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Isolation, Characterization and Tri-lineage Differentiation of Mesenchymal Stem Cells from Human Umbilical Cord Blood

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Since Broxmeyer et al.3)showed experimental evidence that human umbilical cord blood (hUCB) is a rich source of hematopoietic stem/progenitor cells (HSPCs), hUCB has been known as an excellent source of HSPCs for transplan- tation. To date, more than 3,000 cases of hUCB hematopoi- etic stem cells (HSCs) transplantation have been performed

worldwide for patients with various hematological and gene- tic disorders8,21).

MSCs are the best candidates for the tissue engineering and cellular therapy of orthopedic musculoskeletal tissues17). Bone marrow (BM) is well known for a good source of mes- enchymal stem cells (MSCs) at the present time. Despite the fact that bone marrow represents the main available source of MSCs, the use of bone marrow-derived cells is not always acceptable due to the high degree of viral infection and the significant drop in cell number and proliferative/differenti- ation capacity with age. Thus, the search for possible alter- native MSC sources remains to be validated. Umbilical cord blood is a rich source of hematopoietic stem/progenitor cells.

hUCB has the advantage of easy availability and relative

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Isolation, Characterization and Tri-lineage Differentiation of Mesenchymal Stem Cells from Human Umbilical Cord Blood

Sung-Eun Yang, M.D.*, Yoon-Sun Yang, M.D.*, and Chul-Won Ha, M.D.

Department of Orthopedic Surgery, Samsung Medical Center, Sungkyunkwan University, Samsung Biomedical Research Institute, Seoul; Department of Research & Development for Cellular Therapy, Medipost Biomedical Research Institute*, Yongin, Korea

537 537 Address reprint requests to

Chul-Won Ha, M.D.

Department of Orthopedic Surgery, Samsung Medical Center, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, 50 Ilwon-dong, Gangnam-gu, Seoul 135-710, Korea

Tel: +82.2-3410-0275, Fax: +82.2-3410-0084 E-mail: hacw@smc.samsung.co.kr

*This research was supported partly by Grant for Development of Biological Product and Materials funded by the Ministry of Commerce, Industry, and Energy, Republic of Korea.

Purpose: Human umbilical cord blood (hUCB) is well known for a good source of hematopoietic stem cells (HSCs). However, the presence of mesenchymal stem cells (MSCs) in hUCB is still not well approved by many authors. We hereby report the isolation and characterization of MSCs from hUCB, as well as their differentiation into osteogenic, chondrogenic and adipogenic lineages.

Materials and Methods: Mononuclear cells were isolated from each hUCB harvest (n=411) by density gradient centrifugation, and suspended in -minimum essential medium supplemented with 10% fetal bovine serum (FBS). The cell population was expanded by successive sub-cultivation under the same condition. The cell population that showed more than 1,000-fold expansion at fifth to eighth passage was inspected with known surface antigens of MSCs and other cell lineage. The isolated MSCs were cultured in osteogenic, chondrogenic, and adipogenic condition to identify their potential to differentiate into differ- ent mesenchymal cell lineage.

Results: Ninety five out of 411 hUCB units (23.1%) generated the MSC-like cell population during initial cultivation. Nine cell populations (2.2%) showed more than 1,000-fold expansion capacity at fifth to eighth passage. These cells positively expressed all known MSC-related antigens. They did not express any of myeloid, endothelial, or histo-compatibility antigens. All of the MSCs isolated showed the potential to differ- entiate into osteogenic, chondrogenic, and adipogenic lineages.

Conclusion: Our study supports that hUCB does contain MSCs, which can be differentiated into differ- ent cell lineages. We believe hUCB will be a good source of MSCs with the advantage of availability and relative abundance. We think hUCB should not be considered as a medical waste, and it will serve as a good source of cells for tissue engineering and cellular therapy in the future.

Key Words: Umbilical cord blood, Mesenchymal stem cells, Osteogenic differentiation, Chondrogenic differentiation, Adipogenic differentiation

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abundance, however, it is still controversial whether hUCB contains MSCs6,16,30).

In the present study, we report the isolation, expansion, and characterization of MSCs from hUCB, as well as their potential to differentiate into three different mesodermal lin- eages: osteogenic, chondrogenic, and adipogenic lineage.

MATERIALS AND METHODS

1. Isolation and expansion of MSCs

hUCB was collected on delivery from 411 different indi- viduals with informed consent. Mononuclear cells were iso- lated by centrifugation in a Ficoll-Hypaque (Sigma, St. Louis, MO, USA) density gradient centrifugation (1.077 g/cm3).

The separated mononuclear cells were washed, suspended in -minimum essential medium ( -MEM, Gibco BRL, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS, HyClone, Logan, UT, USA). The cells were then seeded in tissue culture plate at a concentration of 5× 106cells/cm2. Cultures were maintained at 37in a humidi- fied atmosphere containing 5% CO2. The culture medium was changed at every 3 days. hUCB-derived mononuclear cells showed fibroblast-like adherent layers during the cul- ture, and the adherent cell reached 80% confluence between 1 to 3 weeks. The cells were trypsinized (0.25% trypsin, HyClone), washed, re-suspended in the same culture medi- um, and sub-cultured at a concentration of 5×104cells/cm2. The cell population from each hUCB harvest was expanded by successive sub-cultivation under the same condition. Fifth to eighth passage cells that showed more than 1,000-fold expansion were studied for immuno-phenotyping of surface antigens and the potential to differentiate into multiple cell lineages.

2. Immuno-phenotyping of hUCB-derived MSC For surface antigen phenotyping, aliquots of the hUCB- derived MSCs after treatment with 0.25% trypsin were washed three times with phosphate-buffered saline (PBS, pH 7.4). Cells were stained with fluorescein isothiocyanate (FITC)- or phycoerythrin (PE)-coupled antibodies. Labeled cells were analyzed by FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA, USA) using the CELLQUEST software (Becton Dickinson, San Jose, CA, USA).

The anti-human antibodies used were CD45-FITC, CD34- FITC, CD14-FITC, HLA-DR-FITC, CD13-PE (Becton

Dickinson, San Jose, CA, USA); CD29-PE, CD44-PE, CD31-FITC, CD106-PE, CD90-FITC, CD51/61-FITC, CD64-FITC (Pharmingen, Los Angeles, CA, USA). As isotypic control, mouse IgG1-FITC, IgG1-PE, IgG2a-FITC (Becton Dickinson), mouse IgG2b-PE, IgG1-FITC, IgG1-PE (Pharmingen) were used. Cells were also immunolabeled with the following anti-human antibodies; SH2, SH3, and SH4. SH2, SH3, and SH4 antibodies made from each hyb- ridoma cell line (American Type Culture Collection, Rock- ville, MD, USA) for indirect assays. As a secondary antibody, anti-mouse IgG-FITC (Jackson ImmunoResearch, West Grove, PA, USA) was used.

3. Osteogenic, chondrogenic, and adipogenic differen- tiation

To induce osteogenic differentiation, cells were cultured in the osteogenic medium. The medium was composed of -MEM supplemented with 10% FBS, 0.1 M dexametha- sone (Sigma), 10 mM -glycerol phosphate (Sigma), and 50 M L-ascorbic acid 2-phosphate (Sigma)10). Medium was changed twice a week.

To induce chondrogenic differentiation, approximately 2

×105cells were placed in 15 mL polypropylene tube and centrifuged at 500×g, 5 minutes, at room temperature to form a pellet. Then, the pallet was treated with chondrogenic medium. Medium was changed twice a week. Chondrogenic medium consisted of serum free, high-glucose Dulbecco’s modified Eagle’s medium (DMEM, Gibco BRL) supple- mented with 100 nM dexamethasone (Sigma), 50 g/mL L- ascorbic acid (Sigma), 100 g/mL sodium pyruvate (Sigma), 40 g/mL L-proline (Sigma), 10 ng/mL transforming growth factor 3 (TGF- 3, Sigma), 500 ng/mL bone morphogenic protein 6 (BMP-6, R&D Systems, Minneapolis, MN, USA), and 50 mg/mL ITS+ premix (Becton Dickinson, 6.25 g/ mL insulin, 6.25 g/mL transferrin, 6.25 ng/mL selenious acid, 1.25 mg/mL bovine serum albumin (BSA), and 5.35 mg/mL linoleic acid22).

To induce adipogenic differentiation, cells were treated with adipogenic medium. Medium changes were carried out twice a week. Adipogenic medium consisted of DMDM (Gibco BRL) supplemented with 10% FBS (Hyclone), 0.5 mM 3-isobutyl-1-methylxanthine (IBMX, Sigma), 1 M dexamethasone (Sigma), 0.2 mM indomethacin (Sigma), and 10 M h-insulin (Sigma)19).

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4. Evaluation of Trilineage Differentiation

The onset of osteogenesis was evaluated by the expression of alkaline phosphatase (ALP), using ALP staining kit (85L- 3R, Sigma) at 1 week interval. Mineralized matrix was asse- ssed by von Kossa staining weekly.

For the evaluation of chondrogenic differentiation, the pallet aggregates were retrieved weekly and frozen in OCT compound (Tissue-Tek, Torrance, CA, USA) for histological and immunohistochemical analyses, and Safranin-O (Sigma) staining for histological evaluation. For immunohistochemi- cal analysis, the frozen sections were fixed for 10 min in me- thanol after a brief immersion in distilled water to remove the OCT compound. Nonspecific antibody binding sites were blocked by incubating the slides in PBS with 5.0%

BSA for 1 hour. Slides were then incubated for an hour at room temperature with type II collagen antibody (Onco-

gene, San Diego, CA, USA) diluted in 0.05 M Tris-HCl with 1.0% BSA (TBS). Reactivity was detected by using the EnVisionTM+ System (DAKO, Carpinteria, CA, USA), Peroxidase (DAB) kit (K4006, DAKO) according to the manufacturer’s instructions.

Adipogenesis were evaluated by Oil-red O (Sigma) stain- ing at every week.

The cells were also analyzed by RT-PCR weekly. Total RNA was extracted from each differentiated cells with a modification of the method of Chomczynski and Sacci.36) Cells were lysed using TRIzol Reagent (Invitrogen, La Jolla, CA, USA), and RNA was prepared as per kit instructions.

The mRNA was reverse transcribed to cDNA in 20 L reaction volume containing 1 gof RNA, 1 Lof 500 g/ mL Oligo dT primer (Invitrogen), 1 L(200 units) of Super- ScriptTMII reverse transcriptase (Invitrogen), 4 Lof 5×

Target gene product Primer sequence Anealing temperature PCR size

GAPDH 5-ACCACAGTCCATGCCATCAC-355°C 452 bp

5-TCCACCACCCTGTTGCTGTA-3

Osteocalcin 5-CATGAGAAGCCCTCACA-3 55°C 310 bp

5-AGAGCGACACCCTAGAC-3

Alkaline 5-TGGAGCTTCAGAAGCTCAACACCA-355°C 453 bp

Phosphatase 5-ATCTCGTTGTCTGAGTACCAGTCC-3

Osteopontin 5-CTAGGCATCACCTGTGCCATACC-355°C 347 bp

5-CAGTGACCAGTTCATCAGATTCATC-3

Collagen type I 5-CCCCCTCCCCAGCCACAAAGA-360°C 360 bp

5-TCTTGGTCGGTGGTGGACTCT-3

Cbfa-I 5-CAGTAGATGGACCTCGGGAA-360°C 395 bp

5-GAGGCAGAAGTCAGAGGTGG-3

Aggrecan 5-TCAGGAGGGCTGGAACAAGTACC-357°C 392 bp

5-GGAGGTGGTAATTGCAGGGAACA-3

Collagen type II 5-TGGAGCTTCAGAAGCTCAACACCA-355°C 225 bp 5-ATCTCGTTGTCTGAGTACCAGTCC-3

Collagen type II 5-TTTCCCAGGTCAAGATGGTC-355°C 377 bp

5-CTTCAGCACCTGTCTCACCA-3

Collagen type IX 5-CCCCCTCCCCAGCCACAAAGA-357°C 159 bp

5-TCTTGGTCGGTGGTGGACTCT-3

Sox-9 5-GGTTGTTGGAGCTTTCCTCA-357°C 401 bp

5-TAGCCTCCCTCACTCCAAGA-3

PPAR 5-TTCAGAAATGCCTTGCAGTG-357°C 599 bp

5-GGGCTCCATAAAGTCACCAA-3

C/EBP 5-CTGGAGCTGACCAGTGACAA-357°C 374 bp

5-CCAAGAATTCTCCCCTCCTC-3

Leptin 5-ACAGAAAGTCACCGGTTTGG-357°C 481 bp

5-TGGCTTAGAGGAGTCAGGGA-3

Lipoprotein lipase 5-TCAATCACAGCAGCAAAACC-357°C 575 bp

5-CCACATCTCCAAGTCCTCTC-3

P2 5-TACTGGGCCAGGAATTTGAC-357°C 240 bp

5-TCAATGCGAACTTCAGTCCA-3Table 1.Primers for RT-PCR

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First-Strand buffer (250 mM Tris-HCl (pH 8.3), 375 mM KCl, 15 mM MgCl2, Invitrogen), 2 Lof 0.5mM dNTPs, 1 Lof 10 mM dTT, and 1 Lof RNaseOUT Ribonucle- ase Inhibitor (Invitrogen).

PCR reactions were performed in 25 Lreaction volume containing 2 Lof cDNA, 2 Lof each 10 pM primer, 0.5 Lof AccuPrimeTMTaq DNA polymerase (Invitrogen), 2.5 Lof 10×AcuuPrimeTMPCR buffer (Invitrogen). Primers used for amplification are listed in Table 1. cDNA was ampli- fied using a ABI GeneAmp PCR System 9600 (Perkin El- mer, Applied Biosystems, Boston, MA, USA) at 95for 30 sec, the annealing temperature for each target gene as described in Table 1 for 30 sec, and 72for 30 sec for 35 cycles, after initial denaturation at 95for 5 min. PCR products were resolved by agarose gel electrophoresis and visualized by ethidium bromide staining.

RESULTS

1. Isolation & Expansion of MSCs

hUCB-derived mononuclear cells seeded in the tissue culture plates gave rise to a population of fibroblast-like adherent cells. By day 21 of primary culture, 95 out of 411 hUCB harvests (23.1%) produced these MSC-like cell pop- ulations. Non-adherent or loosely adherent small round cells were also present in the primary cultures, but disappeared soon after passage 2 or 3.

MSC-like cell colonies derived from hUCB culture mainly consisted of bipolar fibroblast-like cells (Fig. 1A). At day 21 of primary culture, the mean value for total adherent

Fig. 2.Immunophenotypic characterization of the MSCs derived from hUCB. These cells were positive for antigens CD13, CD29, CD44, CD90, SH2, SH3, and SH4. These cells were negative for antigens CD14, CD31, CD34, CD45, CD51/61, CD64, CD106, or HLA-DR. These immunophenotyping was consistent with the known findings for bone marrow MSCs.

Fig. 1.Photomicrographs showing mesenchymal stem cells (MSCs) isolated from human umbilical cord blood at the day 21 (A) and at advanced passage 18 (B). hUCB-derived MSC colonies mainly consisted of bipolar fibroblast-like cells. No significant morphologi- cal changes were observed until passage 18.

A B

Counts

100 101 102103 104 CD13

Counts

100 101 102103 104 CD14

Counts

100 101 102103 104 CD29

Counts

100 101 102103 104 CD31

Counts

100 101 102103 104 CD34

Counts

100 101 102103 104 CD44

Counts

100 101 102103 104 CD45

Counts

100 101 102103 104 CD51-61

Counts

100 101 102103 104 CD64

Counts

100 101 102103 104 CD90

Counts

100 101 102103 104 CD106

Counts

100 101 102103 104 HLA-DR

Counts

100 101 102103 104 SH2

Counts

100 101 102103 104 SH3

Counts

100 101 102103 104 SH4

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cell number was 2.6×106(n=95, range 0.04×105-22.0× 106). After expansion, MSCs-like colonies from nine out of 411 hUCB harvests (2.2%) showed more than 1,000-fold

increase in cell number. These cells could be passaged up to 18 times without displaying significant changes in morpholo- gy (Fig. 1B).

ALP

1 week 2 week 3 week 4 week

1 week 2 week 3 week 4 week

M 0 wk 1 wk 2 wk 3 wk 4 wk

OP OC Col I Cbfa-1 GAPDH

453 bp 347 bp 310 bp 360 bp 398 bp 452 bp

A

B

C

Fig. 3.Osteogenic differentiation of hUCB-derived MSCs. Cul- tured mesenchymal cells formed aggregates and increased their expression of alkaline phosphatase (A, ×100). These cells also formed extracellular calcium matrix, which was demonstrat- ed by von Kossa staining (B, ×100). hUCB-derived MSCs ex- pressed mRNA of ALP, osteopontin, osteocalcin, collagen type I, and Cbfa-I during osteogenic differentiation, which is typical of osteoblastic cells (C).

0 week

2 week 4 week 6 week

2 week 4 week 6 week

M GAPDH (452 bp) Aggrecan (392 bp) Col II(225 bp) Col II(377 bp) CoI IX (159 bp) Sox-9 (401 bp)

4 week A

B C

Fig. 4.Chondrogenic differentiation of hUCB-derived MSCs. Accumulation of sulfated proteoglycans was visualized by Safranin-O staining (A, ×200) at 2 weeks of chondrogenic differentiation. Multiple lacunae with chondrocyte-like cells were also evident in the histological sections. Extensive matrix was rich in type II collagen, which is typical of articular cartilage (B, ×200). mRNA of collagen type II, aggrecan, collagen type IX, and sox-9 was detected by RT-PCR, which is typical of chondrocytes (C).

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2. Immunophenotypic characterization of hUCB -derived MSCs

Immunophenotyping of hUCB-derived MSCs with more than 1,000-fold expanding capacity demonstrated that all of them showed positive expression of the MSCs-related anti- gens, SH2, SH3 and SH4 (Fig. 2). These cells were negative for antigens CD14 (lipopolysaccharide receptor, hematopoi- etic marker), CD31 (PECAM-1), CD34 (gp105-120, hema- topoietic marker), CD45 (leukocyte common antigen), CD 51/61, CD64 (matrix receptor), CD106, or HLA-DR(HLA class II). They were found to be positive for other known antigens of MSC; CD13, CD29 ( 1 integrin), CD44 (matrix receptor for hyaluronate), and CD90(thy-1) (Fig. 2).

3. Osteogenic, chondrogenic, and adipogenic differenti- ations of hUCB-derived MSCs

The MSCs cultured in osteogenic media formed aggre- gates and increased their expression of alkaline phosphatase from the first week through forth week (Fig. 3A). These cells also formed extracellular calcium matrix, which was demonstrated by von Kossa staining (Fig. 3B). The MSCs

also expressed mRNA of bone forming cells, such as alka- line phosphatase (ALP), osteopontin (OP), osteocalcin (OC), collagen type I (Col I), and core binding factor 1 (Cbfa-I) during osteogenic differentiation (Fig. 3C).

During chondrogenic differentiation, the accumulation of sulfated proteoglycans was visualized by Safranin-O stain- ing at 2 weeks of differentiation (Fig. 4A). Multiple lacunae with chondrocyte-like cells were also evident in the histologi- cal sections. The extensive matrix was rich in type II collagen, which is typical of articular cartilage (Fig. 4B). mRNA of collagen type II (Col II), aggrecan, collagen type IX (Col IX), and SOX-9 was detected by RT-PCR at 4 weeks (Fig. 4C)

Adipogenic differentiation was apparent by the accumu- lation of lipid-rich vacuoles within cells, which were visual- ized by staining with Oil red O (Fig. 5A, B). The lipid vac- uoles continued to develop over time, coalesced, and even- tually filled the cells. These cells expressed mRNA of perox- isome proliferation-activated receptor 2 (PPAR 2), lipo- protein lipase (LPL), fatty acid-binding protein aP2, CCAAT/

enhancer-binding protein (C/EBP ), and leptin, which is typical of adipocytes (Fig. 5C).

LPL

1 week 2 week 3 week 4 week

1 week 2 week 3 week 4 week

M 0 wk 1 wk 2 wk 3 wk 4 wk

C/EBP PPAR Leptin P2 GAPDH

575 bp 374 bp 599 bp 481 bp 240 bp 452 bp

A

B

C

Fig. 5.Adipogenic differentiation of hUCB-derived MSCs. Adi- pogenic induction was apparent by the accumulation of lipid-rich vacuoles within cells, which were visualized by staining with Oil red O (A, ×200; B, ×200). These cells expressed mRNA of PPAR 2, LPL, C/EBP , P2, and leptin, which is typical of adipocytes (C).

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Of the nine MSC populations that showed more than 1,000-fold expanding capacity, nine demonstrated osteogenic differentiation, eight demonstrate d chondrogenic differen- tiation, and five demonstrated adipogenic differentiation.

DISCUSSION

hUCB is known to contain a higher proportion of primi- tive hematopoietic cells, including multipotent colony form- ing cells14,20,23,28)as well as in vivo repopulating cells29), than adult bone marrow (BM). hUCB-derived HSCs are reported to have higher proliferation and expansion potentials than their adult BM counterparts9,11,24,26,27,29). In this sense, if hUCB contains MSCs, we may suppose that they have higher poten- tial for proliferation and differentiation than BM-derived MSCs. So far, it is still controversial as to whether hUCB contains MSCs. The possibility of having higher potential is still pretty much in question.

Erices et al. reported that hUCB harvests generate mes- enchymal progenitor cells with large ex vivo expansion capac- ity as well as osteogenic and adipogenic differentiation poten- tials6). On the contrary, Wexler et al.30)and Mareschi et al.16) claimed that adult BM is a reliable source of functional MSC, but hUCB is not. However, a recent study by Lee et al.13) reported that hUCB contains MSCs which can differentiate into bone, cartilage, and fat. They reported that hUCB-de- rived MSCs could even differentiated into neurogenic and hepatogenic lineage.

We performed the present study focusing on the identi- fication, expansion, and phenotypic characterization of the MSCs isolated from human hUCB. Our study demonstrat- ed that the characteristics of the hUCB-derived MSCs were very similar to the features of BM-derived MSCs15,17). Results of our present study support that hUCB does contain MSCs.

In this study, we reveald the presence of MSCs in hUCB by in vitro culture-expansion on a large scale. Our results showed that almost 25% of hUCB harvests generated MSCs-like cell populations. The immunophenotype of these cells was consistent with that reported for BM-derived MSCs15,17). The present study verified that the hUCB-derived MSCs had the capacity to differentiate into different lineages. Whether they even have the potential to differentiate into ectodermal or endomeral lineages still needs to be investigated, and will be one of our next study.

In recent years, the potential use of MSCs has been high-

lighted in tissue engineering, regeneration medicine, and cellular or gene therapy. Interestingly, MSCs have been reported to be transplantable between HLA-incompatible individuals, because they don’t elicit alloreactive lymphocyte proliferate responses and modulate immune responses1,5,12,25). MSCs are thought to be the origin of mesenchymal tissues during the process of normal growth, remodeling, and repair.

So, various tissues have been inspected as a source for MSCs, such as synovium, fat tissue, muscle as well as bone marrow with some successful results2,4,7,18,31). However, these sources are mostly not easy to collect, limited in amount of the ava- ilable tissue, or have the concern of donor site morbidity.

hUCB has the advantage of easy of collection, availability and relative abundance. It is well know that hUCB harvest is not harmful to donor baby and mother. Although the incidence of hUCB harvest producing MSCs is low in our study, some of them showed more than 1,000-fold expand- ing capacity that will be sufficient in cell number to be used for tissue regeneration strategy. So, We believe hUCB can be a useful candidate for source of MSC for future therapeutic options of tissue regeneration. Further studies will be need- ed to select optimal hUCB harvests before or during MSCs cultivation.

The limitation of this study is that the MSCs were eval- uated as a population, rather than a single colony derived cell line. Although many reported studies included this type of method by using MSC population, there has always been a critic on the possibility that the population may be a mix- ture of different progenitor cells of different lineage with or without pure MSCs.

Because we still do not have any specific markers of MSCs, it is not feasible at the present time to specifically isolate the pure MSCs from hUCB, as well as from bone marrow.

The investigation to find out methods to isolate and expand the pure MSCs will be a valuable research in the future.

CONCLUSION

Our study supports that hUCB does contain MSCs, which can be differentiated into different cell lineages. We believe hUCB will be a good source of MSCs with the advantage of availability and relative abundance. We think hUCB should not be considered as a medical waste, and it will serve as a good source of cells for tissue engineering and cellular therapy in the future.

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목 적: 저자들은 인간 제대혈(umbilical cord blood)에서 간엽 줄기 세포를 분리 및 확인할 수 있었고, 이들 세포군에 대하 여 골, 연골 및 지방세포로의 분화가 가능하였기에 이를 보고하고자 한다.

대상 및 방법: 제대혈(n=411)로부터 단핵세포군을 원심분리 방법을 이용하여 분리한 후 배양하였으며, 5-8회 개대배양 시 에 1,000배 이상의 증식을 보인 세포군에 대하여 간엽줄기세포 및 타 세포계열 표면항원 검사를 시행하였다. 분리된 간엽 줄기 세포군에 대하여 골, 연골 및 지방세포로의 분화를 시도하였다.

결 과: 총 411개의 제대혈 중 95개(23.1%)에서 첫 배양 시 간엽 줄기 세포양 세포군(MSC-like cell population)이 확인되었 으며, 5-8회 개대배양 시 1,000배 이상의 증식을 보인 세포군은 9개(2.2%)였다. 이들 세포군은 기존에 알려진 모든 간엽줄 기세포 표면항원검사에 양성, 타 세포계열 표면항원 검사에는 모두 음성이었다. 분리된 간엽 줄기 세포군은 골, 연골 및 지방세포로의 분화가 가능하였다.

결 론: 본 연구의 결과는 인간 제대혈이 서로 다른 세포 계열로 분화할 수 있는 간엽줄기세포를 포함하고 있다는 사실을 지 지하는 것으로 판단된다. 인간 제대혈은 상대적으로 많은 양의 간엽줄기세포를 비교적 용이하게 얻을 수 있다는 장점이 있는 세포 공급원이 될 수 있을 것으로 사료되며, 향후 조직 공학 및 세포 치료를 위한 세포 공급원의 하나가 될 수 있을 것 으로 기대한다.

색인 단어: 제대혈, 간엽 줄기 세포, 골세포 분화, 연골세포 분화, 지방세포 분화

인간 제대혈에서 간엽줄기세포의 분리, 확인 및 골, 연골 및 지방세포로의 분화

양성은*ㆍ양윤선*ㆍ하철원

성균관대의과대학 삼성서울병원 정형외과, 메디포스트(주) 생명공학연구소*

수치

Fig. 1. Photomicrographs showing mesenchymal stem cells (MSCs) isolated from human umbilical cord blood at the day 21 (A) and at advanced passage 18 (B)
Fig. 4. Chondrogenic differentiation of hUCB-derived MSCs. Accumulation of sulfated proteoglycans was visualized by Safranin-O staining (A, ×200) at 2 weeks of chondrogenic differentiation
Fig. 5. Adipogenic differentiation of hUCB-derived MSCs. Adi- Adi-pogenic induction was apparent by the accumulation of lipid-rich vacuoles within cells, which were visualized by staining with Oil red O (A,  ×200; B, ×200)

참조

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