Ichiro Sekiya

Expansion of Human Adult Stem Cells from Bone Marrow Stroma: Conditions that Maximize the Yields of Early Progenitors and Evaluate Their Quality

There is considerable interest in the biology and therapeutic potential of adult stem cells from bone marrow stroma, variously referred to as mesenchymal stem cells or marrow stromal cells (MSCs). Human MSCs can expand rapidly in culture, but the rate of expansion and the yields of multipotential progenitors are inversely related to the plating density and incubation time of each passage. We have defined conditions for optimizing the yields of cultures enriched for early progenitors. Also, we developed a simple method for assessing the quality of the cultures by phase‐contrast microscopy and image analysis or by forward light scatter in a flow cytometer. The cells expanded most rapidly on day 4 after plating, with a minimum average doubling time of about 10 hours for cells initially plated at 10 or 50 cells/cm2. After plating the cells at 1 to 1,000 cells/cm2, the cultures underwent a time‐dependent transition from early progenitors, defined as thin, spindle‐shaped cells (RS‐1A), to wider, spindle‐shaped cells (RS‐1B), and to still wider, spindle‐shaped cells (RS‐1C). Assays for adipogenesis demonstrated that the adipogenic potential of cultures was directly related to their ability to generate single‐cell‐derived colonies and their enrichment for RS‐1A cells. In contrast, cultures enriched for RS‐1B cells showed the greatest potential to differentiate into cartilage in a serum‐free system. The results indicate that, when preparing cultures of human MSCs, it is necessary to compromise between conditions that provide the highest overall yields and those that provide the highest content of early progenitor cells.

Identification of a subpopulation of rapidly self-renewing and multipotential adult stem cells in colonies of human marrow stromal cells

Marrow stromal cells are adult stem cells from bone marrow that can differentiate into multiple nonhematopoietic cell lineages. Previous reports demonstrated that single-cell-derived colonies of marrow stromal cells contained two morphologically distinct cell types: spindle-shaped cells and large flat cells. Here we found that early colonies also contain a third kind of cell: very small round cells that rapidly self-renew. Samples enriched for the small cells had a greater potential for multipotential differentiation than samples enriched for the large cells. Also, the small cells expressed a series of surface epitopes and other proteins that potentially can be used to distinguish the small cells from the large cells. The results suggested it will be important to distinguish the major subpopulations of marrow stromal cells in defining their biology and their potential for cell and gene therapy.

In vitro cartilage formation by human adult stem cells from bone marrow stroma defines the sequence of cellular and molecular events during chondrogenesis

One approach to resolving the complexities of chondrogenesis is to examine simplified systems in vitro. We analyzed cartilage differentiation by human adult stem cells from bone marrow stroma. Marrow stromal cells were cultured as micromass pellets for 21 days in serum-free medium containing transforming growth factor (TGF)-β3, dexamethasone, and bone morphogenetic protein (BMP)-6. Assays for pulse-labeled [3H]DNA and for total DNA indicated that there was little proliferation and a progressive loss of cells in the pellets. There were continuous increases in mRNAs for cartilage matrix (proteoglycans and COL2, -9, -10, and -11), receptors [fibroblast growth factor 2 (FGFR2) and parathyroid hormone-related peptide receptor (PTHrP-R)], and transcription factors (SOX5, -6, and -9) as demonstrated by histochemical and microarray assays. Reverse transcription–PCR assays for 11 mRNAs confirmed the microarray data. SOX4, vascular endothelial growth factor (VEGF), and matrix metalloproteinase 14 (MMP14) increased at day 1 and decreased thereafter, suggesting roles early in chondrogenesis. Also, forkhead, CD10, and MMP13 increased up to day 7 and decreased thereafter, suggesting roles in an intermediate stage of chondrogenesis. In addition, two collagens (COL3A1 and COL16A1), a signaling molecule (WNT11), a homeobox homolog (BAPX1), a receptor (IL-1R1), an IGFs modulator (IGFBP5), and a mettaloproteinase (MMP16) increased progressively up to about day 14, suggesting roles later in chondrogenesis. Our results indicate that the simplicity of the system makes it possible to define in detail the cellular and molecular events during chondrogenesis.

Comparison of rat mesenchymal stem cells derived from bone marrow, synovium, periosteum, adipose tissue, and muscle

Mesenchymal stem cells (MSCs) are increasingly being reported as occurring in a variety of tissues. Although MSCs from human bone marrow are relatively easy to harvest, the isolation of rodent MSCs is more difficult, thereby limiting the number of experiments in vivo. To determine a suitable cell source, we isolated rat MSCs from bone marrow, synovium, periosteum, adipose, and muscle and compared their properties for yield, expansion, and multipotentiality. After two passages, the cells in each population were CD11b (−), CD45 (−), and CD90 (+). The colony number per nucleated cells derived from synovium was 100-fold higher than that for cells derived from bone marrow. With regard to expansion potential, synovium-derived cells were the highest in colony-forming efficiency, fold increase, and growth kinetics. An in vitro chondrogenesis assay demonstrated that the pellets derived from synovium were heavier, because of their greater production of cartilage matrix, than those from other tissues, indicating their superiority in chondrogenesis. Synovium-derived cells retained their chondrogenic potential after a few passages. The Oil Red-O positive colony-rate assay demonstrated higher adipogenic potential in synovium- and adipose-derived cells. Alkaline phosphatase activity was greater in periosteum- and muscle-derived cells during calcification. The yield and proliferation potential of rat MSCs from solid tissues was much better than those from bone marrow. In particular, synovium-derived cells had the greatest potential for both proliferation and chondrogenesis, indicating their usefulness for cartilage study in a rat model.

Adipogenic Differentiation of Human Adult Stem Cells From Bone Marrow Stroma (MSCs)

We assayed gene expressions during adipogenesis of human MSCs. Microarray assays demonstrated time‐dependent increases in expression of 67 genes, including 2 genes for transcription factors that were not previously shown to be expressed during adipogenesis.

Humeral Insertion of the Supraspinatus and Infraspinatus. New Anatomical Findings Regarding the Footprint of the Rotator Cuff

BACKGROUND It is generally believed that the supraspinatus is the most commonly involved tendon in rotator cuff tears. Clinically, however, atrophy of the infraspinatus muscle is frequently observed in patients with even small to medium-size rotator cuff tears. This fact cannot be fully explained by our current understanding of the anatomical insertions of the supraspinatus and infraspinatus. The purpose of this study was to reinvestigate the humeral insertions of these tendons. METHODS The study included 113 shoulders from sixty-four cadavers. The humeral insertion areas of the supraspinatus and infraspinatus were investigated in ninety-seven specimens. In sixteen specimens, all muscular portions of the supraspinatus and infraspinatus were removed, leaving the tendinous portions intact, in order to define the specific characteristics of the tendinous portion of the muscles. Another twenty-six shoulders were used to obtain precise measurements of the footprints of the supraspinatus and infraspinatus. RESULTS The supraspinatus had a long tendinous portion in the anterior half of the muscle, which always inserted into the anteriormost area of the highest impression on the greater tuberosity and which inserted into the superiormost area of the lesser tuberosity in 21% of the specimens. The footprint of the supraspinatus was triangular in shape, with an average maximum medial-to-lateral length of 6.9 mm and an average maximum anteroposterior width of 12.6 mm. The infraspinatus had a long tendinous portion in the superior half of the muscle, which curved anteriorly and extended to the anterolateral area of the highest impression of the greater tuberosity. The footprint of the infraspinatus was trapezoidal in shape, with an average maximum medial-to-lateral length of 10.2 mm and an average maximum anteroposterior width of 32.7 mm. CONCLUSIONS The footprint of the supraspinatus on the greater tuberosity is much smaller than previously believed, and this area of the greater tuberosity is actually occupied by a substantial amount of the infraspinatus.

In vitro chondrogenesis of human synovium-derived mesenchymal stem cells : Optimal condition and comparison with bone marrow-derived cells

There are increasing reports that mesenchymal stem cells (MSCs) are present in various tissues other than bone marrow, including synovium. Here we investigated the optimal conditions for in vitro chondrogenesis of human synovium‐derived MSCs and compared these cells with bone marrow‐derived MSCs, especially in terms of their chondrogenesis potential. Synovium and bone marrow were harvested from six donors during knee operations for ligament injuries. Digested synovium cells or nucleated cells from bone marrow were expanded clonally. A pellet culture system was used for chondrogenesis, and the best combination of up to three cytokines of the seven assessed. Synovium‐derived MSCs plated at a lower density expanded more rapidly. Contrary to previous reports, a combination of TGFβ and dexamethasone was not sufficient to induce chondrogenesis. However, addition of BMP2 to TGFβ and dexamethasone dramatically increased cartilage pellet size and the synthesis of cartilage matrix. The cartilage pellets were also analyzed by electron microscopy and immunohistology. DNA content per pellet decreased during chondrogenesis, indicating the pellet increased its size through the accumulation of newly synthesized extracellular matrix. Sequential chondrogenic gene expression was demonstrated by RT‐PCR. Synovium‐derived MSCs looked similar to the bone marrow‐derived MSCs in their surface epitopes and proliferation potential; however, cartilage pellets from synovium were significantly larger than those from bone marrow in patient‐matched comparisons. We demonstrated that the combination of TGFβ, dexamethasone, and BMP2 was optimal for in vitro chondrogenesis of synovium‐derived MSCs and that the synovium‐derived MSCs have a greater chondrogenesis potential than bone marrow‐derived MSCs.

Comparison of mesenchymal tissues-derived stem cells for in vivo chondrogenesis: suitable conditions for cell therapy of cartilage defects in rabbit

We previously compared mesenchymal stem cells (MSCs) from a variety of mesenchymal tissues and demonstrated that synovium-MSCs had the best expansion and chondrogenic ability in vitro in humans and rats. In this study, we compared the in vivo chondrogenic potential of rabbit MSCs. We also examined other parameters to clarify suitable conditions for in vitro and in vivo cartilage formation. MSCs were isolated from bone marrow, synovium, adipose tissue, and muscle of adult rabbits. Proliferation potential and in vitro chondrogenic potential were compared. Toxicity of the tracer DiI for in vitro chondrogenesis was also examined. MSCs from each tissue were embedded in collagen gel and transplanted into full thickness cartilage defects of rabbits. Cartilage matrix production was compared histologically. The effects of cell density and periosteal patch on the in vivo chondrogenic potential of synovium-MSCs were also examined. Synovium- and muscle-MSCs had a higher proliferation potential than other cells. Pellets from synovium- and bone-marrow-MSCs showed abundant cartilage matrix. DiI had no significant influence on in vitro cartilage formation. After transplantation into cartilage defects, synovium- and bone-marrow-MSCs produced much more cartilage matrix than other cells. When synovium-MSCs were transplanted at a higher cell density and with a periosteal patch, more abundant cartilage matrix was observed. Thus, synovium- and bone-marrow-MSCs had greater in vivo chondrogenic potential than adipose- and muscle-MSCs, but synovium-MSCs had the advantage of a greater proliferation potential. Higher cell density and a periosteum patch were needed to obtain a high production of cartilage matrix by synovium-MSCs.

Isolation and characterization of rapidly self-renewing stem cells from cultures of human marrow stromal cells

BACKGROUND The adult stem cells from BM, known as non-hematopoietic mesenchymal stem cells, or marrow stromal cells (MSCs), readily generate single-cell-derived colonies, but the cultures are known to contain cells with at least two different morphologies and different properties of differentiation. Recently, we tried to identify the earliest progenitors in the cultures. METHODS Human MSCs were plated at very low initial densities of about 3 cells/cm(2), and the growth of colonies was followed by phase microscopy. RESULTS The two kinds of morphologically distinct cells reported by others were readily discerned: large, slowly replicating cells and spindle-shaped, more rapidly replicating cells. In addition, we observed very small cells, with diameters of only about 7 microm, that very rapidly replicated, both symmetrically and asymmetrically. The small rapidly self-renewing (RS) cells had different surface epitopes and profiles of expressed proteins than other cells in the same cultures. They also had a greater capacity for multilineage differentiation. DISCUSSION RS cells are apparently the earliest progenitors and most rapidly replicating cells in cultures of MSCs. They have properties that appear to make them ideal candidates for studying differentiation and probably make them well-suited for cell and gene therapy.

Intra-articular Injected Synovial Stem Cells Differentiate into Meniscal Cells Directly and Promote Meniscal Regeneration Without Mobilization to Distant Organs in Rat Massive Meniscal Defect†‡

Osteoarthritis in the knees, which can be caused by meniscal defect, constitutes an increasingly common medical problem. Repair for massive meniscal defect remains a challenge owing to a lack of cell kinetics for the menisci precursors in knee joint. The synovium plays pivotal roles during the natural course of meniscal healing and contains mesenchymal stem cells (MSCs) with high chondrogenic potential. Here, we investigated whether intra‐articular injected synovium‐MSCs enhanced meniscal regeneration in rat massive meniscal defect. To track the injected cells, we developed transgenic rats expressing dual luciferase (Luc) and LacZ. The cells derived from synovium of the rats demonstrated colony‐forming ability and multipotentiality, both characteristics of MSCs. Hierarchical clustering analysis revealed that gene expression of meniscal cells was closer to that of synovium‐MSCs than to that of bone marrow‐MSCs. Two to 8 weeks after five million Luc/LacZ+ synovium‐MSCs were injected into massive meniscectomized knee of wild‐type rat, macroscopically, the menisci regenerated much better than it did in the control group. After 12 weeks, the regenerated menisci were LacZ positive, produced type 2 collagen, and showed meniscal features by transmission electron microscopy. In in‐vivo luminescence analysis, photons increased in the meniscus‐resected knee over a 3‐day period, then decreased without detection in all other organs. LacZ gene derived from MSCs could not be detected in other organs except in synovium by real‐time PCR. Synovium‐MSCs injected into the massive meniscectomized knee adhered to the lesion, differentiated into meniscal cells directly, and promoted meniscal regeneration without mobilization to distant organs. STEM CELLS 2009;27:878–887