Allogenic mesenchymal stromal cells and their extracellular vesicles in COVID-19 induced ARDS: a randomized controlled trial.

BACKGROUND AND AIMS: The main causes of death in patients with severe Coronavirus disease-2019 (COVID-19) are acute respiratory distress syndrome (ARDS) and multiorgan failure caused by a severe inflammatory cascade. Novel treatment strategies, such as stem-cell-based therapy and their derivatives can be used to relieve inflammation in these cases. In this study, we aimed to evaluate the safety and efficacy of therapy using mesenchymal stromal cells (MSCs) and their derived extracellular vesicles in COVID-19 patients. MATERIALS AND METHODS: COVID-19 patients with ARDS were included in this study and allocated into two study and control groups using block randomization. While all patients received recommended treatment based on guidelines from the national advisory committee for COVID-19 pandemic, the two intervention groups received two consecutive injections of MSCs (100 × 106 cells) or one dose of MSCs (100 × 106 cells) followed by one dose of MSC-derived extracellular vesicles (EVs). Patients were assessed for safety and efficacy by evaluating clinical symptoms, laboratory parameters, and inflammatory markers at baseline and 48 h after the second intervention. RESULTS: A total number of 43 patients (the MSC alone group = 11, MSC plus EV group = 8, and control group = 24) were included in the final analysis. Mortality was reported in three patients in the MSC alone group (RR: 0.49; 95% CI 0.14-1.11; P = 0.08); zero patient in the MSC plus EV group (RR: 0.08; 95% CI 0.005-1.26; P = 0.07) and eight patients in the control group. MSC infusion was associated with a decrease in inflammatory cytokines such as IL-6 (P = 0.015), TNF-α (P = 0.034), IFN-γ (P = 0.024), and CRP (P = 0.041). CONCLUSION: MSCs and their extracellular vesicles can significantly reduce the serum levels of inflammatory markers in COVID-19 patients, with no serious adverse events. Trial registration IRCT, IRCT registration number: IRCT20200217046526N2. Registered 13th April 2020, http://www.irct.ir/trial/47073 .

A bioscaffold of decellularized whole osteochondral sheet improves proliferation and differentiation of loaded mesenchymal stem cells in a rabbit model.

As a Natural decellularized extracellular matrix, osteochondral tissue is the best scaffold for the restoration of osteoarthritis defects. Bioscaffolds have the most similarly innate properties like biomechanical properties and the preserved connection of the bone-to-cartilage border. Although, their compacity and low porosity particularly, are proven to be difficulties of decellularization and cell penetration. This study aims to develop a new bioscaffold of decellularized osteochondral tissue (DOT) that is recellularized by bone marrow-derived mesenchymal stem cells (BM-MSCs), as a biphasic allograft, which preserved the interface between the cartilage section and subchondral bone of the joint. Whole osteochondral tissues of rabbit knee joints were sheeted in cartilaginous parts in 200-250 µm sections while connected to the subchondral bone and then fully decellularized. The BM-MSCs were seeded on the scaffolds in vitro; some constructs were subcutaneously implanted into the back of the rabbit. The cell penetration, differentiation to bone and cartilage, viability, and cell proliferation in vitro and in vivo were evaluated by qPCR, histological staining, MTT assay, and immunohistochemistry. DNA content analysis and SEM assessments confirmed the decellularization of the bioscaffold. Then, histological and SEM evaluations indicated that the cells could successfully penetrate the bone and cartilage lacunas in implanted grafts. MTT assay confirmed cell proliferation. Prominently, gene expression analysis showed that seeded cells differentiated into osteoblasts and chondrocytes in both bone and cartilage sections. More importantly, seeded cells on the bioscaffold started ECM secretion. Our results indicate that cartilage-to-bone border integrity was largely preserved. Additionally, ECM-sheeted DOT could be employed as a useful scaffold for promoting the regeneration of osteochondral defects.

Dexamethasone loaded injectable, self-healing hydrogel microspheresbased on UPy-functionalized Gelatin/ZnHAp physical network promotes bone regeneration.

Biopolymer-based injectable hydrogels provide great potential as bone tissue engineering (BTE) scaffolds on account of biocompatibility, and pore interconnectivity that enables delivery of cells and/or signaling molecules for bone repair. Recently, Gelatin hydrogels based on H-bonds were considered in response to concerns around the chemical crosslinking agents. In this study, a self-healing gelatin hydrogel with remarkable compressive and self-healing properties was prepared via formation of quadruple hydrogen bonds between ureidopyrimidinon functional groups, which were substituted on NH2 groups of gelatin(GelUPy). Degree of substitution controls properties of the resulting hydrogel from a shape- memory hydrogel (100% substitution), to a hydrogel (about 80%), to this self-healing hydrogel (about 40%). We report a strategy that adopts an emulsion synthesis approach to delivery of dexamethasone and Ca/Zn ions from injectable self-healing GelUPy hydrogel (GelUPy-ZnHApUPy-DEX), to induce osteogenic differentiation of adipose-derived stem cells, in vitro, and enhance bone regeneration in a cranial bone defect in a rat model. We show that key properties of the composite hydrogels, including mechanical properties, and release behavior of DEX are a match to the requirements of BTE. Overall, our results demonstrate that this self-healing gelatin approach is a promising strategy to enhance bone regeneration through a minimally invasive procedure.

A Roadmap for the Production of a GMP-Compatible Cell Bank of Allogeneic Bone Marrow-Derived Clonal Mesenchymal Stromal Cells for Cell Therapy Applications.

BACKGROUND: Allogeneic mesenchymal stromal cells (MSCs) have been used extensively in various clinical trials. Nevertheless, there are concerns about their efficacy, attributed mainly to the heterogeneity of the applied populations. Therefore, producing a consistent population of MSCs is crucial to improve their therapeutic efficacy. This study presents a good manufacturing practice (GMP)-compatible and cost-effective protocol for manufacturing, banking, and lot-release of a homogeneous population of human bone marrow-derived clonal MSCs (cMSCs). METHODS: Here, cMSCs were isolated based on the subfractionation culturing method. Afterward, isolated clones that could reproduce up to passage three were stored as the seed stock. To select proliferative clones, we used an innovative, cost-effective screening strategy based on lengthy serial passaging. Finally, the selected clones re-cultured from the seed stock to establish the following four-tired cell banking system: initial, master, working, and end of product cell banks (ICB, MCB, WCB, and EoPCB). RESULTS: Through a rigorous screening strategy, three clones were selected from a total of 21 clones that were stored during the clonal isolation process. The selected clones met the identity, quality, and safety assessments criteria. The validated clones were stored in the four-tiered cell bank system under GMP conditions, and certificates of analysis were provided for the three-individual ready-to-release batches. Finally, a stability study validated the EoPCB, release, and transport process of the frozen final products. CONCLUSION: Collectively, this study presents a technical and translational overview of a GMP-compatible cMSCs manufacturing technology that could lead to the development of similar products for potential therapeutic applications.

Application of bone and cartilage extracellular matrices in articular cartilage regeneration.

Articular cartilage has an avascular structure with a poor ability for self-repair; therefore, many challenges arise in cases of trauma or disease. It is of utmost importance to identify the proper biomaterial for tissue repair that has the capability to direct cell recruitment, proliferation, differentiation, and tissue integration by imitating the natural microenvironment of cells and transmitting an orchestra of intracellular signals. Cartilage extracellular matrix (cECM) is a complex nanostructure composed of divergent proteins and glycosaminoglycans (GAGs), which regulate many functions of resident cells. Numerous studies have shown the remarkable capacity of ECM-derived biomaterials for tissue repair and regeneration. Moreover, given the importance of biodegradability, biocompatibility, 3D structure, porosity, and mechanical stability in the design of suitable scaffolds for cartilage tissue engineering, demineralized bone matrix (DBM) appears to be a promising biomaterial for this purpose, as it possesses the aforementioned characteristics inherently. To the best of the authors' knowledge, no comprehensive review study on the use of DBM in cartilage tissue engineering has previously been published. Since so much work is needed to address DBM limitations such as pore size, cell retention, and so on, we decided to draw the attention of researchers in this field by compiling a list of recent publications. This review discusses the implementation of composite scaffolds of natural or synthetic origin functionalized with cECM or DBM in cartilage tissue engineering. Cutting-edge advances and limitations are also discussed in an attempt to provide guidance to researchers and clinicians.

Fabrication and characterization of an injectable reinforced composite scaffold for cartilage tissue engineering: an in vitro study.

There are limitations in current medications of articular cartilage injuries. Although injectable bioactive hydrogels are promising options, they have decreased biomechanical performance. Researchers should consider many factors when providing solutions to overcome these challenges. In this study, we created an injectable composite hydrogel from chitosan and human acellular cartilage extracellular matrix (ECM) particles. In order to enhance its mechanical properties, we reinforced this hydrogel with microporous microspheres composed of the same materials as the structural building blocks of the scaffold. Articular cartilage from human donors was decellularized by a combination of physical, chemical, and enzymatic methods. The decellularization efficiency was assessed by histological analysis and assessment of DNA content. We characterized the composite constructs in terms of storage modulus, gelation time, biocompatibility, and differentiation potential. The results showed that mechanical behavior increased with an increase in microsphere content. The sample that contained 10% microsphere had an enhanced storage modulus of up to 90 kPa. Biocompatibility and preliminary differentiation investigations revealed that this composite hydrogel might have potential benefits for cartilage tissue engineering.

Cartilage Repair by Mesenchymal Stem Cell-Derived Exosomes: Preclinical and Clinical Trial Update and Perspectives.

Osteoarthritis (OA) and other degenerative joint diseases are characterized by articular cartilage destruction, synovial inflammation, sclerosis of subchondral bone, and loss of extracellular matrix (ECM). Worldwide, these diseases are major causes of disability. Cell therapies have been considered to be the best therapeutic strategies for long-term treatment of articular cartilage diseases. It has been suggested that the mechanism of stem cell-based therapy is related to paracrine secretion of extracellular vesicles (EVs), which are recognized as the main secretion factors of stem cells. EVs, and in particular the subclass exosomes (Exos), are novel therapeutic approaches for treatment of cartilage lesions and OA. The results of recent studies have shown that EVs isolated from mesenchymal stem cells (MSCs) could inhibit OA progression. EVs isolated from various stem cell sources, such as MSCs, may contribute to tissue regeneration of the limbs, skin, heart, and other tissues. Here, we summarize recent findings of preclinical and clinical studies on different MSC-derived EVs and their effectiveness as a treatment for damaged cartilage. The Exos isolation techniques in OA treatment are also highlighted.

M2c Macrophages enhance phalange regeneration of amputated mice digits in an organ co-culture system.

Objectives: Delayed anti-inflammatory responses and scar-formation are the main causes for inability of injured body parts such as phalanges to regrow in mammals. Salamanders can regenerate fully scar-free body structures, followed by the appearance of anti-inflammatory responses at the injured site immediately after amputation. This study aimed to evaluate the local regenerative effects of direct amplified anti-inflammatory signals on regeneration of amputated mice digit tips using M2c-macrophages in a co-cultured organ system for the first time. Materials and Methods: We used the amputated digits from the paws of 18.5E day old C57BL/6J mice. Monocytes were obtained from peripheral blood and co-cultured with amputated digits, which subsequently enhanced the M2c macrophage phenotype induced by IL-10. We also examined the regenerative effects of IL-10 and transcription growth factor-beta 1 (TGF-ß1). Results: The regrowth of new tissue occurred 10 days post-amputation in all groups. This regrowth was related to enhanced Msh homeobox-1 (Msx1), Msh homeobox-2 (Msx2), and bone morphogenic protein-4 (Bmp4) genes. Increased expression of fibroblast growth factor-8 (Fgf-8) also increased the proliferation rate. Histological analyses indicated that epidermal-closure occurred at 3-dpa in all groups. We observed full digit tip regeneration in the co-cultured group. Particularly, there was new tissue regrowth observed with 40 µg/ml of IL-10 and 120 µg/ml of TGF-ß. In contrast, the control group had no remarkable digit elongation. Conclusion: We propose that a direct amplified anti-inflammatory response at the digit injury site can regenerate epithelial and mesenchymal tissues, and might be useful for limb regeneration without scar formation in adult mammals.

Which Protocol is better for Treatment of Ectopic Pregnancy by Methotrexate? Single-dose or Multiple-dose.

BACKGROUND: Ectopic pregnancy (EP) is the most common cause of death in the first trimester of pregnancy. Methotrexate (MTX) is an acceptable treatment in the cases with the lack of tube rupture or no important one, which has reduced surgical treatment. Despite numerous studies, there is still no consensus about medications. The present study is aimed to evaluate the single- and multiple-dose of MTX among these patients. MATERIALS AND METHODS: This clinical trial study was done on 108 EP patients who were selected for the systemic MTX treatment and divided into two groups. For the single-dose group, MTX was administered once and ß human chorionic gonadotropin (ßHCG) levels were measured first and then on days 4 and 7. In the multi-dose group, 1 mg/kg MTX was injected on days 1, 3, 5, and 7. In both groups, MTX was prescribed following these days if ßHCG was not reduced. In the two groups, ßHCG levels were assessed after 1 week. The success rate of treatment and complications were followed up and recorded up to 6 weeks after treatment. RESULTS: The success rate in the single-dose and multiple-dose MTX group was 47% and 51%. The MTX level in the single dose group decreased from 2532 ± 1154 mIU/mL to 1341 ± 553 mIU/mL and in the multiple dose group from 2671 ± 2685 mIU/mL to 1313 ± 605 mIU/mL (P < 0.05). Although a significant decrease was observed in each of the two groups over time, no significant difference was found between the two groups (P > 0.05). CONCLUSION: Single and multi-dose regimen did not show a significant difference in terms of the success of treatment. Therefore, given that the lower dose of the drug associated with lower the risk of complications, it is safe to choose the single-dose regimen.

Correction to: Photobiomodulation with single and combination laser wavelengths on bone marrow mesenchymal stem cells: proliferation and differentiation to bone or cartilage.

In the originally published article, the name of the 3rd and 4th authors were labeled incorrectly. The correct names are Mohammadreza Baghaban Eslaminejad and Leila Taghiyar. Also, affiliation 4 has been corrected.

New insight into functional limb regeneration: A to Z approaches.

Limb/digit amputation is a common event in humans caused by trauma, medical illness, or surgery. Although the loss of a digit is not lethal, it affects quality of life and imposes high costs on amputees. In recent years, the increasing interest in limb regeneration has led to enhanced scientific knowledge. However, the limited ability to develop functional limb regeneration in the clinical setting suggests that a challenging issue remains in limb regeneration. Recently, the emergence of regenerative engineering is a promising field to address this challenge and close the gap between science and clinical applications. Cell signalling and molecular mechanisms involved in the limb regeneration process have been extensively studied; however, there is still insufficient data on cell therapy and tissue engineering for limb regeneration. In this review, we intend to focus on therapeutic approaches for limb regeneration that are closely related to gene, immune, and stem cell therapies, as well as tissue engineering approaches that take into consideration the peculiar developmental properties of the limbs. In addition, we attempt to identify the challenges of these strategies for limb regeneration studies in terms of clinical settings and as a road map to accomplish the goal of functional human limb regeneration.

Regenerative Medicine Applications of Mesenchymal Stem Cells.

A major research challenge is to develop therapeutics that assist with healing damaged tissues and organs because the human body has limited ability to restore the majority of these tissues and organs to their original state. Tissue engineering (TE) and regenerative medicine (RM) promises to offer efficient therapeutic biological strategies that use mesenchymal stem cells (MSCs). MSCs possess the capability for self-renewal, multilineage differentiation, and immunomodulatory properties that make them attractive for clinical applications. They have been extensively investigated in numerous preclinical and clinical settings in an attempt to overcome their challenges and promote tissue regeneration and repair. This review explores the exciting opportunities afforded by MSCs, their desirable properties as cellular therapeutics in RM, and implicates their potential use in clinical practice. Here, we attempt to identify challenges and issues that determine the clinical efficacy of MSCs as treatment for skeletal and non-skeletal tissues.

Isolation, Characterization and Osteogenic Potential of Mouse Digit Tip Blastema Cells in Comparison with Bone Marrow-Derived Mesenchymal Stem Cells In Vitro.

OBJECTIVES: Limb regeneration mediated by blastema cells (BlCs) in mammals is limited to the digit tips of neonates. Due to the lack of access to BlCs in adults and the difficulty in isolating and expanding BlCs from neonates, the use of a cellular population with similar features of BlCs would be a valuable strategy to direct a non-regenerative wound towards regeneration. In this study, we have initially isolated and cultured BlCs, and explored their characteristics in vitro. Next, we compared the capability of bone marrow-derived mesenchymal stem cells (BM-MSCs) as an alternative accessible cell source to BlCs for regeneration of appendages. MATERIALS AND METHODS: In this experimental study, BM-MSCs were isolated from BM and we obtained BlCs from the neonatal regenerating digit tip of C57B/6 mice. The cells were characterized for expressions of cell surface markers by flow cytometry. Quantitative-reverse transcription polymerase chain reaction (qRT-PCR) and lineage-specific staining were used to assess their ability to differentiate into skeletal cell lineages. The colony forming ability, proliferation, alkaline phosphatase (ALP) activity, calcium content, and osteogenic gene expression were evaluated in both BMMSCs and BlCs cultures at days 7, 14, and 21. RESULTS: qRT-PCR analysis revealed that the cells from both sources readily differentiated into mesodermal lineages. There was significantly higher colony forming ability in BM-MSCs compared to BlCs (P<0.05). Alizarin red staining (ARS), calcium, and the ALP assay showed the same degree of mineral deposition in both BlCs and BM-MSCs. Gene expression levels of osteblastic markers indicated similar bone differentiation capacity for both BlCs and BM-MSCs at all time-points. CONCLUSIONS: Characteristics of BlCs in vitro appear to be similar to BM-MSCs. Therefore, they could be considered as a substitute for BlCs for a regenerative approach with potential use in future clinical settings for regenerating human appendages.

Msh homeobox 1 (Msx1)- and Msx2-overexpressing bone marrow-derived mesenchymal stem cells resemble blastema cells and enhance regeneration in mice.

Amputation of the proximal region in mammals is not followed by regeneration because blastema cells (BCs) and expression of regenerative genes, such as Msh homeobox (Msx) genes, are absent in this animal group. The lack of BCs and positional information in other cells is therefore the main obstacle to therapeutic approaches for limb regeneration. Hence, this study aimed to create blastema-like cells (BlCs) by overexpressing Msx1 and Msx2 genes in mouse bone marrow-derived mesenchymal stem cells (mBMSCs) to regenerate a proximally amputated digit tip. We transduced mBMSCs with Msx1 and Msx2 genes and compared osteogenic activity and expression levels of several Msx-regulated genes (Bmp4, Fgf8, and keratin 14 (K14)) in BlC groups, including MSX1, MSX2, and MSX1/2 (in a 1:1 ratio) with those in mBMSCs and BCs in vitro and in vivo following injection into the amputation site. We found that Msx gene overexpression increased expression of specific blastemal markers and enhanced the proliferation rate and osteogenesis of BlCs compared with mBMSCs and BCs via activation of Fgf8 and Bmp4 Histological analyses indicated full regrowth of digit tips in the Msx-overexpressing groups, particularly in MSX1/2, through endochondral ossification 6 weeks post-injection. In contrast, mBMSCs and BCs formed abnormal bone and nail. Full digit tip was regenerated only in the MSX1/2 group and was related to boosted Bmp4, Fgf8, and K14 gene expression and to limb-patterning properties resulting from Msx1 and Msx2 overexpression. We propose that Msx-transduced cells that can regenerate epithelial and mesenchymal tissues may potentially be utilized in limb regeneration.

Effects of Photobiomodulation and Mesenchymal Stem Cells on Articular Cartilage Defects in a Rabbit Model.

OBJECTIVE: The aim of this study was to evaluate the effectiveness of the application of cultured autologous bone marrow mesenchymal stem cells (BMSCs) with scaffold and low-level laser therapy (LLLT) on the repair of articular cartilage defects in rabbits. BACKGROUND DATA: For healing of the articular cartilage defects, although positive effects of BMSCs and LLLT have been demonstrated, their combination effect is still unknown; therefore, we investigated combining these two techniques has a synergistic effect. MATERIALS AND METHODS: After bone marrow aspiration from 10 rabbits, BMSCs were isolated, cultured in monolayer, suspended on a type I collagen scaffold and then implanted onto a full-thickness osteochondral defect (4 mm in diameter), artificially made on the patellar groove of both knees in the same rabbits. Then a knee was selected randomly in each rabbit as the experimental group, and subjected to Ga-Al-As (810 nm) laser irradiation with energy density of 4 J/cm2 every other day for 3 weeks. As the control group, the other knee did not receive LLLT. After this period, animals were euthanized and osteochondral defects were evaluated by histomorphometric methods. RESULTS: No significant difference in new cartilage formation and inflammation was found between the groups (p > 0.05). However, there was significantly more new bone formation in the experimental group (p < 0.05). CONCLUSIONS: In terms of our research, although better healing in osteochondral defects was seen when combining BMSCs and LLLT compared with the use of BMSCs alone, this improvement was predominantly caused by new bone formation rather than new cartilage formation.

Intra-articular injection of autologous mesenchymal stem cells in six patients with knee osteoarthritis.

BACKGROUND: Osteoarthritis (OA) is a progressive disorder of the joints caused by gradual loss of articular cartilage, which naturally possesses a limited regenerative capacity. In the present study, the potential of intra-articular injection of mesenchymal stem cells (MSCs) has been evaluated in six osteoarthritic patients. METHODS: Six female volunteers, average age of 54.56 years, with radiologic evidence of knee OA that required joint replacement surgery were selected for this study. About 50 ml bone marrow was aspirated from each patient and taken to the cell laboratory, where MSCs were isolated and characterized in terms of some surface markers. About 20-24 × 10(6) passaged-2 cells were prepared and tested for microbial contamination prior to intra-articular injection. RESULTS: During a one-year follow-up period, we found no local or systemic adverse events. All patients were partly satisfied with the results of the study. Pain, functional status of the knee, and walking distance tended to be improved up to six months post-injection, after which pain appeared to be slightly increased and patients' walking abilities slightly decreased. Comparison of magnetic resonance images (MRI) at baseline and six months post-stem cell injection displayed an increase in cartilage thickness, extension of the repair tissue over the subchondral bone and a considerable decrease in the size of edematous subchondral patches in three out of six patients. CONCLUSION: The results indicated satisfactory effects of intra-articular injection of MSCs in patients with knee OA.

Quantitative analysis of the proliferation and differentiation of rat articular chondrocytes in alginate 3D culture.

BACKGROUND: While articular chondrocytes are among those appropriate candidates for cartilage regeneration, the cell dedifferentiation during monolayer culture has limited their application. Several investigations have indicated the usefulness of alginate, but the topic of proliferation and differentiation of chondrocytes in alginate culture has still remained controversial. METHODS: Rat articular chondrocytes were released by enzymatic digestion, plated at 5 x 104 cells/cm2 and culture-expanded. Passaged-5 cells were then cultivated in alginate as 2-mm beads for a period of two months during which the expansion rate and the level of cell differentiation were determined by [3-(A, 5- dimethylthiazolyl- 2-yl)-1, 5-diphenyl tetrazolium bromide] assay and real-time PCR analysis respectively and compared with those of chondrocytes in monolayer culture. RESULTS: Average population doubling time in alginate cultures (10.04 +/- 0.9 days) tended to be significantly (P<0.05) higher than that in monolayer cultures (2.94 +/- 0.3 days). The period of alginate culture could be subdivided into expansion phase (Days 0-40); during which proliferation appeared to be high and differentiation phase (Days 40-60) during which the expression of cartilage-specific genes including collagen II and aggrecan tended to be upregulated. During both the proliferation and differentiation phases, the expression of collagen I was low. At chondrocytes monolayer cultures, the proliferating cells appeared to have a very low expression level of cartilage-specific genes and a high expression level of collagen I gene during the entire culture period (P<0.05). CONCLUSION: It seems that alginate provides conditions in which rat articular chondrocytes are able to undergo proliferation and differentiation in certain time point of cultivation period.

Murine mesenchymal stem cells isolated by low density primary culture system.

Murine mesenchymal stem cells (mMSC) and the difficult task of isolation and purification of them have been the subject of rather extensive investigation. The present study sought to isolate these cells from two different mouse strains, one outbred and the other inbred, primarily through a relatively simple but novel approach, the most important feature of which was the low density primary culture of bone marrow cells. For this purpose, mononuclear cells from either NMRI or BALB/c bone marrow were plated at about 500 cells per well of 24-well plates and incubated for 7 days. At this point, the fibroblastic clones that had emerged were pooled together and expanded through several subcultures. To investigate the mesenchymal nature, we differentiated the cells into the osteoblastic, chondrocytic and adipocytic lineages in different subcultures up to passage 10. According to the results, 1 week after culture initiation, several clones each comprising several fibroblastic cells appeared in each plate. The cells from different passages were capable of differentiating into corresponding skeletal tissues. In the present investigation, the best culture condition for maximum proliferation and also the expression of certain surface marker on isolated cells were examined. In this term the two murine strains showed some differences.