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BOR - Papers in Press, published online ahead of print October 1, 2003.
Biol Reprod 2003, 10.1095/biolreprod.103.020271
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BIOLOGY OF REPRODUCTION 70, 415–418 (2004)
DOI: 10.1095/biolreprod.103.020271
© 2004 by the Society for the Study of Reproduction, Inc.

Reproductive Technology

Nuclear Transfer of Adult Bone Marrow Mesenchymal Stem Cells: Developmental Totipotency of Tissue-Specific Stem Cells from an Adult Mammal1

Yoko Kato3, Hideaki Imabayashi4, Taisuke Mori4, Tetsuya Tani3, Masanori Taniguchi5, Mikihiko Higashi5, Michio Matsumoto5, Akihiro Umezawa4, and Yukio Tsunoda2,3

Laboratory of Animal Reproduction,3 College of Agriculture, Kinki University, Nara 631-8505, Japan Department of Pathology,4 Keio University School of Medicine, Tokyo 160-8582, Japan Kumamoto Prefecture Livestock Stations and Animal Public Health Center,5 Kumamoto 861-1113, Japan


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Recent studies have demonstrated that somatic stem cells have a flexible potential greater than previously expected when they are transplanted into different tissues. On the other hand, recent studies also have revealed that these potentials might occur because of spontaneous cell fusion with recipient cells. The nuclei of somatic cells could have been reprogrammed when they were artificially or spontaneously fused with mouse embryonic stem (ES) cells. The resultant hybrid cells acquired a developmental pluripotency that the original somatic cells did not have but that ES cells did. LaBarge and Blau (Cell 2002; 111:589–601) demonstrated that adult bone marrow-derived cells contributed to muscle tissue in a stepwise biological progression. This means that bone marrow-derived cells became satellite cells of mononucleate muscle stem cells after the first irradiation-induced damage to the mouse, and after the second irradiation-induced damage, multinucleate myofibers appeared from the bone marrow-derived cells. Considered together, the differentiation potential of the somatic stem cell nucleus itself remains unclear. Although the pluripotency of somatic stem cell populations has been evaluated, the developmental totipotency of the nuclei of somatic stem cells, whether or not they fused with other cells, has not been shown, except in only one study concerning fetal neural cells (never in adult stem cells). Here, we showed the developmental totipotency of adult bovine mesenchymal stem cells by nuclear transfer.

assisted reproductive technology, developmental biology, early development, embryo, gamete biology


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Somatic stem cells were recently identified in hematopoietic [1], hepatic [2], epidermal [3], gastrointestinal [4], neural [5, 6], muscular [6], and bone marrow [6–8] tissues. Many researchers have demonstrated the developmental pluripotency of somatic stem cells of other tissues. Bone marrow-derived stem cells can be trans-differentiated into multilineage cells, such as muscle [9] of mesoderm, lung [10] and liver [10, 11] of endoderm, and brain [12–15] and skin [10] of ectoderm. Moreover, adult neural stem cells generated all germ layers in chimera [16]. Thus, somatic stem cells were evaluated to determine the extent of their potential for trans-differentiation in other tissues. It has been suggested that somatic stem cells are more desirable than embryonic stem (ES) cells for cell therapeutics because of ethical considerations and possible immunologic rejection of ES cells. The developmental totipotency of somatic stem cells, however, remains unknown.

Recent studies demonstrated that somatic stem cells have a more flexible potential than expected when put into different tissues. On the other hand, recent studies also revealed the possibility that this high potential might develop because of spontaneous cell fusion with recipient ES cells [17, 18]. Somatic cell nuclei can be reprogrammed when artificially [17] or spontaneously [17, 18] fused with mouse ES cells. The resulting hybrid cells acquire a developmental pluripotency that the original somatic cells do not have, but that ES cells do. LaBarge and Blau [19], however, demonstrated that adult bone marrow-derived cells contributed to muscle tissue in a stepwise biological progression. This finding indicates that bone marrow-derived cells became satellite cells of mononucleate muscle stem cells after the first irradiation-induced damage, and after the second irradiation-induced damage, multinucleate myofibers appeared from the bone marrow-derived cells.

Thus, the differentiation potential of somatic stem cell nuclei remains unclear. Although the pluripotency of somatic stem cells has been evaluated by fusion with other cells, the developmental totipotency of somatic stem cell nuclei has not been demonstrated in adult stem cells; however, it has been demonstrated in one report using fetal neural cells [20].

In the present study, we report the developmental totipotency of adult bovine mesenchymal stem cells by nuclear transfer. To our knowledge, this is the first report of developmental totipotency of tissue-specific stem cells derived from the adult mesodermal cell lineage. The present study clearly demonstrates that pluripotent bovine mesenchymal stem cells derived from adult bone marrow have developmental totipotency by nuclear transfer.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
All experiments and protocols were carried out in strict accordance with the Guiding Principles for the Care and Use of Research Animals promulgated by the Kinki University Committee on Animal Research and Bioethics.

Bone marrow mesenchymal stem cells were cultivated from female and male bovine stromal cells isolated from a femur, sternum, and rib [14, 21–23] by flushing the bone. Collected cells were cultured in 20% fetal bovine serum (FBS) supplemented with Dulbecco modified Eagle medium (DMEM; high glucose) for several days in vitro and subcultured several times before use.

Recipient ooplasm was obtained from enucleated metaphase II-phase oocytes that were derived from in vitro culture for 22–24 h (TCM-199, catalog no. 31100-027, supplemented with 10% FBS; Gibco BRL, Invitrogen, Carlsbad, CA) of immature germinal vesicle-phase oocytes residing in ovarian follicles. In some case, ovaries that had been preserved overnight at a low temperature were used for collection of oocytes because of a national guideline for avoiding bovine spongiform encephalopathic infection from slaughterhouses in Japan. The best temperature at which to preserve ovaries overnight was previously determined. Judging by the in vitro maturation rate of immature oocytes and the in vitro developmental potential after in vitro fertilization of in vitro-matured oocytes, 10°C preserves ovaries overnight in saline solution (Matsushita et al., unpublished observation). Thus, we preserved the ovaries at 10°C until the infection test for bovine spongiform encephalopathy was completed.

Nuclear transfer methods were performed as described previously [24–26]. Quiescent donor cells, which were produced by serum starvation or contact inhibition, were used. A single donor cell was electrically fused with oocytes immediately after enucleation using two direct current (DC) pulses of 150 V/mm for 25 µsec in Zimmerman fusion medium [24]. Two fusion pulses were administered at 15-min intervals until fusion was achieved. Fused oocytes were further stimulated by electrical pulses (DC pulses of 20 V/mm for 20 µsec) to confirm activation. Reconstituted oocytes were immediately treated with cycloheximide (10 µg/ml) in CR1-aa medium with 3 mg of bovine serum albumin (fatty acid-free) for 5–6 h. After treatment, oocytes were cultured in cycloheximide-free medium. On the third day (first day = day of nuclear transfer), embryos were transferred to CR1-aa medium supplemented with 10% FBS and cocultured with mouse embryonic fibroblast cells, which are routinely used for mouse ES cell culture [27]. On the seventh day of culture, they were moved to modified DMEM supplemented with 10% FBS.

To determine the pluripotency of the donor cells, the cells were induced to differentiate into osteogenic precursor cells or adipocytes [17, 28]. Cells were stained with von Kossa and alkaline phosphatase for the osteogenic precursor cells, Oil-Red-O for adipocytes, and antibodies against nestin and glial fibrillary acidic protein (GFAP) for neuroectodermal differentiation. Moreover, specific antibodies were used to check the differentiation status and stem cell specificity, such as CD29, CD44, CD166, CD14, CD31, CD34, CD45, and CD117, which are routinely used as markers for human mesenchymal stem cells.

Data were analyzed using the chi-square and Student t-tests.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Some cells induced for specific differentiation were positive for von Kossa or alkaline phosphatase, indicating differentiation into osteogenic progenitor cells. In another culture, cells were positive for Oil-Red-O, indicating that they differentiated into adipocytes. Some cells were positive for nestin and GFAP antibodies, demonstrating successful neuroectodermal differentiation [18, 28].

When several antibodies used as markers of mesenchymal stem cells in human and mouse were examined, cells were positive for CD29, CD44, and CD166 and negative for CD14, CD31, CD34, CD45, and CD117. These results were consistent with those from human mesenchymal stem cells. The cells were negative, however, against CD90, CD105, and CD140a, which are usually positive for human and mouse mesenchymal stem cells.

We preliminarily examined whether oocytes collected from preserved ovaries have the same ability to mature in vitro to support development after in vitro fertilization and nuclear transfer with cumulus cells. We concluded that oocytes from ovaries preserved at 10°C overnight had an ability similar to that of those in the nonpreserved group (Matsushita et al., unpublished observation). When bovine mesenchymal stem cells were fused with enucleated metaphase II-phase oocytes with or without preservation and then electrically activated, they developed to the blastocyst stage in vitro after 8–9 days (Table 1). The proportion of blastocysts after nuclear transfer of mesenchymal stem cells was significantly decreased in the preserved group (7% vs. 39%), but after transfer to the foster mother, two recipients with embryos from preserved oocytes and one from nonpreserved oocytes were pregnant at Day 40. One female each from the preserved and nonpreserved groups was aborted between 80 and 120 days and between 40 and 80 days, respectively. One healthy offspring was obtained from the preserved group (Table 2). The results of the microsatellite marker analysis verified that donor cells were the source of the genetic material used to produce the cloned bovine.


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  		TABLE 1. In vitro development of nuclear transferred oocytes with adult bovine mesenchymal stem cells


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  		TABLE 2. In vivo development after transfer to recipient females


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Recently, Jiang et al. [8] reported that mesenchymal stem cells derived from adult mouse bone marrow have pluripotency in vitro. Those authors also demonstrated that mesenchymal stem cells differentiated into almost all tissues of adult mouse after injection of a single cell into blastocysts. This finding that tissue-specific stem cells could act like ES cells was extremely important. On the other hand, two different groups [17, 18] recently demonstrated that the high potential of somatic stem cells residing in other tissues might develop because of spontaneous cell fusion with other cells. These authors demonstrated that these stem cells have the ability to fuse with ES cells spontaneously in vitro. Once fused, the resulting hybrid cells acquire the developmental pluripotency of the ES cells. A similar observation was reported when somatic cells were artificially fused with ES cells [29]. Thus, it has remained unclear whether somatic stem cell nuclei have the ability to trans-differentiate. The results of the present study, however, clearly indicate that adult bovine mesenchymal stem cells have developmental totipotency after nuclear transfer.

Recently developed animal cloning technology using adult somatic cells by nuclear transfer [30] has several advantages, one of which is that the resulting animals are exactly the same as the provider of the donor cells. Compared with nuclear transfer of germ line cells, somatic cells are easily collected and cultured without serious injury to animals. Also, somatic cells do not undergo the essential modification of imprinted genes that occurs during the production of germ cells. When primordial germ cells (PGCs) were used as donors for nuclear transfer, the resulting fetuses were inviable at Day 10.5 because of the erasing and abnormal expression of imprinted genes that occur when PGCs differentiate into functional germ cells [31, 32]. In contrast, the results of nuclear transfer using ES cells largely depend on the cell lines, number of passages, and background of the mouse strains of the ES cells used [33–36]. The ES cells derived from inner-cell-mass cells of a blastocyst, which include a subset of cells that are PGCs, should develop into a fetus. Therefore, some ES cells must also undergo modification of imprinted genes during in vitro culture. Thus, somatic cells have advantages for nuclear transfer, and a solid nuclear transfer system using somatic cells must be established. For this reason, it is essential to determine the somatic cell type best suited for nuclear transfer to produce normal young.

Because the efficiency of somatic cell cloning remains low and peri- and postnatal death of young remains high [37], it is still too early to apply this method in human clinics or farm animal production without further basic research to solve the problems of abnormal development. Previously [25], we evaluated the cell type most appropriate for efficient cloning in bovines. We demonstrated that among the cell types tested, cumulus cells are good candidate nuclear donors, judging by the incidence of peri- and postnatal death of young. Even in cumulus cells, however, the potential after nuclear transfer depends on the cell line [25]. It is unclear both why and how cell lines affect developmental totipotency after nuclear transfer. Although cumulus cells are good cloning candidates, they can be obtained only from female animals. On the other hand, mesenchymal stem cells can be obtained from both female and male living animals. In the present study, we examined whether this cell type is a suitable nuclear donor candidate. As the results show, adult mesenchymal stem cells are good cloning candidates (one healthy clone was obtained).

Yamazaki et al. [20] demonstrated the developmental totipotency of embryonic neural cells in the cerebral cortex of mouse postimplantation embryos after nuclear transfer. They reported that the cloning efficiency of premature and early postmitotic neural cells from the ventricular side of the cortex was higher than that of postmitotic differentiated neurons from the pial side of the cortex and also higher than that of other somatic cells. Those authors concluded that for cloning, premature somatic stem cells in fetuses are better than differentiated somatic stem cells. This hypothesis is consistent with the previous success of Hochedlinger and Jaenisch [38]. Those authors obtained cloned mice derived from terminally differentiated, mature B and T cells using a two-step method, but they never succeeded with a simple nuclear transfer. In the present study, we demonstrated that adult bovine stem cells have preserved developmental totipotency.

Moreover, in most studies, the derivation of somatic cells used as donors was not clear. For example, most cells cultivated from tissues such as skin and liver were probably fibroblast cells, the viability of which might be higher than that of other specific cell types in tissues. In the present study, we examined the characteristics of bone marrow cells in detail using specific antibodies and stains. Bovine bone marrow mesenchymal stem cells had developmental pluripotency in vitro. They differentiated into not only osteogenic cells but also adipocytes, as examined by the in vitro culture experiments. Because no markers have been established for bovine mesenchymal stem cells [28], we used several markers for human mesenchymal stem cells. We do not know why CD90, CD105, and CD140a, which are positive markers for human mesenchymal stem cells, were negative in this investigation, but species-specific effects of these antibodies might be involved. Whether the cloned offspring were derived from such pluripotent cells is unclear, because it is technically impossible to determine precisely. However, as a group, our cell population was pluripotent, and the cloned animal was born from pluripotent mesenchymal stem cells.

In conclusion, the present study clearly demonstrates that bone marrow mesenchymal stem cells can be isolated and identified from adult bovine and that adult mesenchymal stem cells have developmental totipotency after nuclear transfer.


   ACKNOWLEDGMENTS
 
We thank all staff members at the Kumamoto Prefecture Livestock Stations and Animal Public Health Center for embryo transfer, assistance, and management of recipient animals.


   FOOTNOTES
 
1 Supported by grants from the Programme for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN) and partly supported by a grant from the Ministry of Education, Science, and Culture (14034259, 12358014, 13308050, 15039233); the Ministry of Agriculture, Forestry and Fisheries (Cloning); Ito Kinen Foundation; and Nakajima International Interchange Foundation. Back

2 Correspondence. FAX: 81 7 4243 115; tsunoda{at}nara.kindai.ac.jp Back

Received: 12 June 2003.

First decision: 1 July 2003.

Accepted: 30 September 2003.


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 DISCUSSION
 REFERENCES
 

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