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Characterization and differentiation of equine umbilical cord-derived matrix cells
Biochemical and Biophysical Research Communications 362 (2007) 347–353 www.elsevier.com/locate/ybbrc
Characterization and differentiation of equine umbilical cord-derived matrix cells Steven M. Hoynowski a, Madeline M. Fry a, Bryn M. Gardner a, Matthew T. Leming a, Jeanell R. Tucker a, Linda Black b, Theodore Sand b,*, Kathy E. Mitchell a,* a
Department of Pharmacology and Toxicology, University of Kansas, 1251 Wescoe Hall Drive, Malott Rm 5064, Lawrence, KS 66045-7582, USA b Vet-Stem, Inc., 12860 Danielson Court, Suite B, Poway, CA 92064, USA Received 12 July 2007 Available online 13 August 2007
Abstract Stem cells are being evaluated in numerous human clinical trials and are commercially used in veterinary medicine to treat horses and dogs. Stem cell differentiation, homing to disease sites, growth and cytokine factor modulation, and low antigenicity contribute to their therapeutic success. Bone marrow and adipose tissue are the two most common sources of adult-derived stem cells in animals. We report on the existence of an alternative source of primitive, multipotent stem cells from the equine umbilical cord cellular matrix (Wharton’s jelly). Equine umbilical cord matrix (EUCM) cells can be cultured, cryogenically preserved, and differentiated into osteo-, adipo-, chondrogenic, and neuronal cell lineages. These results identify a source of stem cells that can be non-invasively collected at birth and stored for future use in that horse or used as donor cells for treating unrelated horses. 2007 Elsevier Inc. All rights reserved. Keywords: Wharton’s jelly; Stem cells; Mesenchymal cells; Equine; Adipogenic differentiation; Osteogenic differentiation; Chondrogenic differentiation; Neuronal differentiation; Oct-4; Umbilical cord matrix
Adult stem cells that are immunologically compatible can be harvested from a variety of sources including adipose tissue [1], skin tissue [2], bone marrow [3,4], and synovial membrane [5] and have no ethical issues related to their use. In addition to stem cells from the embryo and adult, stem cells can be found in extraembryonic tissues including umbilical cord and placental blood [6], amniotic fluid [7], and umbilical cord matrix [8–11]. Stem cells from extraembryonic sources have the advantages of being obtained by non-invasive procedures and low immunogenicity. For example, umbilical cord blood cells express low levels of HLA antigens and have been used for bone marrow replacement when HLA-
* Corresponding authors. Fax: +1 858 748 2005 (T. Sand), +1 785 864 5219 (K.E. Mitchell). E-mail addresses: [email protected] (T. Sand), [email protected] (K.E. Mitchell).
0006-291X/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2007.07.182
matched donors cannot be found [12]. Clinical trials are currently underway using stem cells from extraembryonic sources for treating a number of diseases [13,14] and for tissue engineering [15,16]. These preliminary studies show the promise of stem cells derived from extraembryonic tissues and yet little is known about their basic properties. We have demonstrated that cells isolated from porcine umbilical cord matrix differentiated in vitro into cells that resembled neurons morphologically and expressed neuronal and glial-specific proteins [8]. We also demonstrated that porcine umbilical cord matrix cells survive, migrate, and express markers for mature neurons when transplanted into rat brain [9]. Importantly, the implanted umbilical cord matrix cells showed no evidence of forming teratomas [9]. Rat umbilical cord matrix cells show similar properties [17]. When injected into rat brains in a model of whole brain ischemia, not only do they survive and migrate, they show a protective effect against damage to
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CA1 hippocampal neurons and improve overall neurological outcome [17]. These results indicate that umbilical cord matrix cells are a potential source of multipotent stem cells and may support many therapeutic and biotechnological roles [18]. Here, we describe the isolation and properties of equine-derived umbilical cord matrix cells (EUCM). Materials and methods Isolation of equine umbilical cord matrix cells and cell culture. Vet-Stem (Poway, CA) provided equine umbilical cords obtained in accordance with standard veterinary practice. The umbilical cord tissue was processed as previously described [19]. When culture confluency reached 50–80% the cells were harvested using 0.05% trypsin/0.53 mM EDTA solution and replated into larger culture flasks at a 1:2 split ratio. Cells were enumerated by dilution (1:2) in trypan blue (ICN, Aurora, OH) and counted with a hemacytometer using a Nikon Eclipse TS100 microscope to enumerate viable cells for CD, CFU-F, flow cytometry, or differentiation assays. Cultures were maintained at the stated confluency (50–80%) for propagation in a 37 C humidified incubator with 5% CO2. For cryogenic preservation, cells were suspended at 1 · 106 cells/mL in 10% DMSO and 90% FBS, and frozen in liquid nitrogen. Cell cycle analysis. EUCM cells were trypsinized and resuspended at 1– 2 · 106 cells/mL and fixed with 70% ethanol for 1 h at 4 C, then washed twice with cold PBS. Cells were labeled with 100 lL of 0.5 mg/mL propidium iodide (Roche, Indianapolis) (106 cells in 1 mL) followed by treatment with 1 U of DNase-free RNase A (Fisher, Fair Lawn, NJ) (106 cells in 1 mL) and incubated for 3 h at 4 C. CFU-F. The CFU frequency (CFU-F) was determined by means of limiting dilution. Nucleated cells (n = 4) isolated from equine umbilical cords were seeded in six-well plates at the following densities: 25, 50, 100, 200, 400, 800 cells/cm2. Cells were maintained in stromal medium that was changed every 2–3 days. After 10 days, the cells were washed and stained with a-Naphthyl Acetate Kit (Sigma–Aldrich, St. Louis, MO) according to manufacturer’s instructions. Positively stained colonies that contained >50 cells were counted as CFU’s. Proliferation studies. Cells from EUCM (n = 4 cords) were counted and seeded in T25 flasks. The cells from each flask were harvested and enumerated over eight passages starting with passage 1 (P1). The mean of the counts was calculated by the following formula: CD = ln(Nf/Ni)/ln 2 and DT = CT/CD, where DT is the cell-doubling time, CD is cell-doubling number, and CT is cell culture time. The proliferative rate was cal-
culated from each passage, where Nf is the final number of cells and Ni the initial number of cells [20]. Adipogenic, chondrogenic, osteogenic, and neuronal differentiation. EUCM cells were expanded in culture for three to four passages to obtain sufficient cells for differentiation into mesenchymal lineages. EUCM cells were counted and seeded at 2 · 105 cells per well in six-well plates 1 day prior to differentiation. Cells were then washed twice with PBS and differentiation media were added as described by Wang et al. [21]. The differentiation media were changed every 3 days. EUCM cells were induced to become neural stem cells and neuronal cells as described previously [22,23]. Histochemical staining. Differentiated cells were washed twice with PBS, fixed with 4% paraformaldehyde for 10 min at 37 C, and then washed thoroughly with PBS. To assess differentiation, cells were stained with Alizarin Red for osteogenic, Toluidine Blue for chondrogenic, and Oil Red O for adipogenic differentiation and imaged with a Nikon Eclipse TE 2000U and Photometrix Cool Snapcf digital camera using MetaMorph (Universal Imaging, Downingtown, PA) imaging software. Immunocytochemistry and alkaline phosphatase detection. Cultured EUCM cells were fixed with 4% paraformaldehyde in PBS for 10 min and then washed in PBS. Cells were permeabilized with 0.2% Triton X-100 in PBS for 5 min, washed, blocked in 0.2% Triton X-100, 2% normal serum in PBS for 30 min, followed by incubation with primary antibody against Oct-4 (1:50) (Abcam, Cambridge, MA) for 1 h, washed three times with PBS, and incubated with chicken anti-rabbit Alexa Fluor 488-conjugated secondary antibody (1:200) for 30 min (Molecular Probes, Eugene, Oregon). Excess secondary antibody was removed by repeated washing with PBS. Alkaline phosphatase activity was detected using an Alkaline Phosphatase Detection Kit (Chemicon, Temecula, CA) according to manufacturer’s instruction. Images were obtained with a 510 Zeiss laser scanning microscope under a 40· oil-immersion lens. Flow cytometry. EUCM cells at 200–400 · 105 cells/mL were fixed and permeabilized using BD Cytofix/Cytoperm Plus Kit (BD Biosciences, San Diego, CA) according to manufacturer’s instructions. Cells were labeled with primary antibodies: CD54, CD133, Oct-4 (Abcam, Cambridge, MA); CD34, CD45, CD73, and CD105 (BD Biosciences, San Jose, CA); CD90, CD146, HLA-ABC, SSEA-3, and TRA-1-60 (Chemicon, Temecula, CA); c-Kit, c-Myc, and SSEA-4 (Santa Cruz Biotechnology, Santa Cruz, CA.) for 1 h, followed by incubation with secondary Alexa Fluor 488 conjugates (1:100) for 30 min (Molecular Probes, Eugene, Oregon). Labeled cells were washed twice in ice cold PBS and analyzed using a FACSCalibur flow cytometer (Beckman Coulter, Miami, FL). Ten thousand cells (no gating) were collected and analyzed in the FL1 channel. All analyses were based on control cells incubated with isotype-specific IgGs to establish the background signal.
Fig. 1. Equine umbilical cord cells in culture and oct-4/alkaline phosphatase staining. (A) Photomicrograph of undifferentiated EUCM cells in culture demonstrating a stellate appearance (magnification 30·). (B) Analysis of expression of pluripotent stem cell markers, Oct-3/4 and alkaline phosphatase, by cultured undifferentiated EUCM cells. Alkaline phosphatase expression (pink) in the cytoplasm is expressed in cells that also have nuclear Oct-4 expression (green punctuate) (magnification 63·).
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Results Equine umbilical cord matrix is a reservoir of primitive stem cells Undifferentiated EUCM cells demonstrate a stellate appearance in culture (Fig. 1A) and were found to express Oct-4, an essential transcription factor for maintaining the primitive pluripotent state of embryonic stem (ES) cells, as shown by the punctate nuclear expression pattern (Fig. 1B). In addition, Oct-4-expressing EUCM cells showed high levels of cytoplasmic alkaline phosphatase activity, which has been associated with undifferentiated cells (Fig. 1). Taken together, expression of these two proteins suggests a primitive phenotype for cultured EUCM cells. Culture and growth characteristics of EUCM cells The population doubling time of EUCM cells in culture showed an initial lag time (Fig. 2). By passage three, the population doubling time became relatively constant through passage eight with a mean of 3.6 ± 0.09 days (means ± SD, n = 4) (Fig. 2A). Cell cycle analysis indicated the existence of a large subset (76%) of quiescent cells (G0/ G1) along with a smaller population (20%) of proliferating cells (S + G2/M) (Fig. 2B) [24,25]. Morphology and growth characteristics of the cultured EUCM cells did not change significantly over the eight passages. The CFU-F assay was performed according to previously described methods [26]. The CFU-F range was 1:337 ± 93.9 (n = 4) (means ± SD) for P1. Characterization of phenotype The expression of a number of markers associated with stem cells from embryonic and adult stem cells was assessed by flow cytometry in cultured EUCM cells (Fig. 3). EUCM cells showed expression of the embryonic markers: Oct-4, SSEA-4, and c-Kit, as well as weak expression of SSEA-3 and TRA-1-60 (<10%). Expression of c-Myc, a regulator of early gene expression and cell cycle progression in a variety of proliferating cells, was detected as well. EUCM cells also expressed a number of antigens associated with pluripotent adult stem cells, including CD54, CD90, CD105, and CD146. Expression of HLA-ABC, HLA-1AG (not shown), and MHC-II (not shown) was not detectable. While HLAABC immunoreactivity was detected in other equine adult stem cells (data not shown), positive expression of HLA1AG and MHC-II was not confirmed for equine. The reagents used for CD34, CD45, and CD133 also were not confirmed for equine, so the negative reactions for these markers are not necessarily true negatives. Adipogenic, chondrogenic, and osteogenic differentiation of EUCM cells EUCM cells cultured by previously described methods [21], demonstrated to differentiate human MSC into mes-
Fig. 2. Growth characteristics of EUCM cells in culture. (A) Celldoubling time of passaged umbilical cord-derived stem cells were compiled from multiple EUCM cell cultures and are shown as the means (n = 4) with error bars indicating ±SD. (B) The histogram indicates a representative cell cycle study of EUCM cells from P3 during log-phase growth.
enchymal cell lineages, resulted in the differentiation of EUCM cells into adipogenic, chondrogenic, and osteogenic cell types as shown in Fig. 4. EUCM cells cultured in osteogenic medium formed mineralized bone nodules in approximately 2 weeks and stained positively with Alizarin Red (Fig. 4A). EUCM cells treated by the chondrogenic differentiation protocol started with cell aggregates in culture that became transformed into spherical masses over time, which were stained by Toluidine Blue (Fig. 4B). EUCM cells grown in adipogenic medium formed intracellular lipid globules (indicated by Oil Red O staining) within 1 week, which enlarged over time after treatment with differentiation medium (Fig. 4C).
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Fig. 3. Phenotype of cultured EUCM cells. Flow cytometry analysis of the protein expression by cultured EUCM cells (P3 to P4) labeled with antibodies against antigens: CD34, CD45, CD54, CD73, CD90, CD105, CD133, CD146, c-Kit, c-Myc, HLA-ABC, Oct-4, SSEA-3, SSEA-4, and TRA-1-60. Shaded histograms indicate fluorescence intensity of EUCM cells labeled with isotype control antibody only; open histograms, positive reactivity with the indicated antibodies. Histograms represent relative number of cells vs. fluorescence intensity.
Neuronal differentiation of EUCM cells EUCM cells were treated to become neural stem cells and neuronal cells as described previously [22,23]. After treatment, the EUCM cells adopted a morphology typical of neurons with axon- and dendrite-like processes and the appearance of primitive networks of processes typical of those observed in primary neurons in culture (Fig. 4D). This observation parallels that made for porcine and
human UCM cells [23], in which UCM cells were found to differentiate into neural cells with a similar morphologic appearance as shown in Fig. 4D. Discussion The Wharton’s jelly or matrix is the gelatinous connective tissue from the umbilical cord and is composed of myofibroblast-like stromal cells, collagen fibers, and prote-
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Fig. 4. EUCM cells differentiate into osteocyte, chondrocyte, adipocyte, and neuronal-like cells. The panels display representative photomicrographs of: (A) osteoblasts detected by Alizarin Red staining (magnification 30·); (B) chondrocytes detected by toluidine blue staining (magnification 15·); (C) adipocytes detected by Oil Red O staining (magnification 30·); (D) brightfield photomicrograph of neuron-like cells derived from EUCM with multiple axon- and dendrite-like processes typical of primary neurons in culture (magnification 20·).
oglycans [27]. Following enzymatic digestion of the cord tissue, adherent EUCM cells were expanded in culture until sufficient numbers were available for characterization and differentiation studies. The focus of this study was to characterize the equine umbilical cord matrix cells and their differentiation capacity. Adherent cells from EUCM were found to have the capacity to proliferate extensively in vitro and to maintain their morphological and growth characteristics over the passages studied. The doubling times (Fig. 2A) and CFU-F are similar to those observed from cells of the perivascular region of the human umbilical cord [28]. EUCM cells can also be cryogenically stored and brought back into culture with no obvious changes in their growth or phenotypic characteristics (data not shown). EUCM cells grown in the absence of differentiation media have the myofibroblast-like stellar morphology observed for UCM cells from other species as well as bone marrow stromal and other tissue-specific MSC [23]. EUCM cells appear to be primitive ES-like cells based on expression of markers such as Oct-4, SSEA-3, SSEA-4, and TRA-1-60 as shown by flow cytometry (Fig. 3). Of particular interest is the expression of Oct-4, an essential transcription factor for maintaining the primitive pluripotent state of ES cells. In addition, Oct-4expressing EUCM cells showed high levels of alkaline phosphatase activity, which has been associated with undifferentiated cells (Fig. 1) [29,30]. Additional support for the existence of primitive cells in EUCM was the finding that a large subset of the cultured cells remained in
the quiescent state (Go/G1), a trait related to self-renewal ability (Fig. 2B) [31]. EUCM cells also exhibit properties of tissue-specific MSC-like cells. A number of markers commonly used to define MSC populations were assessed [11]. EUCM cells expressed the MSC-associated antigens CD54, CD90, and CD105. They also express CD146, a marker for pericytes, that has been associated with a number of MSCs [32]. A lack of reactivity with the hematopoietic stem cell markers, CD34, CD45, and CD133, might indicate that the EUMC lack hematopoietic progenitors or that the human-directed reagents do not cross-react with the corresponding equine epitopes. To evaluate further the potential MSC phenotype of EUCM cells, they were treated in osteo-, chondro-, and adipogenic differentiation media. The results suggest that these cells are capable of differentiating into the expected MSC-derived cell types (Fig. 4A–C). This is consistent with findings in which human UCM cells have been shown to produce collagen Type I and II- and glucosaminoglycanexpressing cells when grown on a three-dimensional matrix, which is consistent with cartilage formation [19]. This suggests that in addition to their primitive ES-cell-like characteristics, that EUCM cells can readily form mesenchymal lineage cells, such as bone, cartilage, and fat. The ES-cell and MSC-like characteristics of the EUCM cells as demonstrated herein indicate that they have a pluripotent phenotype. This is supported by preliminary findings that show differentiation of EUCM cells into neuronlike cells (Fig. 4D). Importantly, this suggests that EUCM cells are capable of differentiation into multiple germ lay-
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ers: mesoderm and ectoderm. Future work includes studies to determine the capacity of EUCM cells to differentiate into endodermal cell types and to characterize the EUCM-derived neuron-like cells. An important property to evaluate in stem cells to assess their usefulness in allogeneic regenerative medicine is the expression of markers related to their immunogenicity. In the present study, we evaluated expression of HLA-ABC, HLA-1AG, and MHC-II. There was no significant expression of any of these antigens in EUCM cells. Expression of these antigens in other equine cell types was confirmed only for HLA-ABC. Therefore, the problem of using a reagent developed for human cells means that the reaction with equine cells could be truly negative or the reagent does not cross-react with the equine epitope. These markers were found to be negative in studies of HUCM cells obtained in a similar manner to the EUMC cells [9]. However, autologous use of EUMC cells side steps the need for low antigenicity. In summary, these data demonstrate that EUCM cells cultured in stromal media give rise to a population of cells with functional features similar to MSCs, but with a population of cells that reflect a more primitive cell type possessing self-renewal properties. EUCM cells also have the capacity to proliferate extensively, and to differentiate into several cells types in vitro, all characteristics that point to an important role for EUCM cells in stem cell-based therapies in the horse. Disclosures The authors indicate no potential conflicts of interest. References [1] P.A. Zuk, M. Zhu, P. Ashjian, D.A. De Ugarte, J.I. Huang, H. Mizuno, Z.C. Alfonso, J.K. Fraser, P. Benhaim, M.H. Hedrick, Human adipose tissue is a source of multipotent stem cells, Mol. Biol. Cell 13 (2002) 4279–4295. [2] J.G. Toma, M. Akhavan, K.J. Fernandes, F. Barnabe-Heider, A. Sadikot, D.R. Kaplan, F.D. Miller, Isolation of multipotent adult stem cells from the dermis of mammalian skin, Nat. Cell Biol. 3 (2001) 778–784. [3] Y. Jiang, B.N. Jahagirdar, R.L. Reinhardt, R.E. Schwartz, C.D. Keene, X.R. Ortiz-Gonzalez, M. Reyes, T. Lenvik, T. Lund, M. Blackstad, J. Du, S. Aldrich, A. Lisberg, W.C. Low, D.A. Largaespada, C.M. Verfaillie, Pluripotency of mesenchymal stem cells derived from adult marrow, Nature 418 (2002) 41–49. [4] M.F. Pittenger, A.M. Mackay, S.C. Beck, R.K. Jaiswal, R. Douglas, J.D. Mosca, M.A. Moorman, D.W. Simonetti, S. Craig, D.R. Marshak, Multilineage potential of adult human mesenchymal stem cells, Science 284 (1999) 143–147. [5] C. De Bari, F. Dell’Accio, P. Tylzanowski, F.P. Luyten, Multipotent mesenchymal stem cells from adult human synovial membrane, Arthritis Rheum. 44 (2001) 1928–1942. [6] G. Kogler, S. Sensken, J.A. Airey, T. Trapp, M. Muschen, N. Feldhahn, S. Liedtke, R.V. Sorg, J. Fischer, C. Rosenbaum, S. Greschat, A. Knipper, J. Bender, O. Degistirici, J. Gao, A.I. Caplan, E.J. Colletti, G. Almeida-Porada, H.W. Muller, E. Zanjani, P. Wernet, A new human somatic stem cell from placental cord blood with intrinsic pluripotent differentiation potential, J. Exp. Med. 200 (2004) 123–135.
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Characterization and differentiation of equine umbilical cord-derived matrix cells Steven M. Hoynowski a, Madeline M. Fry a, Bryn M. Gardner a, Matthew T. Leming a, Jeanell R. Tucker a, Linda Black b, Theodore Sand b,*, Kathy E. Mitchell a,* a
Department of Pharmacology and Toxicology, University of Kansas, 1251 Wescoe Hall Drive, Malott Rm 5064, Lawrence, KS 66045-7582, USA b Vet-Stem, Inc., 12860 Danielson Court, Suite B, Poway, CA 92064, USA Received 12 July 2007 Available online 13 August 2007
Abstract Stem cells are being evaluated in numerous human clinical trials and are commercially used in veterinary medicine to treat horses and dogs. Stem cell differentiation, homing to disease sites, growth and cytokine factor modulation, and low antigenicity contribute to their therapeutic success. Bone marrow and adipose tissue are the two most common sources of adult-derived stem cells in animals. We report on the existence of an alternative source of primitive, multipotent stem cells from the equine umbilical cord cellular matrix (Wharton’s jelly). Equine umbilical cord matrix (EUCM) cells can be cultured, cryogenically preserved, and differentiated into osteo-, adipo-, chondrogenic, and neuronal cell lineages. These results identify a source of stem cells that can be non-invasively collected at birth and stored for future use in that horse or used as donor cells for treating unrelated horses. 2007 Elsevier Inc. All rights reserved. Keywords: Wharton’s jelly; Stem cells; Mesenchymal cells; Equine; Adipogenic differentiation; Osteogenic differentiation; Chondrogenic differentiation; Neuronal differentiation; Oct-4; Umbilical cord matrix
Adult stem cells that are immunologically compatible can be harvested from a variety of sources including adipose tissue [1], skin tissue [2], bone marrow [3,4], and synovial membrane [5] and have no ethical issues related to their use. In addition to stem cells from the embryo and adult, stem cells can be found in extraembryonic tissues including umbilical cord and placental blood [6], amniotic fluid [7], and umbilical cord matrix [8–11]. Stem cells from extraembryonic sources have the advantages of being obtained by non-invasive procedures and low immunogenicity. For example, umbilical cord blood cells express low levels of HLA antigens and have been used for bone marrow replacement when HLA-
* Corresponding authors. Fax: +1 858 748 2005 (T. Sand), +1 785 864 5219 (K.E. Mitchell). E-mail addresses: [email protected] (T. Sand), [email protected] (K.E. Mitchell).
0006-291X/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2007.07.182
matched donors cannot be found [12]. Clinical trials are currently underway using stem cells from extraembryonic sources for treating a number of diseases [13,14] and for tissue engineering [15,16]. These preliminary studies show the promise of stem cells derived from extraembryonic tissues and yet little is known about their basic properties. We have demonstrated that cells isolated from porcine umbilical cord matrix differentiated in vitro into cells that resembled neurons morphologically and expressed neuronal and glial-specific proteins [8]. We also demonstrated that porcine umbilical cord matrix cells survive, migrate, and express markers for mature neurons when transplanted into rat brain [9]. Importantly, the implanted umbilical cord matrix cells showed no evidence of forming teratomas [9]. Rat umbilical cord matrix cells show similar properties [17]. When injected into rat brains in a model of whole brain ischemia, not only do they survive and migrate, they show a protective effect against damage to
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CA1 hippocampal neurons and improve overall neurological outcome [17]. These results indicate that umbilical cord matrix cells are a potential source of multipotent stem cells and may support many therapeutic and biotechnological roles [18]. Here, we describe the isolation and properties of equine-derived umbilical cord matrix cells (EUCM). Materials and methods Isolation of equine umbilical cord matrix cells and cell culture. Vet-Stem (Poway, CA) provided equine umbilical cords obtained in accordance with standard veterinary practice. The umbilical cord tissue was processed as previously described [19]. When culture confluency reached 50–80% the cells were harvested using 0.05% trypsin/0.53 mM EDTA solution and replated into larger culture flasks at a 1:2 split ratio. Cells were enumerated by dilution (1:2) in trypan blue (ICN, Aurora, OH) and counted with a hemacytometer using a Nikon Eclipse TS100 microscope to enumerate viable cells for CD, CFU-F, flow cytometry, or differentiation assays. Cultures were maintained at the stated confluency (50–80%) for propagation in a 37 C humidified incubator with 5% CO2. For cryogenic preservation, cells were suspended at 1 · 106 cells/mL in 10% DMSO and 90% FBS, and frozen in liquid nitrogen. Cell cycle analysis. EUCM cells were trypsinized and resuspended at 1– 2 · 106 cells/mL and fixed with 70% ethanol for 1 h at 4 C, then washed twice with cold PBS. Cells were labeled with 100 lL of 0.5 mg/mL propidium iodide (Roche, Indianapolis) (106 cells in 1 mL) followed by treatment with 1 U of DNase-free RNase A (Fisher, Fair Lawn, NJ) (106 cells in 1 mL) and incubated for 3 h at 4 C. CFU-F. The CFU frequency (CFU-F) was determined by means of limiting dilution. Nucleated cells (n = 4) isolated from equine umbilical cords were seeded in six-well plates at the following densities: 25, 50, 100, 200, 400, 800 cells/cm2. Cells were maintained in stromal medium that was changed every 2–3 days. After 10 days, the cells were washed and stained with a-Naphthyl Acetate Kit (Sigma–Aldrich, St. Louis, MO) according to manufacturer’s instructions. Positively stained colonies that contained >50 cells were counted as CFU’s. Proliferation studies. Cells from EUCM (n = 4 cords) were counted and seeded in T25 flasks. The cells from each flask were harvested and enumerated over eight passages starting with passage 1 (P1). The mean of the counts was calculated by the following formula: CD = ln(Nf/Ni)/ln 2 and DT = CT/CD, where DT is the cell-doubling time, CD is cell-doubling number, and CT is cell culture time. The proliferative rate was cal-
culated from each passage, where Nf is the final number of cells and Ni the initial number of cells [20]. Adipogenic, chondrogenic, osteogenic, and neuronal differentiation. EUCM cells were expanded in culture for three to four passages to obtain sufficient cells for differentiation into mesenchymal lineages. EUCM cells were counted and seeded at 2 · 105 cells per well in six-well plates 1 day prior to differentiation. Cells were then washed twice with PBS and differentiation media were added as described by Wang et al. [21]. The differentiation media were changed every 3 days. EUCM cells were induced to become neural stem cells and neuronal cells as described previously [22,23]. Histochemical staining. Differentiated cells were washed twice with PBS, fixed with 4% paraformaldehyde for 10 min at 37 C, and then washed thoroughly with PBS. To assess differentiation, cells were stained with Alizarin Red for osteogenic, Toluidine Blue for chondrogenic, and Oil Red O for adipogenic differentiation and imaged with a Nikon Eclipse TE 2000U and Photometrix Cool Snapcf digital camera using MetaMorph (Universal Imaging, Downingtown, PA) imaging software. Immunocytochemistry and alkaline phosphatase detection. Cultured EUCM cells were fixed with 4% paraformaldehyde in PBS for 10 min and then washed in PBS. Cells were permeabilized with 0.2% Triton X-100 in PBS for 5 min, washed, blocked in 0.2% Triton X-100, 2% normal serum in PBS for 30 min, followed by incubation with primary antibody against Oct-4 (1:50) (Abcam, Cambridge, MA) for 1 h, washed three times with PBS, and incubated with chicken anti-rabbit Alexa Fluor 488-conjugated secondary antibody (1:200) for 30 min (Molecular Probes, Eugene, Oregon). Excess secondary antibody was removed by repeated washing with PBS. Alkaline phosphatase activity was detected using an Alkaline Phosphatase Detection Kit (Chemicon, Temecula, CA) according to manufacturer’s instruction. Images were obtained with a 510 Zeiss laser scanning microscope under a 40· oil-immersion lens. Flow cytometry. EUCM cells at 200–400 · 105 cells/mL were fixed and permeabilized using BD Cytofix/Cytoperm Plus Kit (BD Biosciences, San Diego, CA) according to manufacturer’s instructions. Cells were labeled with primary antibodies: CD54, CD133, Oct-4 (Abcam, Cambridge, MA); CD34, CD45, CD73, and CD105 (BD Biosciences, San Jose, CA); CD90, CD146, HLA-ABC, SSEA-3, and TRA-1-60 (Chemicon, Temecula, CA); c-Kit, c-Myc, and SSEA-4 (Santa Cruz Biotechnology, Santa Cruz, CA.) for 1 h, followed by incubation with secondary Alexa Fluor 488 conjugates (1:100) for 30 min (Molecular Probes, Eugene, Oregon). Labeled cells were washed twice in ice cold PBS and analyzed using a FACSCalibur flow cytometer (Beckman Coulter, Miami, FL). Ten thousand cells (no gating) were collected and analyzed in the FL1 channel. All analyses were based on control cells incubated with isotype-specific IgGs to establish the background signal.
Fig. 1. Equine umbilical cord cells in culture and oct-4/alkaline phosphatase staining. (A) Photomicrograph of undifferentiated EUCM cells in culture demonstrating a stellate appearance (magnification 30·). (B) Analysis of expression of pluripotent stem cell markers, Oct-3/4 and alkaline phosphatase, by cultured undifferentiated EUCM cells. Alkaline phosphatase expression (pink) in the cytoplasm is expressed in cells that also have nuclear Oct-4 expression (green punctuate) (magnification 63·).
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Results Equine umbilical cord matrix is a reservoir of primitive stem cells Undifferentiated EUCM cells demonstrate a stellate appearance in culture (Fig. 1A) and were found to express Oct-4, an essential transcription factor for maintaining the primitive pluripotent state of embryonic stem (ES) cells, as shown by the punctate nuclear expression pattern (Fig. 1B). In addition, Oct-4-expressing EUCM cells showed high levels of cytoplasmic alkaline phosphatase activity, which has been associated with undifferentiated cells (Fig. 1). Taken together, expression of these two proteins suggests a primitive phenotype for cultured EUCM cells. Culture and growth characteristics of EUCM cells The population doubling time of EUCM cells in culture showed an initial lag time (Fig. 2). By passage three, the population doubling time became relatively constant through passage eight with a mean of 3.6 ± 0.09 days (means ± SD, n = 4) (Fig. 2A). Cell cycle analysis indicated the existence of a large subset (76%) of quiescent cells (G0/ G1) along with a smaller population (20%) of proliferating cells (S + G2/M) (Fig. 2B) [24,25]. Morphology and growth characteristics of the cultured EUCM cells did not change significantly over the eight passages. The CFU-F assay was performed according to previously described methods [26]. The CFU-F range was 1:337 ± 93.9 (n = 4) (means ± SD) for P1. Characterization of phenotype The expression of a number of markers associated with stem cells from embryonic and adult stem cells was assessed by flow cytometry in cultured EUCM cells (Fig. 3). EUCM cells showed expression of the embryonic markers: Oct-4, SSEA-4, and c-Kit, as well as weak expression of SSEA-3 and TRA-1-60 (<10%). Expression of c-Myc, a regulator of early gene expression and cell cycle progression in a variety of proliferating cells, was detected as well. EUCM cells also expressed a number of antigens associated with pluripotent adult stem cells, including CD54, CD90, CD105, and CD146. Expression of HLA-ABC, HLA-1AG (not shown), and MHC-II (not shown) was not detectable. While HLAABC immunoreactivity was detected in other equine adult stem cells (data not shown), positive expression of HLA1AG and MHC-II was not confirmed for equine. The reagents used for CD34, CD45, and CD133 also were not confirmed for equine, so the negative reactions for these markers are not necessarily true negatives. Adipogenic, chondrogenic, and osteogenic differentiation of EUCM cells EUCM cells cultured by previously described methods [21], demonstrated to differentiate human MSC into mes-
Fig. 2. Growth characteristics of EUCM cells in culture. (A) Celldoubling time of passaged umbilical cord-derived stem cells were compiled from multiple EUCM cell cultures and are shown as the means (n = 4) with error bars indicating ±SD. (B) The histogram indicates a representative cell cycle study of EUCM cells from P3 during log-phase growth.
enchymal cell lineages, resulted in the differentiation of EUCM cells into adipogenic, chondrogenic, and osteogenic cell types as shown in Fig. 4. EUCM cells cultured in osteogenic medium formed mineralized bone nodules in approximately 2 weeks and stained positively with Alizarin Red (Fig. 4A). EUCM cells treated by the chondrogenic differentiation protocol started with cell aggregates in culture that became transformed into spherical masses over time, which were stained by Toluidine Blue (Fig. 4B). EUCM cells grown in adipogenic medium formed intracellular lipid globules (indicated by Oil Red O staining) within 1 week, which enlarged over time after treatment with differentiation medium (Fig. 4C).
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Fig. 3. Phenotype of cultured EUCM cells. Flow cytometry analysis of the protein expression by cultured EUCM cells (P3 to P4) labeled with antibodies against antigens: CD34, CD45, CD54, CD73, CD90, CD105, CD133, CD146, c-Kit, c-Myc, HLA-ABC, Oct-4, SSEA-3, SSEA-4, and TRA-1-60. Shaded histograms indicate fluorescence intensity of EUCM cells labeled with isotype control antibody only; open histograms, positive reactivity with the indicated antibodies. Histograms represent relative number of cells vs. fluorescence intensity.
Neuronal differentiation of EUCM cells EUCM cells were treated to become neural stem cells and neuronal cells as described previously [22,23]. After treatment, the EUCM cells adopted a morphology typical of neurons with axon- and dendrite-like processes and the appearance of primitive networks of processes typical of those observed in primary neurons in culture (Fig. 4D). This observation parallels that made for porcine and
human UCM cells [23], in which UCM cells were found to differentiate into neural cells with a similar morphologic appearance as shown in Fig. 4D. Discussion The Wharton’s jelly or matrix is the gelatinous connective tissue from the umbilical cord and is composed of myofibroblast-like stromal cells, collagen fibers, and prote-
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Fig. 4. EUCM cells differentiate into osteocyte, chondrocyte, adipocyte, and neuronal-like cells. The panels display representative photomicrographs of: (A) osteoblasts detected by Alizarin Red staining (magnification 30·); (B) chondrocytes detected by toluidine blue staining (magnification 15·); (C) adipocytes detected by Oil Red O staining (magnification 30·); (D) brightfield photomicrograph of neuron-like cells derived from EUCM with multiple axon- and dendrite-like processes typical of primary neurons in culture (magnification 20·).
oglycans [27]. Following enzymatic digestion of the cord tissue, adherent EUCM cells were expanded in culture until sufficient numbers were available for characterization and differentiation studies. The focus of this study was to characterize the equine umbilical cord matrix cells and their differentiation capacity. Adherent cells from EUCM were found to have the capacity to proliferate extensively in vitro and to maintain their morphological and growth characteristics over the passages studied. The doubling times (Fig. 2A) and CFU-F are similar to those observed from cells of the perivascular region of the human umbilical cord [28]. EUCM cells can also be cryogenically stored and brought back into culture with no obvious changes in their growth or phenotypic characteristics (data not shown). EUCM cells grown in the absence of differentiation media have the myofibroblast-like stellar morphology observed for UCM cells from other species as well as bone marrow stromal and other tissue-specific MSC [23]. EUCM cells appear to be primitive ES-like cells based on expression of markers such as Oct-4, SSEA-3, SSEA-4, and TRA-1-60 as shown by flow cytometry (Fig. 3). Of particular interest is the expression of Oct-4, an essential transcription factor for maintaining the primitive pluripotent state of ES cells. In addition, Oct-4expressing EUCM cells showed high levels of alkaline phosphatase activity, which has been associated with undifferentiated cells (Fig. 1) [29,30]. Additional support for the existence of primitive cells in EUCM was the finding that a large subset of the cultured cells remained in
the quiescent state (Go/G1), a trait related to self-renewal ability (Fig. 2B) [31]. EUCM cells also exhibit properties of tissue-specific MSC-like cells. A number of markers commonly used to define MSC populations were assessed [11]. EUCM cells expressed the MSC-associated antigens CD54, CD90, and CD105. They also express CD146, a marker for pericytes, that has been associated with a number of MSCs [32]. A lack of reactivity with the hematopoietic stem cell markers, CD34, CD45, and CD133, might indicate that the EUMC lack hematopoietic progenitors or that the human-directed reagents do not cross-react with the corresponding equine epitopes. To evaluate further the potential MSC phenotype of EUCM cells, they were treated in osteo-, chondro-, and adipogenic differentiation media. The results suggest that these cells are capable of differentiating into the expected MSC-derived cell types (Fig. 4A–C). This is consistent with findings in which human UCM cells have been shown to produce collagen Type I and II- and glucosaminoglycanexpressing cells when grown on a three-dimensional matrix, which is consistent with cartilage formation [19]. This suggests that in addition to their primitive ES-cell-like characteristics, that EUCM cells can readily form mesenchymal lineage cells, such as bone, cartilage, and fat. The ES-cell and MSC-like characteristics of the EUCM cells as demonstrated herein indicate that they have a pluripotent phenotype. This is supported by preliminary findings that show differentiation of EUCM cells into neuronlike cells (Fig. 4D). Importantly, this suggests that EUCM cells are capable of differentiation into multiple germ lay-
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ers: mesoderm and ectoderm. Future work includes studies to determine the capacity of EUCM cells to differentiate into endodermal cell types and to characterize the EUCM-derived neuron-like cells. An important property to evaluate in stem cells to assess their usefulness in allogeneic regenerative medicine is the expression of markers related to their immunogenicity. In the present study, we evaluated expression of HLA-ABC, HLA-1AG, and MHC-II. There was no significant expression of any of these antigens in EUCM cells. Expression of these antigens in other equine cell types was confirmed only for HLA-ABC. Therefore, the problem of using a reagent developed for human cells means that the reaction with equine cells could be truly negative or the reagent does not cross-react with the equine epitope. These markers were found to be negative in studies of HUCM cells obtained in a similar manner to the EUMC cells [9]. However, autologous use of EUMC cells side steps the need for low antigenicity. In summary, these data demonstrate that EUCM cells cultured in stromal media give rise to a population of cells with functional features similar to MSCs, but with a population of cells that reflect a more primitive cell type possessing self-renewal properties. EUCM cells also have the capacity to proliferate extensively, and to differentiate into several cells types in vitro, all characteristics that point to an important role for EUCM cells in stem cell-based therapies in the horse. Disclosures The authors indicate no potential conflicts of interest. References [1] P.A. Zuk, M. Zhu, P. Ashjian, D.A. De Ugarte, J.I. Huang, H. Mizuno, Z.C. Alfonso, J.K. Fraser, P. Benhaim, M.H. Hedrick, Human adipose tissue is a source of multipotent stem cells, Mol. Biol. Cell 13 (2002) 4279–4295. [2] J.G. Toma, M. Akhavan, K.J. Fernandes, F. Barnabe-Heider, A. Sadikot, D.R. Kaplan, F.D. Miller, Isolation of multipotent adult stem cells from the dermis of mammalian skin, Nat. Cell Biol. 3 (2001) 778–784. [3] Y. Jiang, B.N. Jahagirdar, R.L. Reinhardt, R.E. Schwartz, C.D. Keene, X.R. Ortiz-Gonzalez, M. Reyes, T. Lenvik, T. Lund, M. Blackstad, J. Du, S. Aldrich, A. Lisberg, W.C. Low, D.A. Largaespada, C.M. Verfaillie, Pluripotency of mesenchymal stem cells derived from adult marrow, Nature 418 (2002) 41–49. [4] M.F. Pittenger, A.M. Mackay, S.C. Beck, R.K. Jaiswal, R. Douglas, J.D. Mosca, M.A. Moorman, D.W. Simonetti, S. Craig, D.R. Marshak, Multilineage potential of adult human mesenchymal stem cells, Science 284 (1999) 143–147. [5] C. De Bari, F. Dell’Accio, P. Tylzanowski, F.P. Luyten, Multipotent mesenchymal stem cells from adult human synovial membrane, Arthritis Rheum. 44 (2001) 1928–1942. [6] G. Kogler, S. Sensken, J.A. Airey, T. Trapp, M. Muschen, N. Feldhahn, S. Liedtke, R.V. Sorg, J. Fischer, C. Rosenbaum, S. Greschat, A. Knipper, J. Bender, O. Degistirici, J. Gao, A.I. Caplan, E.J. Colletti, G. Almeida-Porada, H.W. Muller, E. Zanjani, P. Wernet, A new human somatic stem cell from placental cord blood with intrinsic pluripotent differentiation potential, J. Exp. Med. 200 (2004) 123–135.
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