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Mesenchymal stem cells (MSCs) offer promise as therapeutic aids in the repair of tendon, ligament, and bone damage suffered by sport horses. The objective of the study was to identify and characterize stem-like cells from newborn foal umbilical cord blood (UCB). UCB was collected and MSC isolated using human reagents. The cells exhibit a fibroblast-like morphology and express the stem cell markers Oct4, SSEA-1, Tra1-60 and Tra1-81. Culture of the cells in tissue-specific differentiation media leads to the formation of cell types characteristic of mesodermal and endodermal origins. Chondrogenic differentiation reveals proteoglycan and glycosaminoglycan synthesis as measured histochemically and Sox9 and collagen 2A1 gene transcription. Osteocytes capable of mineral deposition, osteonectin and Runx2 transcription were evident. Hepatogenic cells formed from UCBs express albumin and cytokeratin 18. Multinucleated myofibers that express desmin were observed indicating partial differentiation into mature muscle cells. Interestingly, conventional human protocols for UCB differentiation into adipocytes were unsuccessful in foal UCB and adult horse adipose-derived MSC. These results demonstrate that equine UCB can be induced to form multiple cell types that underlie their value for regenerative medicine in injured horses. In addition, this work suggests that subtle differences exist between equine and human UCB stem cells. J. Cell. Physiol. 215: 329–336, 2008. © 2007 Wiley-Liss, Inc.
Bone marrow (BM) derived mesenchymal stem cells (MSCs) are the conventional model of choice for adult stem cell based therapeutics in humans due to their mutlilineage differentiation capabilities. Their relative ease of expansion in vitro without loss of plasticity makes MSCs an attractive repair aid for damaged or diseased heart, bone and vascular tissues [for review see Giordano et al. (2007)]. However, enthusiasm for the use of MSCs as cytotherapeutics is tempered by their age-dependent decline in absolute numbers and the invasive nature of their harvest (Stenderup et al., 2003). To counter these problems, umbilical cord blood (UCB) MSCs may represent a viable alternative. Several reports define a clonogenic population of cells from the umbilicus that differentiate into both mesenchymal and non-mesenchymal tissue derivatives (McGuckin et al., 2003; Aoki et al., 2004; Baal et al., 2004; Bonanno et al., 2004; Peled et al., 2004; Ruzicka et al., 2004; He et al., 2005; Holm et al., 2006; Martin-Rendon et al., 2007). The identity of these cells as circulating stem-like progenitors versus endothelial progenitors detached from the umbilicus remains debatable (Kogler et al., 2006).
A hierarchy in stem cell plasticity exists such that embryonic stem (ES) cells are pluripotent and adult MSCs are more limited in their differentiation capacity (Feinberg, 2007). UCB stem cells likely fall in the area between the two. The three classes of stem cells demonstrate variable stage specific embryonic antigen (SSEA) and tumor rejection antigen (Tra) surface marker protein expression patterns as well as differences in transcriptional circuitry. SSEA-3 and SSEA-4 are prevalent on the surface of human ES cells; these undifferentiated cells do not express SSEA-1 (Thomson et al., 1998; Reubinoff et al., 2000; Henderson et al., 2002). By contrast, mouse blastocyst inner cell mass cells and ES cells express SSEA-1 but not SSEA-3 or SSEA-4 (Henderson et al., 2002; Tielens et al., 2006). The keratan sulfate proteoglycan markers, Tra1-60 and Tra1-81, are localized within the extracellular matrix of human ES cells (Henderson et al., 2002; Stojkovic et al., 2004). Key to the establishment and maintenance of the undifferentiated state of ES cells are the coordinated activities of Oct4, nanog, and Sox2 (Boyer et al., 2005). This combination of surface markers and plasticity genes represent the minimal defining components of a naïve ES cell. By comparison, human BM derived MSCs are more limited in their expression of the central ES indicators likely owing to the heterogeneity of the population. SSEA4 is present on the surface of BM-MSC; the cells lack Oct4 but can be induced to form multiple lineages (Gang et al., 2007). Culture of BM-MSC in FGF2 supplemented media results in Oct4 and nanog transcription suggesting that a premature phenotype reminiscent of ES cells can be established (Battula et al., 2007). UCB stem cells are unique in that they possess an intermediate phenotype that more closely resembles ES cells. SSEA-3, SSEA-4, Tra1-60, Tra1-81, Oct4, and Nanog are present in this population (McGuckin et al., 2005; Zhao et al., 2006; Markov et al., 2007; Sun et al., 2007).
BM-MSC isolated from adult horses differentiate along the chondrogenic and osteogenic lineages comparable to their human counterparts (Fortier et al., 1998; Worster et al., 2001; Koerner et al., 2006). However, a reduced level of success exists for the formation of adipocytes from BM aspirates (Koerner et al., 2006; Vidal et al., 2006). Because human UCB stem cells exhibit a heightened degree of plasticity, we chose to identify a comparable cell entity in newborn foal cord blood as an alternative to BM-MSC. Using conventional human purification methods, culture conditions and differentiation protocols, an equine UCB cell population was discovered that possesses stem cell-like markers and multilineage differentiation capabilities. The isolation and characterization of these cells represent a first-step toward their application in cytotherapeutic repair of sport horse injuries.
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There is widespread interest in tendon, ligament and cartilage repair in horses through the use of directed stem cell transplantation methods. To date, published reports of multipotent cells isolated from BM, peripheral blood, and UCB exist (Fortier et al., 1998; Saito et al., 2002; Koerner et al., 2006; Li et al., 2006). Cells from each of these sources display limited differentiation into mesodermal cell types with predominant induction of chondrogenic and osteogenic precursors. Beyond these two cell types, vast differences in differentiation efficiencies and alternate cellular identities exist. The disparities may be attributed to tissue source or suboptimal culture conditions; both possibilities necessitate further study. Alternatively, the transcriptional regulators that govern pluripotency may be absent or inactive thereby, limiting plasticity. Key to the ES cell-like nature is expression of Oct4, Sox2, nanog, c-myc, and Klf4 (Takahashi and Yamanaka, 2006). Foal UCB stem cells maintained in a growth factor rich medium expressed Oct4, SSEA-1, Tra1-60 and Tra1-81, all stem cell marker proteins. However, repeated attempts to detect nanog and Sox2 mRNA were unsuccessful. The absence of these transcription factors may contribute to the restricted types of cells generated and their incomplete differentiation (myoblasts). Interestingly, the ability of UCB stem cells to express these embryonic markers sets them apart from adult MSCs. Surface expression of SSEA-1 and SSEA-4 were not evident in equine AD-MSCs. The lack of SSEA markers points to a hierarchy in plasticity that may account for some of the differences in differentiation capabilities. Efforts to define culture medias that support nanog, Sox2, and Klf4 expression may lead to an increased range of differentiated lineages from UCB stem cells.
Stem cells isolated from the umbilical cord matrix of pigs develop a morphology that resembles that reported by others for equine UCB stem cells (Carlin et al., 2006; Koch et al., 2007). In both examples, the majority of the cells attached to the cultureware surface and possessed a flat, spindle-shaped, fibroblast-like morphology. A lesser population formed light-refractile colonies that grew upward from the substratum surface in a manner consistent with transformed fibroblast foci. These colonies of small cells with a high nuclear to cytoplasmic volume were evident in our cultures of newborn foal UCB stem cells only after reaching confluency. Our UCB stem cells were maintained as a monolayer and passaged at approximately 60% confluency thereby, selecting against the development of these cell clusters that appear to grow independent of contact inhibition. While the identity of this cell population remains less clear, it is possible that these colonies represent a more primitive progenitor cell. Indeed, these cell clusters resemble those found in cultures of mouse ES cells. As such, one would predict that confluent equine UCB cultures that contain both the fibroblast-like and light-refractile cell colonies would express the plasticity genes, nanog and Sox2. However, expression of SSEA-1, Tra1-60, Tra1-81 and alkaline phosphatase, in a manner consistent with equine inner cell mass-derived ES cells, provides encouraging evidence that our monolayer cells are naive and undifferentiated (Takahashi and Yamanaka, 2006).
Equine UCB stem cells, in our hands, are not direct equivalents to human UCB stem cells but do possess many similarities. Human UCB stem cells can be isolated directly from the blood and frozen without expansion (Lee et al., 2005). This aspect of enrichment and storage remains elusive in our equine UCB cells. Partial purification by negative immunoselection and density gradient centrifugation produces a cell population that survives immediate cryopreservation very poorly. This may be due to the small numbers of stem cells and/or heightened sensitivity of these cells to plasma membrane perturbation. Culture of the fresh isolates for 3–5 days allows for the removal of contaminating lymphocytes and cellular debris and expansion of the putative stem cell population, which can be stored in liquid nitrogen and subsequently recovered. Direct enrichment of the UCB stem cell population by affinity purification with CD133 antibodies may provide an alternative to both cell heterogeneity and cryopreservation issues.
The capacity of foal UCB stem cells to initiate hepatocyte-specific gene transcription demonstrates an endodermal developmental potential. Reports exist demonstrating hepatocyte formation from human UCB stem cells and MSC isolates from BM (Hong et al., 2005; Talens-Visconti et al., 2006). However, this is the first report of hepatocyte formation using equine multipotential cells. Putative stem cells from the inner cell mass of equine blastocysts undergo spontaneous differentiation in vitro to yield cell derivatives of the three germ layers with endoderm defined by RT-PCR detection of α-fetoprotein (Li et al., 2006). The ability of newborn foal UCB stem cells to form liver cells is encouraging as it provides additional evidence for a population with plasticity characteristics that more closely resemble an ES cell than an adult stem cell. Additional endoderm-derived cell types of clinical importance include pancreatic and cardiogenic. Human UCB stem cells can be induced to form heart cells following a two-step differentiation protocol that involves 5-azacytidine treatment (Kadivar et al., 2006). Culture with the hypomethylating agent suggests that UCB stem cells are more restricted in their differentiation capabilities than ES cells and require chemical-induced reprogramming. In our hands, treatment of foal UCB stem cells with 5-azacytidine did not induce the expression of myosin immunopositive cells. Because our antibody (MF20) recognizes all forms of sarcomeric myosin, this result provides indirect evidence that a full-fledged cardiocyte is not created in response to epigenetic modification. However, a more comprehensive analysis of growth factor, morphogen and substratum requirements for UCB stem cell differentiation into cardiocytes is warranted.
Induction of the myogenic gene program has proven difficult in MSC originating from multiple animal and tissue sources. Exposure of rat BM-derived MSCs to 5-azacytidine caused differentiation into elongated, multinucleated myofibers (Wakitani et al., 1995). However, reprogramming the equine UCB transcriptome with this chemical did not induce the myogenic gene program (data not shown). A similar result was noted by others (Chan et al., 2006). Others reported that human UCB stem cells formed limited numbers of desmin immunopositive cells following in vitro differentiation (Nunes et al., 2007). These cells were devoid of the myogenic regulatory factors (MRFs) as measured by RT-PCR. Interestingly, injection of the putative stem cells into mdx mice resulted in engraftment suggesting that components within the muscle niche are essential for myogenic progression. One of those proteins is likely galectin-1, a glycoprotein of the basal lamina. Chan et al. (2006) demonstrated that culture of human fetal MSC in media containing galectin-1 initiated both biochemical and morphological differentiation into myocytes. These cells formed large, multinucleated fibers that expressed contractile proteins and the MRFs. We used a similar approach with some degree of success. Supplementation of foal UCB cell culture medium with purified galectin-1 caused myogenic lineage establishment as determined by desmin immunocytochemistry. However, a large percentage of the myoblasts were fusion-defective. In addition, we were unable to detect gene transcripts for MyoD, an early MRF, or myogenin, an MRF required for fusion and contractile gene expression. In accordance with our failure to amplify members of the MRFs, we did not detect myosin heavy chain or troponin T by immunocytochemical methods. The constraints to full activation of the myogenic program may be attributed to the absence of complementary soluble proteins. The source of galectin-1 used by Chan was spent media from COS cells that produce and secrete the glycoprotein. Thus, additional proteins within the galectin-1 supplement may have aided induction of myogenesis. Alternatively, specie-specific differences may underlie the discrepant results.
Given the relative ease of adipocyte formation by human and rodent MSC, the inefficiency of adipogenesis in equine UCB stem cells was surprising. Less than 1% of cells contained Oil Red O reactive lipid droplets following application of conventional adipogenic induction protocols. Koerner reported a similar result using BM-derived and peripheral blood-derived MSC isolated from adult horses (Koerner et al., 2006). A very small number of adipocytes were found and the cytoplasmic lipid droplets within said cells were miniscule. By contrast, robust lipid formation is evident by Oil Red O histology in equine UBCs cultured in a similar adipocyte induction media (Koch et al., 2007). The discrepancy between these various reports may be attributed to the heterogeneity of the starting population and/or culture conditions. Koch reported the presence of dome-like, clusters of small cells as well as a fibroblast-like cell type (Koch et al., 2007). While we observe the same morphologies, care was taken to maintain the adherent monolayer exclusive of the foci-like colonies. Future efforts will concentrate on resolving the identity of these divergent cellular phenotypes and their contribution to plasticity.