Is it possible to obtain "true endothelial progenitor cells" by in vitro culture of bone marrow mononuclear cells?

In vitro-cultured bone marrow cells have been shown to contain some low-density lipoprotein (LDL) uptake-positive cells. Although a small portion of LDL uptake-positive cells had expression for endothelial markers, all of them demonstrated a phagocytosis function similar to monocyte/macrophages and expression of the panleukocyte surface marker CD45 and monocyte marker CD14. These LDL uptake-positive cells did not show significant proliferative capacity and died out gradually in long-term culture. In contrast, the bone marrow-derived LDL uptake-negative cells showed strong proliferation and expression of typical mesenchymal surface markers CD29 and CD44. Although cultured under endothelial promoting conditions, these mesenchymal stem cells (MSCs) did not show any sign of differentiation toward endothelial cells. In conclusion, adult bone marrow-derived LDL uptake-positive cells that have been reported so far actually are monocytes/macrophages that can express some endothelial markers but are not "true endothelial progenitor cells" (EPCs). MSCs, which are the only cell type that shows strong proliferation during long-term adherent culture for bone marrow cells, do not differentiate toward the endothelial lineage when grown under endothelial promoting conditions.     

Human heart, spleen, and perirenal fat-derived mesenchymal stem cells have immunomodulatory capacities

Mesenchymal stem cells (MSCs) have important tissue repair functions and show potent immunosuppressive capacities in vitro. Although usually isolated from the bone marrow, MSCs have been identified in other tissues, including the skin and liver. In the present study, we isolated and characterized MSCs from human heart, spleen, and perirenal adipose tissue. MSCs from these different tissue sites were similar to those derived from bone marrow in that they expressed comparable levels of the cell-surface markers CD90, CD105, CD166, and HLA class I, were negative for CD34, CD45, HLA class II, CD80, and CD86 expression, and were capable of osteogenic and adipogenic differentiation. Like bone marrow-derived MSCs, MSCs from these different tissue sources inhibited the proliferation of alloactivated peripheral blood mononuclear cells (PBMCs), giving 85%, 79%, 79%, and 81% inhibition, respectively. Also in line with bone marrow-derived MSCs they inhibited proliferative responses of PBMCs to phytohemagglutinin, a nonspecific stimulator of lymphocyte proliferation, and reduced-memory T lymphocyte responses to tetanus toxoid. The results of this study demonstrate that MSCs from various tissues have similar immunophenotypes, in vitro immunosuppressive properties, and differentiation potential.     

Stromal cells from term fetal membrane are highly suppressive in allogeneic settings in vitro

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) have immunosuppressive properties and have been used to treat steroid-refractory acute graft-versus-host disease (GVHD) in stem cell transplant patients. Cells with similar capacities can also be found in term placental tissue. We have isolated stromal cells from term fetal membrane (FMSCs), umbilical cords (UCSCs) and placental villi (PVSCs) as well as from bone marrow and compared their immunoregulatory capacity in allogeneic settings. We found that FMSCs and UCSCs suppressed proliferation significantly in mixed lymphocyte reactions (MLRs), whereas PVSCs showed inconsistent suppressive effects. When added to MLR cultures, FMSCs suppressed the production of interferon (IFN)-γ and interleukin (IL)-17, whereas UCSCs and PVSCs promoted the production of IL-17 instead. Secretion of IL-10 was increased after addition of FMSCs and UCSCs. In this setting, BM-MSCs had no significant effect on secretion of IFN-γ, IL-17 or IL-10 in MLR cultures. When analysing the expression of adhesion markers, we noted that FMSCs expressed the highest levels of CD29 (β1), CD49d (α4) and CD54 (ICAM-1) compared to the other types of stromal cells. Thus, our data indicate that stromal cells isolated from term fetal membrane have great immunosuppressive capacity in terms of proliferation and production of proinflammatory cytokines from alloreactive T cells, and also promote anti-inflammatory IL-10. They express high levels of integrins that may be of importance in homing to inflamed tissues. Fetal membrane may provide a valuable source of cells with immunosuppressive properties and could possibly be used for treatment of acute GVHD and other inflammatory disorders.     

Searching for alternative sources of postnatal human mesenchymal stem cells: candidate MSC-like cells from umbilical cord

Mesenchymal stem cells (MSCs) have the capability for renewal and differentiation into various lineages of mesenchymal tissues. These features of MSCs attract a lot of attention from investigators in the context of cell-based therapies of several human diseases. 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/differentiation capacity with age. Thus, the search for possible alternative MSC sources remains to be validated. Umbilical cord blood is a rich source of hematopoietic stem/progenitor cells and does not contain mesenchymal progenitors. However, MSCs circulate in the blood of preterm fetuses and may be successfully isolated and expanded. Where these cells home at the end of gestation is not clear. In this investigation, we have made an attempt to isolate MSCs from the subendothelial layer of umbilical cord vein using two standard methodological approaches: the routine isolation of human umbilical vein endothelial cell protocol and culture of isolated cells under conditions appropriate for bone-marrow-derived MSCs. Our results suggest that cord vasculature contains a high number of MSC-like elements forming colonies of fibroblastoid cells that may be successfully expanded in culture. These MSC-like cells contain no endothelium- or leukocyte-specific antigens but express alpha-smooth muscle actin and several mesenchymal cell markers. Therefore, umbilical cord/placenta stroma could be regarded as an alternative source of MSCs for experimental and clinical needs.     

Fluids from immune privileged sites endow macrophages with the capacity to induce antigen-specific immune deviation via a mechanism involving transforming growth factor-β

The eye, brain, and fetoplacental unit within the pregnant uterus are immunologically privileged sites that contain unique fluids with suspected immunoinhibitory properties. Aqueous humor, which is normally present within the anterior chamber (AC) of the eye, has been shown to suppress antigen-driven T cell activation, and to contain significant amounts of transforming growth factor β-2 (TGF-β). Antigens injected into the AC of normal mice induce a deviant form of systemic immunity, termed anterior chamber-associated immune deviation (ACAID), which is characterized by a selective inability to display antigen-specific delayed hypersensitivity. It has recently been reported that eye-derived macrophages appear in the blood following AC injection of a soluble antigen, and that these cells can induce antigen-specific ACAID when injected into naive, syngeneic recipients. Moreover, antigen-pulsed peritoneally derived macrophages that are exposed in vitro to aqueous humor, supernatants of cultured iris and ciliary body cells (I/CD), or TGF-β have been found to assume ACAID-inducing properties. In the present experiments, amniotic fluid and cerebrospinal fluid from mice, rats and humans, as well as supernatants from cultured I/CB cells, have been examined for their capacity to confer ACAID-inducing properties on peritoneal macrophages. It was found that each of these biologic fluids, but neither normal mouse serum nor rat thoracic duct lymph, was able to endow antigen-pulsed peritoneal macrophages with ACAID-inducing properties. Moreover, fluids from these immune privileged sites were found to contain latent and/or active TGF-β, as determined by bioassay, using neutralizing anti-TGF-β antibodies. It is concluded (a) that the immune privileged states of the eye, the brain, and the fetoplacental unit share common features, and possess unique fluids with a similar capacity to force macrophages to present antigens in a “deviant” manner and (b) that this capacity is mediated, at least in part, by TGF-β. These results are discussed in terms of the potential physiologic and pathophysiologic significance of immune privilege in these three specialized tissues.     

Histochemical localization of zinc and copper in rat ocular tissues

Zinc and copper were histochemically localized in rat ocular tissues. Zinc was demonstrated by the dithizone method, and copper by the rubeanic acid and rhodanine methods. The retinal photoreceptor's outer segment showed the presence of zinc, but no zinc reaction was seen in other parts of the retina or other ocular tissues, even in rats fed excess zinc. It seems that photoreceptor cells contain the highest concentration of zinc. Copper was seen in the corneal epithelium and endothelium, iris, ciliary body, lens epithelium, retinal outer nuclear layer, photoreceptor inner and outer segments, retinal pigment epithelium, choroid, sclera, and optic nerve. These trace elements seems to play roles as components of some metalloenzymes and may have other functions till now unknown.     

BK-Type K(Ca) channels in two parasympathetic cell types: differences in kinetic properties and developmental expression

The intrinsic electrical properties of identified choroid and ciliary neurons of the chick ciliary ganglion were examined by patch-clamp recording methods. These neurons are derived from a common pool of mesencephalic neural crest precursor cells but innervate different target tissues and have markedly different action potential waveforms and intrinsic patterns of repetitive spike discharge. Therefore it is important to determine whether these cell types express different types of plasma membrane ionic channels, and to ascertain the developmental stages at which these cell types begin to diverge. This study has focused on large-conductance Ca(2+)-activated K(+) channels (K(Ca)), which are known to regulate spike waveform and repetitive firing in many cell types. Both ciliary ganglion cell types, identified on the basis of size and somatostatin immunoreactivity, express a robust macroscopic K(Ca) carried by a kinetically homogeneous population of large-conductance (BK-type) K(Ca) channels. However, the kinetic properties of these channels are different in the two cell types. Steady-state fluctuation analyses of macroscopic K(Ca) produced power spectra that could be fitted with a single Lorentzian curve in both cell types. However, the resulting corner frequency was significantly lower in choroid neurons than in ciliary neurons, suggesting that the underlying K(Ca) channels have a longer mean open-time in choroid neurons. Consistent with fluctuation analyses, significantly slower gating of K(Ca) channels in choroid neurons was also observed during macroscopic activation and deactivation at membrane potentials positive to -30 mV. Differences in the kinetic properties of K(Ca) channels could also be observed directly in single-channel recordings from identified embryonic day 13 choroid and ciliary neurons. The mean open-time of large-conductance K(Ca) channels was significantly greater in choroid neurons than in ciliary neurons in excised inside-out patches. The developmental expression of functional K(Ca) channels appears to be regulated differently in the two cell types. Although both cell types acquire functional K(Ca) at the same developmental stages (embryonic days 9-13), functional expression of these channels in ciliary neurons requires target-derived trophic factors. In contrast, expression of functional K(Ca) channels proceeds normally in choroid neurons developing in vitro in the absence of target-derived trophic factors. Consistent with this, extracts of ciliary neuron target tissues (striated muscle of the iris/ciliary body) contain K(Ca) stimulatory activity. However, K(Ca) stimulatory activity cannot be detected in extracts of the smooth muscle targets of choroid neurons.     

Proliferation of C3HXCBA Hybrid Lymphocytes in the Spleens of Irradiated CBA Mice: Inhibition of this Phenomenon by Pretreatment of the Hosts with C3HXCBA Spleen Cells

Lymphocytes from C3HXCBA hybrid mice proliferate intensely in the spleens of irradiated CBA mice. Presentment of the CBA hosts with C3HXCBA spleen cells, known to specifically reduce the T-cell reactivity of the hosts against the strongly stimulatory Mls-antigen determined by the C3H-genome, abolished the capacity of the host to promote proliferation of C3HXCBA lymphocytes. In contrast, proliferation of C3HXCBA bone marrow cells, as measured by splenic ⁵⁹Fe incorporation, was unaffected by such pretreatment. By examining the capacity of spleen cell populations of (C3HXCBA)XCBA back-cross mice to inhibit lymphocyte proliferation, as described above, and to express the C3H-delermincd Mls-antigen, it was concluded that these two trails are associated.     

Cell transplantation in wounded mixed connective tissues

Direct transplantation of multipotent precursor cells into the periodontium could provide a therapeutic approach for restoring periodontal tissues destroyed by periodontitis or trauma. To improve the understanding of cell migration, proliferation, and differentiation, we used a rodent model combining orthodontic tooth movement and transplantation of Lac-Z-positive murine-cultured periodontal ligament (PL) or femur-derived bone marrow precursor cells into a defined mandibular wound site, thus promoting tissue regeneration in wounded periodontium. Our results show that in orthodontically traumatized tissues, transplanted PL and bone marrow cells migrated systemically, contributing to the repopulation of sites with reduced cell/matrix density. The transplanted PL cells proliferated in adjacent alveolar bone marrow spaces, thus migrating to vascular tissues in the PL. The capillary walls in the PL serve as delivery sites for these cells and other marrow-derived hematopoietic cells, including monocytes. The transplanted marrow cells, extracted from femur of transgenic (TgR) mice exhibited similar behavior to those of transplanted PL cells, showing high proliferative activity in alveolar marrow as well as intensive repopulating capacity in wounded periodontium. On the other hand, the buccal skin fibroblasts failed to migrate and home effectively and thus the transplantation of these cells had no effect on periodontium regeneration. Based on these results, we conclude that the transplanted PL and bone marrow cells migrate systemically and following a cyclical process of growth and development and differentiate into PL fibroblasts, osteoblasts, and cementoblasts, thereby contributing to periodontal regeneration.     

Bone marrow cells engraft within the epidermis and proliferate in vivo with no evidence of cell fusion

In adults, bone marrow-derived cells (BMDC) can contribute to the structure of various non-haematopoietic tissues, including skin. However, the physiological importance of these cells is unclear. This study establishes that bone marrow-derived epidermal cells are proliferative and, moreover, demonstrates for the first time that BMDC can localize to a known stem cell niche: the CD34-positive bulge region of mouse hair follicles. In addition, engraftment of bone marrow cells into the epidermis is significantly increased in wounded skin, bone marrow-derived keratinocytes can form colonies in the regenerating epidermis in vivo, and the colony-forming capacity of these cells can be recapitulated in vitro. In some tissues this apparent plasticity is attributed to differentiation, and in others to cell fusion. Evidence is also provided that bone marrow cells form epidermal keratinocytes without undergoing cell fusion. These data suggest a functional role for bone marrow cells in epidermal regeneration, entering known epidermal stem cell niches without heterokaryon formation.     

Vascular Endothelial Cells Present Alloantigens to Unprimed Lymphocytes

Antigen-presenting cells (APC) were removed from canine peripheral blood by carbonyl iron treatment and adherence to plastic and to nylon-wool. This treatment resulted in low proliferation in mixed lymphocyte cultures (MLC) and lack of generation of cell-mediated cytotoxicity (CMC) in depleted cell suspensions compared with untreated cell suspension. The proliferative response could be restored to normal by the addition of low numbers of autologous arterial or venous endothelial cells to the MLC of depleted cell suspensions. Cytotoxicity against phytohaemagglutinin-stimulated lymphoblasts of the stimulator was generated in the untreated MLC and also in the MLC of APC-depleted cell suspensions with endothelial cells added. It is concluded that arterial and venous endothelial cells can substitute for APC in the proliferative response of autologous lymphocytes against alloantigen and in the generation of CMC. Therefore, endothelial cells have an alloantigen-presenting capacity.     

Studies on the induction of anterior chamber-associated immune deviation (ACAID). III. Induction of ACAID depends upon intraocular transforming growth factor-beta.

Delayed hypersensitivity (DH), the prototypical form of cell-mediated immune responsiveness, is mediated with the participation of considerable nonspecific inflammation which necessarily disrupts the anatomic integrity of involved and adjacent tissues. Damage of this type is of minor consequence to many visceral and cutaneous organs, but is of devastating consequence for organs such as the eye and the brain. At least in the case of the eye, the organ is remarkably adept at regulating the immune system's ability to respond to intraocular antigens by selectively down-regulating both the induction and expression of delayed hypersensitivity while leaving other effector modalities intact. This ability of the eye to selectivity down-regulate systemic DH responses to intracamerally inoculated antigens is known as anterior chamber-associated immune deviation (ACAID) and is mediated in part by antigen-specific regulatory T cells. Recent work suggests that macrophages (M phi) that reside in the iris and ciliary body can migrate out of an antigen-bearing eye and activate regulatory T cells within the spleen. In an effort to understand the mechanism by which intraocular M phi interact with antigen in the anterior chamber of the eye (AC) and subsequently induce splenic regulatory cells in ACAID, we have investigated what role, if any, the AC microenvironment itself plays in ACAID induction. The results reveal that CD45- parenchymal iris/ciliary cells secrete a soluble factor(s) locally and into the aqueous humor which endows resident, mature M phi with ACAID-inducing capabilities. Mice receiving infusions of these altered, antigen-pulsed M phi are incapable of mounting a significant DH response following immunization with antigen in adjuvant. Importantly, the ACAID-inducing effect is achieved when conventional, extraocular M phi are exposed in vitro to a soluble factor present in aqueous humor or culture SN from iris and ciliary body cells. Further investigations into the identity of this factor reveal it to be transforming growth factor-beta (TGF-beta). The role of TGF-beta in the generation of ACAID, as well as the implications of these findings to an understanding of immunologic privilege in general, are discussed.     

小鼠骨髓来源Flk-1+间充质干细胞分离制备与生物学特点

背景：骨髓间充质干细胞具有广泛的应用前途,其三系分化的能力和免疫诱导特性使其具有再生医学的应用前景。 目的：从小鼠骨髓中研究分离制备Flk-1⁺间充质干细胞的新方法,检测了这类细胞的生物特性。 方法：以小鼠骨髓为研究对象,分离单个核细胞,体外培养并 扩增原始间充质干细胞,检测它们的细胞周期、表型和多系分化的能力。 结果与结论：小鼠来源的骨髓来源的原始间充质干细胞呈成纤维样生长,大部分细胞处于G₀/G₁期,并且高表达Flk1,CD13,CD29,CD44,小鼠骨髓来源Flk-1⁺间充质干细胞在光镜下均呈成纤维样或多角型贴壁生长,在体外相应的诱导液环境中均可以向三系分化。提示小鼠骨髓来源Flk-1⁺间充质干细胞具有多向分化潜能。     

Retinal stem cells: mechanism of differentiation and therapeutic application

Retinal dystrophies are rarely curable diseases and several avenues of research are being pursued, such replacement therapies and pharmacological treatment. Among them, the transplantation of functional retinal cells has been envisaged in order to restore vision in patients who have these diseases by repopulating the damaged retina and/or by rescuing retinal neurons from further degeneration. Over the past few years, identification and characterization of stem cells has opened new avenues in cell-replacement therapy. Since retinal stem cells are already present during embryonic development, they persist in the adult mammalian eye only in the ciliary marginal zone, even a stem cell potential has been described for the Müller glia in the retina. This result opened possibilities of regeneration by mobilizing endogenous stem cells to respond to injury. Regarding the transplantation studies, in all experiments using different types of stem cells (retinal progenitors, neural stem cells, bone marrow-derived stem cells and ES cells), despite their incorporation within the host's retina, the transplanted cells failed to express retina-specific markers and to establish synaptic connections. Therefore, the true potential of the different stem cells in retina repair can only be realized with more information about mechanisms that regulate their proliferation and differentiation; and by development of techniques that allow their prospective identification and enrichment.     

Dual effect of ciliary body cells on T lymphocyte proliferation

The interaction between organ-resident cells from the anterior uvea of the eye and T helper (Th) cells was investigated. Cells from Lewis rat ciliary body processes (CB cells), grown in tissue culture using an explant technique, could be induced to express major histocompatibility complex class II (Ia) antigens by incubation with rat interferon-gamma. Ia+ CB cells only poorly functioned as antigen-presenting cells (APC) for a syngeneic, uveitogenic Th cell line specific for the retinal soluble antigen (SAg). Moreover, if added to an Ag-driven lymphocyte proliferation assay in the presence of conventional APC, the rat CB cells had an inhibiting effect on Th proliferation. This inhibitory activity was not species specific, since similar effects were observed with bovine and human ciliary epithelial cells. The suppressive activity of CB cells was composed of a soluble factor, as well as a membrane-associated inhibitor. The soluble activity did not appear to be related to transforming growth factor-beta (TGF-beta), since no reversal of inhibition by a neutralizing antibody to TGF-beta was found. Part of the soluble inhibitory activity could be reversed by indomethacin treatment. The membrane-associated component was trypsin sensitive, suggesting a protein molecule. After abrogation of the inhibitory capacity by trypsin treatment and fixation by glutaraldehyde, CB cells effectively presented SAg to Th cells. These data suggest that CB cells are capable of mediating both Ag presentation and inhibition of Th cell proliferation.     

Sensory supply of the anterior uvea: A light and electron microscope study

Summary We have studied the ultrastructural features of sensory nerve fibers in the ciliary body and the iris and their parent trigeminal ganglion cells, using intra-axonally transported horseradish peroxidase as a tracer. Unmyelinated nerve fibers of ipsilateral trigeminal origin were found in stroma of both the ciliary body and the iris. Most appeared in bundles of nerve fibers; but some were found singly. Two distinct types of varicosities were found, one containing both a few irregularly shaped vesicles and mitochondria, the other containing mitochondria only. The thin profiles of the axons between these varicosities or endings contained neurotubules and filaments. The trigeminal ganglion cells supplying the anterior eye were of a relatively small size (15–50 m in diameter) and confined to the anteromedial part of the ipsilateral ganglion. These cells could not be classified according to the arrangement of their subcellular organelles. The morphological characteristics of the sensory nerve fibers and ganglion cells supplying the anterior uvea are consistent with the view that pain may be the predominant sensation mediated by these neurons.     

Morphological variations of pigment granules in eyes of the rhesus monkey

Melanin granules of rhesus monkey eyes develop in four stages. Early in stage I, they are small, spherical or elongated, membrane-limited vesicles containing parallel arrays of membranes oriented along the long axis and having regular periodicity. Melanization of these membranes, which is indicated by increased thickness and electron opacity, leads to the formation of stages II and III granules. In stage IV, their internal structure is completely obliterated by electron-dense melanin. During this process of development, melanosomes are transformed from thin, long vesicles into large ovoid bodies. By 60 days of gestation, melanosomes have developed in the pigmented epithelium of the iris, ciliary body, and retina, those in the retina being more mature and fully pigmented than the others. Stromal pigment cells of the uveal tract develop later than those of the pigmented epithelium. Pigment first appears in the iris at 140 days, then in the ciliary body, and lastly (150 days) in the choroid, where only well-developed melanosomes are found. Melanosomes of the stromal pigment cells as well as those of the pigmented epithelium may have their origin in the iris. Early melanosomes (stage II) are present almost exclusively in the iridial cells whereas mature forms are found in the ciliary and retinal cells.     

Human Mesenchymal Stem Cells Derived from Bone Marrow Display a Better Chondrogenic Differentiation Compared with Other Sources

Mesenchymal stem cells (MSCs) are multipotent cells capable of differentiation into several mesodermal lineages. These cells have been isolated from various tissues, such as adult bone marrow, placenta, and fetal tissues. The comparative potential of these cells originating from different tissues to differentiate into the chondrogenic lineage is still not fully defined. The aim of our study was to investigate the chondrogenic potential of MSCs isolated from different sources. MSCs from fetal and adult tissues were phenotypically characterized and examined for their differentiation capacity, based on morphological criteria and expression of extracellular matrix components. Our results show that both fetal and adult MSCs have chondrogenic potential under appropriate conditions. The capacity of bone marrow-derived MSCs to differentiate into chondrocytes was reduced on passaging of cells. MSCs of bone marrow origin, either fetal or adult, exhibit a better chondrogenesis than fetal lung- and placenta-derived MSCs, as demonstrated by the appearance of typical morphological features of cartilage, the intensity of toluidine blue staining, and the expression of collagen type II, IX, and X after culture under chondrogenic conditions. As MSCs represent an attractive tool for cartilage tissue repair strategies, our data suggest that bone marrow should be considered the preferred MSC source for these therapeutic approaches.     

Human mesenchymal stem cells derived from bone marrow display a better chondrogenic differentiation compared with other sources

Mesenchymal stem cells (MSCs) are multipotent cells capable of differentiation into several mesodermal lineages. These cells have been isolated from various tissues, such as adult bone marrow, placenta, and fetal tissues. The comparative potential of these cells originating from different tissues to differentiate into the chondrogenic lineage is still not fully defined. The aim of our study was to investigate the chondrogenic potential of MSCs isolated from different sources. MSCs from fetal and adult tissues were phenotypically characterized and examined for their differentiation capacity, based on morphological criteria and expression of extracellular matrix components. Our results show that both fetal and adult MSCs have chondrogenic potential under appropriate conditions. The capacity of bone marrow-derived MSCs to differentiate into chondrocytes was reduced on passaging of cells. MSCs of bone marrow origin, either fetal or adult, exhibit a better chondrogenesis than fetal lung- and placenta-derived MSCs, as demonstrated by the appearance of typical morphological features of cartilage, the intensity of toluidine blue staining, and the expression of collagen type II, IX, and X after culture under chondrogenic conditions. As MSCs represent an attractive tool for cartilage tissue repair strategies, our data suggest that bone marrow should be considered the preferred MSC source for these therapeutic approaches.     

The mechanical coupling of adult marrow stromal stem cells during cardiac regeneration assessed in a 2-D co-culture model

Postnatal cardiomyocytes undergo terminal differentiation and a restricted number of human cardiomyocytes retain the ability to divide and regenerate in response to ischemic injury. However, whether these neo-cardiomyocytes are derived from endogenous population of resident cardiac stem cells or from the exogenous double assurance population of resident bone marrow-derived stem cells that populate the damaged myocardium is unresolved and under intense investigation. The vital challenge is to ameliorate and/or regenerate the damaged myocardium. This can be achieved by stimulating proliferation of native quiescent cardiomyocytes and/or cardiac stem cell, or by recruiting exogenous autologous or allogeneic cells such as fetal or embryonic cardiomyocyte progenitors or bone marrow-derived stromal stem cells. The prerequisites are that these neo-cardiomyocytes must have the ability to integrate well within the native myocardium and must exhibit functional synchronization. Adult bone marrow stromal cells (BMSCs) have been shown to differentiate into cardiomyocyte-like cells both in vitro and in vivo. As a result, BMSCs may potentially play an essential role in cardiac repair and regeneration, but this concept requires further validation. In this report, we have provided compelling evidence that functioning cardiac tissue can be generated by the interaction of multipotent BMSCs with embryonic cardiac myocytes (ECMs) in two-dimensional (2-D) co-cultures. The differentiating BMSCs were induced to undergo cardiomyogenic differentiation pathway and were able to express unequivocal electromechanical coupling and functional synchronization with ECMs. Our 2-D co-culture system provides a useful in vitro model to elucidate various molecular mechanisms underpinning the integration and orderly maturation and differentiation of BMSCs into neo-cardiomyocytes during myocardial repair and regeneration.