Study and culture of haematopoietic progenitor cells from peripheral blood in rats, hamsters and mice

The aim of this work was to isolate and cultivate a subpopulation of pluripotent stem cells present in peripheral blood of different animal species, frequently used in laboratory studies (mice, rats and hamsters). Pluripotent stem cells (PSCs), already described in human beings, are fibroblast-like cells that exhibit a CD34 marker, specific for haematopoietic stem cells. Commonly used human commercial media were investigated for culturing animal PSCs. These findings suggest that this simple and standardized methodology may be applicable in several fields such as the study of the pharmacological effects of drugs on the haematopoietic line and the study of new strategies in cellular therapy for some human diseases.     

Isolation and development of haematopoietic progenitor cells from peripheral blood of adult and newborn pigs

Pluripotent stem cells (PSCs), already described in human beings, are fibroblast-like cells that exhibit a CD34 marker specific for haematopoietic stem cells. In this work we have demonstrated the presence of PSCs in the peripheral blood of pigs, a species frequently used in transplantation studies as an animal model for human diseases. Differentiation into haematopoietic colonies (granulomacrophagic colonies, erythroid colonies and mixed colonies) has been carried out with the peripheral blood of adult and newborn pigs, using solely human commercial media. Peripheral blood mononuclear cells (PBMNCs) were cultured in semisolid methylcellulose based media enriched with recombinant human cytokines, achieving granulomacrophagic-colony forming unit (GM-CFU) and mixed-colony forming unit (Mix-CFU) growth with erythroblastic lineage proliferation in the presence of erythropoietin (Epo). In all the samples CFU growth was associated with the presence of recombinant human cytokine. No evidence of proliferation in control plates without cytokines was found. From liquid medium culture, a population of macrophages and CD34+ fibroblast like cells were retrieved 21 days after sowing. These findings allow us to think about the direct application of this simple and standardised method in several work fields such as the study of pharmacological effects of many drugs over the haematopoietic line and in the study of new strategies in cellular therapy for some human diseases.     

Pluripotent cell derivation from male germline cells by suppression of Dmrt1 and Trp53

Diploid germ cells are thought to have pluripotency potential. We recently described a method to derive pluripotent stem cells (PSCs) from cultured spermatogonial stem cells (SSCs) by depleting Trp53 and Dmrt1, both of which are known suppressors of teratomas. In this study, we used this technique to analyze the effect of this protocol in deriving PSCs from the male germline at different developmental stages. We collected primordial germ cells (PGCs), gonocytes and spermatogonia, and the cells were transduced with lentiviruses expressing short hairpin RNA against Dmrt1 and/or Trp53. We found that PGCs are highly susceptible to reprogramming induction and that only Trp53 depletion was sufficient to induce pluripotency. In contrast, gonocytes and spermatogonia were resistant to reprogramming by double knockdown of Dmrt1 and Trp53. PSCs derived from PGCs contributed to chimeras produced by blastocyst injection, but some of the embryos showed placenta-only phenotypes suggestive of epigenetic abnormalities of PGC-derived PSCs. These results show that PGCs and gonocytes/spermatogonia have distinct reprogramming potential and also suggest that fresh and cultured SSCs do not necessarily have the same properties.     

Flow cytometric analysis of bone marrow leukocytes in neonatal dogs

Dogs represent both an important veterinary species and a convenient model for allogeneic hematopoietic stem cell transplantation. Even though anti-canine CD34 antibodies have recently become available, little is known about hematopoietic lineages in dogs, partially because CD34- cells have been ignored in all analyses performed so far. In this study, we have focused on the bone marrow mononuclear compartment to provide an additional piece of information on the phenotype of CD34+ progenitors and to identify the dominant CD34- population. We have shown that, in contrast to the adults, mature lymphocytes are scarce in neonatal dog bone marrow. Using cross-reactive antibodies against CD79alpha we have shown that the B lineage of hematopoiesis strongly prevails. CD34+ cells were shown to be positive for MHC class II and SWC3, a member of the signal regulatory protein family.     

Isolation and Differentiation of Mesenchymal Stem Cells From Bovine Umbilical Cord Blood

Currently, mesenchymal stem cells (MSCs) are used in veterinary clinical applications. Bone marrow and adipose tissue are the most common sources of stem cells derived from adult animals. However, cord blood which is collected non‐invasively is an alternative source of stem cells other than bone marrow and adipose tissue. Moreover, high availability and lower immunogenicity of umbilical cord blood (UCB) haematopoietic stem cells compared to other sources of stem cell therapy such as bone marrow have made them a considerable source for cell therapy, but MSCs is not highly available in cord blood and their immunogenicity is poorly understood. In this study, the cells with spindle morphology from 7 of 9 bovine UCB samples were isolated and cultured. These mesenchymal stromal cells were successfully differentiated to osteocytes, chondrocytes and adipocytes. In addition, Oct‐4 and SH3 were determined by RT‐PCR assay. It is the first report of isolation, culture, characterization and differentiation of bovine umbilical stem cells.     

Development of New Stem Cell-based Technologies for Carnivore Reproduction Research

New reproductive technologies based on stem cells offer several potential benefits to carnivore species. For example, development of lines of embryonic stem cells in cats and dogs would allow for the generation of transgenic animal models, which could be used to advance both veterinary and human health. Techniques such as spermatogonial stem cell transplantation (SSCT) and testis xenografting offer new approaches to propagate genetically valuable individual males, even if they should die before producing sperm. These techniques might therefore have application to the conservation of endangered species of carnivores, as well as to biomedical research. Recently, our laboratory has successfully performed SSCT in the dog, with a recipient dog producing sperm of donor genetic origin. Testis xenografting has been used to produce sperm from pre-pubertal testis tissue from both cats and ferrets. These early steps reinforce the need not only for research on stem cell technologies, but also for additional research into complementary technologies of assisted reproduction in carnivores, so that the widest array of research and clinical benefits can be realized.     

Evaluation of the potential use of adipose-derived mesenchymal stromal cells in the treatment of canine atopic dermatitis: a pilot study

Stem cells and their potential therapeutic uses in human and veterinary medicine have generated considerable interest. These cells have a number of potentially unique immunologic properties; most notable are their reported regenerative and antiinflammatory capabilities. The aim of this prospective pilot study was to evaluate the efficacy of intravenously administered autogenous adipose-derived mesenchymal stem cells (AD-MSCs) in the treatment of canine atopic dermatitis. AD-MSCs administered intravenously at a dose of 1.3 million cells/kg did not significantly reduce the clinical signs of canine atopic dermatitis or the owner-assessed pruritus level.     

犬造血干细胞移植的作用机制及应用研究进展

造血干细胞(hematopoietic stem cell, HSC)位于造血系统的顶端, 是造血系统中唯一具有多能性和自我更新能力的细胞, 可以分化为各种功能的血细胞, 维持血液系统的建立和动态平衡, 是目前研究最为透彻、临床应用最为成熟的成体干细胞。造血干细胞的这些重要特性, 使得基于造血干细胞在治病机制研究和临床应用中的研究取得了很大进展。以造血干细胞为基础的再生医学治疗是目前治疗恶性血液病和遗传病的首选方法, 如造血干细胞治疗犬白细胞黏附缺陷综合征、犬遗传性贫血、犬淋巴瘤、犬白细胞减少症、犬X连锁严重联合免疫缺陷病等, 但由于白细胞抗原匹配的供体稀缺、可获得的造血干细胞数量有限等原因, 无法满足临床需求, 因此, 如何通过体外培养获得满足临床需要的造血干细胞成为近年来学者们研究的热点问题。作者参照现有研究成果对造血干细胞的来源和体外分离培养、治病机制进行系统的描述, 并对造血干细胞移植治疗遗传性溶血性贫血、白细胞黏附缺陷综合征、淋巴瘤、白细胞减少症、X连锁严重联合免疫缺陷病的临床研究进行综述, 以期为今后将造血干细胞广泛应用于临床治疗提供参考依据。     

Further insights into the characterization of equine adipose tissue-derived mesenchymal stem cells

Adipose tissue-derived stem cells (ADSCs) represent a promising subpopulation of adult stem cells for tissue engineering applications in veterinary medicine. In this study we focused on the morphological and molecular biological properties of the ADSCs. The expression of stem cell markers Oct4, Nanog and the surface markers CD90 and CD105 were detected using RT-PCR. ADSCs showed a proliferative potential and were capable of adipogenic and osteogenic differentiation. Expression of Alkaline phosphatase (AP), phosphoprotein (SPP1), Runx2 and osteocalcin (OC) mRNA were positive in osteogenic lineages and peroxisome proliferator activated receptor (Pparγ2) mRNA was positive in adipogenic lineages. ADSCs show stem cell and surface marker profiles and differentiation characteristics that are similar to but distinct from other adult stem cells, such as bone marrow-derived mesenchymal stem cells (BM-MSCs). The availability of an easily accessible and reproducible cell source may greatly facilitate the development of stem cell based tissue engineering and therapies for regenerative equine medicine.     

造血干细胞研究进展

造血干细胞是具有自我更新、高度增殖和多向分化潜能的细胞群体,在人体造血系统中起着至关重要的作用。本文介绍了造血干细胞的生物学特征、表面标志以及造血干细胞在干细胞移植、细胞治疗和基因治疗等方面的临床应用和前景。     

Classification of hematopoietic system neoplasia in the dog

This article contains an overview of the classification of neoplasia of the hematopoietic system in the dog. The objective of this article is to convey an understanding of currently used classification schemes and to correlate this classification with modern concepts of hematopoietic system biology. This article will be concerned with those neoplastic disease processes that are derived from the mesodermally derived pluripotent hematopoietic stem cells and their progeny.     

Morphological description of limbal epithelium: searching for stem cells crypts in the dog,cat, pig,cow, sheep and horse

The cornea provides protection and transparency to the eye, allowing an optimal sharpness view. In some pathological conditions the cornea is able to regenerate thanks to the presence of a stem cells reservoir present at the level of the transition area between cornea and sclera (limbus). Corneal cell therapies in Veterinary Medicine are really limited due to the lacking of knowledge about the anatomy of the limbal area, the putative presence of stem cells and their identification in domestic species. The aim of this study was to provide an overview of the main distinctive structural features of the sclero-corneal junction and conjunctival-corneal junction areas in some species of veterinary importance, using optic microscope observations of histological sections. The resulting data were compared with cornea from humans adapting protocols already used to identify stem cells by means of a specific cellular marker. We tested the expression of ΔNp63α isoform in the cornea basal cells, trying to correlate the distribution profile with areas of highly proliferative turnover. The results obtained from this study represent a first step towards the identification of a corneal stem cells reservoir in different animals.     

In vitro neuronal and osteogenic differentiation of mesenchymal stem cells from human umbilical cord blood

Mesenchymal stem cells (MSCs) have the capabilities for self-renewal and differentiation into cells with the phenotypes of bone, cartilage, neurons and fat cells. These features of MSCs have attracted the attention of investigators for using MSCs for cell-based therapies to treat several human diseases. Because bone marrow-derived cells, which are a main source of MSCs, are not always acceptable due to a significant drop in their cell number and proliferative/differentiation capacity with age, human umbilical cord blood (UCB) cells are good substitutes for BMCs due to the immaturity of newborn cells. Although the isolation of hematopoietic stem cells from UCB has been well established, the isolation and characterization of MSCs from UCB still need to be established and evaluated. In this study, we isolated and characterized MSCs. UCB-derived mononuclear cells, which gave rise to adherent cells, exhibited either an osteoclast or a mesenchymal-like phenotype. The attached cells with mesenchymal phenotypes displayed fibroblast-like morphologies, and they expressed mesenchym-related antigens (SH2 and vimentin) and periodic acid Schiff activity. Also, UCB-derived MSCs were able to transdifferentiate into bone and 2 types of neuronal cells, in vitro. Therefore, it is suggested that the MSCs from UCB might be a good alternative to bone marrow cells for transplantation or cell therapy.     

Isolation and characterization of equine amniotic membrane-derived mesenchymal stem cells

Recent studies have shown that mesenchymal stem cells (MSCs) are able to differentiate into multi-lineage cells such as adipocytes, chondroblasts, and osteoblasts. Amniotic membrane from whole placenta is a good source of stem cells in humans. This membrane can potentially be used for wound healing and corneal surface reconstruction. Moreover, it can be easily obtained after delivery and is usually discarded as classified waste. In the present study, we successfully isolated and characterized equine amniotic membrane-derived mesenchymal stem cells (eAM-MSCs) that were cultured and maintained in low glucose Dulbecco''s modified Eagle''s medium. The proliferation of eAM-MSCs was measured based on the cumulative population doubling level (CPDL). Immunophenotyping of eAM-MSCs by flow cytometry showed that the major population was of mesenchymal origin. To confirm differentiation potential, a multi-lineage differentiation assay was conducted. We found that under appropriate conditions, eAM-MSCs are capable of multi-lineage differentiation. Our results indicated that eAM-MSCs may be a good source of stem cells, making them potentially useful for veterinary regenerative medicine and cell-based therapy.     

Stem cells and veterinary medicine: tools to understand diseases and enable tissue regeneration and drug discovery

New sophisticated laboratory techniques, as well as established interactions between basic science, researchers and veterinarians, have led to an exponential increase in our understanding of the animal body in health and disease. The advent of animal cloning, the identification and characterization of stem cells, and publication of the various mammalian genomes has afforded the opportunity to exploit these technologies to better understand disease and develop new therapies. In human medicine, these medical advances are already being translated into clinical practice, the promise being that previously untreatable or incurable chronic diseases will become a thing of the past. In parallel, the veterinary profession is looking to these technologies to explore novel therapies for chronic diseases, such as osteoarthritis in companion animals, and is applying these technologies to enhance food animal production. This review focuses on the emerging area of stem cell biology and explores the potential applications of stem cell technologies to veterinary medicine.     

Transplantation of CD6-depleted peripheral blood stem cells after DLA-haploidentical bone marrow transplantation contributes to engraftment and tolerance in a preclinical model of stem cell transplantation

Human leukocyte antigen (HLA)-haploidentical stem cell transplantation is an opportunity for nearly all patients lacking an HLA matched stem cell donor. However, graft rejection and graft-versus-host disease (GvHD) as well as infectious complications still result in high treatment-related mortality. Here, we used the dog as a preclinical model for the study of tolerance induction with the aim to optimize and to improve a clinical protocol of haploidentical stem cell transplantation. For this purpose CD6-depleted peripheral blood stem cells (PBSCs) were transfused 6d after transplantation of unmodified bone marrow from dog leukocyte antigen (DLA)-haploidentical littermate donors in order to induce immune tolerance. Besides hematopoietic stem cells CD6-depleted PBSC contain, NK cells and a minority of suppressive CD8-positive cells that may suppress activated T lymphocytes. Recipients were conditioned with, cyclophosphamide and antithymocyte globulin (ATG) preceded by a transfusion of donor buffy coat and either 1, 2 or 3 × 3.3 Gy total body irradiation (TBI). Postgrafting immunosuppression was limited to 30 d of cyclosporine and methotrexate. The additional administration of CD6-depleted PBSCs after unmodified marrow could not prevent GvHD, but it may improve engraftment and chimerism after conditioning with 2 × 3.3 Gy TBI. Reasons for incomplete suppression and possible improvements for clinical applications are discussed.