithelial cells that will undergo differentiation. The rapid and continual turnover of skin throughout life depends on a cell population with unique characteristics: the stem cells. These cells are relatively undifferentiated, retain a high capacity for self-renewal throughout their lifetime, have a large proliferative potential, and are normally slow cycling. The long-term regeneration of grafted cultured epidermis indicates that epidermal stem cells are maintained in cultures. In animals they can be identified with 3H-thymidine or bromodeoxyuridine based on their property of slow cycling. The development of markers such as keratin 19 also permits their study in human tissues. In this chapter, protocols to study skin stem cells using their property of slow cycling and their expression of keratin 19 will be described in detail. The methods include the double labeling of tissues for keratin 19 and label-retaining cells (auto radiography of 3H-thymidine) in situ. The labeling of keratin 19 by immunofluorescence of by flow cytometry is described for cells in vitro.
Animals↗
The challenge of promoting integration: conceptualization, implementation, and assessment of a pilot care delivery model for patients with type 2 diabetes.
BACKGROUND: The Côte-des-Neiges diabetes pilot project strove to conceptualize, implement, and assess an integrated health care system for Type 2 diabetes. Using a disease management and population-based approach, a multidisciplinary team sought to (1). organize health care in an integrative framework, (2). promote behavior changes in patients to foster self-care, (3). introduce tools to allow family physicians to modify their practices, and (4). encourage local community action to support patients and providers. METHODS: Information from a needs assessment helped guide the development of the care model, which was implemented over a 1-year period. A preliminary assessment was undertaken using qualitative methods. Data were collected through in-depth interviews, focus groups, participant observation, and document analysis. RESULTS: (1). Physicians and patients appreciated having access to a multidisciplinary team and related services, and personalized communication was preferred to computerized links. (2). Patients also perceived the benefit of individualized assessment and self-care educational sessions allowing them to participate in their illness management. (3). A diabetes care flow sheet altered the management strategies of physicians. (4). Limited time prevented full development of networking efforts to promote community mobilization. CONCLUSIONS: Approaches to chronic diseases such as diabetes require integrative health care strategies to support patients and providers in their community. In spite of time constraints, patients perceived the value of education with increasing involvement in their illness, physicians reported changes in their practice, and steps were initiated to mobilize community resources.
Adult↗
Normal human Merkel cells are present in epidermal cell populations isolated and cultured from glabrous and hairy skin sites.
The Merkel cell is a highly specialized cell that primarily acts as a slowly adapting mechanoreceptor. Merkel cells are scarce in normal skin but can be identified by the expression of distinct keratin filaments. Merkel cells constitute a very unique population and many questions still remain as to their origin, number, proliferative capacity, and functions in cutaneous biology. The dissociation of epidermal cells from skin is a widely used technique to extract and culture keratinocytes. We took advantage of a two-step extraction method to quantify keratin-20-expressing Merkel cells among total cutaneous cells obtained from either hairy or glabrous skin biopsies. Flow cytometry analysis revealed that keratin-20-labeled Merkel cells represent between 3.6% and 5.7% of freshly dissociated basal epidermal cells. No significant differences were seen between samples derived from glabrous palmar and hairy anatomic sites, from children and adult, respectively. We also report on the presence of Merkel cells in primary and first subcultures of epidermal cells indicating their capacity to remain viable after extraction from skin of various anatomic sites. To our knowledge, this is the first demonstration of nontumorigenic human Merkel cells in culture in vitro. The persistence of a small number of Merkel cells in culture suggests that, with the development of appropriate culture conditions, these cells could be amplified and further studied to unravel long-standing questions relative to their paracrine function or epithelial origin.
Adult↗
Reconstructed human skin produced in vitro and grafted on athymic mice.
BACKGROUND: The best alternative to a split-thickness graft for the wound coverage of patients with extensive burns should be in vitro reconstructed autologous skin made of both dermis and epidermis and devoid of exogenous extracellular matrix proteins and synthetic material. We have designed such a reconstructed human skin (rHS) and present here its first in vivo grafting on athymic mice. METHODS: The rHS was made by culturing newborn or adult keratinocytes on superimposed fibrous sheets obtained after culturing human fibroblasts with ascorbic acid. Ten days after keratinocyte seeding, reconstructed skins were either cultured at the air-liquid interface or grafted on athymic mice. We present the macroscopic, histologic, and phenotypic properties of such tissues in vitro and in vivo after grafting on nude mice. RESULTS: After maturation in vitro, the reconstructed skin exhibited a well-developed human epidermis that expressed differentiated markers and basement membrane proteins. Four days after grafting, a complete take of all grafts was obtained. Histological analysis revealed that the newly generated epidermis of newborn rHS was thicker than that of adult rHS after 4 days but similar 21 days after grafting. The basement membrane components (bullous pemphigoid antigens, laminin, and type IV and VII collagens) were detected at the dermo-epidermal junction, showing a continuous line 4 days after grafting. Ultrastructural studies revealed that the basement membrane was continuous and well organized 21 days after transplantation. The macroscopic aspect of the reconstructed skin revealed a resistant, supple, and elastic tissue. Elastin staining and elastic fibers were detected as a complex network in the rHS that contributes to the good elasticity of this new reconstructed tissue. CONCLUSIONS: This new rHS model gives supple and easy to handle skins while demonstrating an adequate wound healing on mice. These results are promising for the development of this skin substitute for permanent coverage of burn wounds.
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Tissue reorganization in response to mechanical load increases functionality.
In the rapidly growing field of tissue engineering, the functional properties of tissue substitutes are recognized as being of the utmost importance. The present study was designed to evaluate the effects of static mechanical forces on the functionality of the produced tissue constructs. Living tissue sheets reconstructed by the self-assembly approach from human cells, without the addition of synthetic material or extracellular matrix (ECM), were subjected to mechanical load to induce cell and ECM alignment. In addition, the effects of alignment on the function of substitutes reconstructed from these living tissue sheets were evaluated. Our results show that tissue constructs made from living tissue sheets, in which fibroblasts and ECM were aligned, presented higher mechanical resistance. This was assessed by the modulus of elasticity and ultimate strength as compared with tissue constructs in which components were randomly oriented. Moreover, tissue-engineered vascular media made from a prealigned living tissue sheet, produced with smooth muscle cells, possessed greater contractile capacity compared with those produced from living tissue sheets that were not prealigned. These results show that the mechanical force generated by cells during tissue organization is an asset for tissue component alignment. Therefore, this work demonstrates a means to improve the functionality (mechanical and vasocontractile properties) of tissues reconstructed by tissue engineering by taking advantage of the biomechanical forces generated by cells under static strain.
Biocompatible Materials↗