Priming mesenchymal stem cells boosts stem cell therapy to treat myocardial infarction

Cardiovascular diseases are the number one cause of death globally and are projected to remain the single leading cause of death. Treatment options abounds, although efficacy is limited. Recent studies attribute discrete and ephemeral benefits to adult stem cell therapies, indicating the urge to improve stem cell based–therapy. In this study, we show that priming mesenchymal stem cells (MSC) towards cardiomyogenic lineage enhances their beneficial effects in vivo as treatment option for acute phase myocardial infarction. MSC were primed using cardiomyogenic media for 4 days, after which peak expression of key cardiomyogenic genes are reached and protein expression of Cx‐43 and sarcomeric α‐actinin are observed. MSC and primed MSC (pMSC) were characterized in vitro and used to treat infarcted rats immediately after left anterior descending (LAD) occlusion. Echocardiography analysis indicated that MSC‐treated myocardium presented discrete improvement in function, but it also showed that pMSC treatment lead to superior beneficial results, compared with undifferentiated MSC. Seven days after cell injection, MSC and pMSC could still be detected in the myocardium. Connexin‐43 expression was quantified through immunoblotting, and was superior in pMSC, indicating that this could be a possible explanation for the superior performance of pMSC therapy.     

Allogeneic mesenchymal stem cell infusion for the stabilization of focal segmental glomerulosclerosis

Focal segmental glomerulosclerosis (FSGS) is the most frequent acquired renal condition resulting in end stage kidney disease in children. We describe a cell therapy treatment with human allogeneic bone marrow mesenchymal stem cells (MSC) in a 13-year-old patient developing recurrent FSGS after renal transplantation, which was not responding to conventional therapy.This treatment relied on the following measurements:clinical and laboratory evaluation of renal function, proteome array, biopsy, short tandem repeat assay.Before MSC treatment, the patient needed weekly plasmapheresis to achieve proteinuria-to-creatininuria ratio below 5. After three MSC infusions without adverse events, the patient has a stable renal function and the proteinuria target was reached without plasmapheresis. In addition, some circulating inflammatory factors decreased and their levels were still low after one year.This is the first report of an MSC treatment in an FSGS patient. Even though different factors may have contributed to the clinical results, after MSC infusion a stable reduction in the serum level of several inflammatory factors has been registered and the patient does not need anymore plasmapheresis to keep proteinuria under control.In addition, this encouraging single case let us identify some putative efficacy biomarkers that could be of clinical interest in chronic kidney diseases.     

Bone marrow-derived mesenchymal stem cells inhibit vascular smooth muscle cell proliferation and neointimal hyperplasia after arterial injury in rats

We investigated whether mesenchymal stem cell (MSC)-based treatment could inhibit neointimal hyperplasia in a rat model of carotid arterial injury and explored potential mechanisms underlying the positive effects of MSC therapy on vascular remodeling/repair. Sprague-Dawley rats underwent balloon injury to their right carotid arteries. After 2 days, we administered cultured MSCs from bone marrow of GFP-transgenic rats (0.8 × 10⁶ cells, n = 10) or vehicle (controls, n = 10) to adventitial sites of the injured arteries. As an additional control, some rats received a higher dose of MSCs by systemic infusion (3 × 10⁶ cells, tail vein; n = 4). Local vascular MSC administration significantly prevented neointimal hyperplasia (intima/media ratio) and reduced the percentage of Ki67 + proliferating cells in arterial walls by 14 days after treatment, despite little evidence of long-term MSC engraftment. Notably, systemic MSC infusion did not alter neointimal formation. By immunohistochemistry, compared with neointimal cells of controls, cells in MSC-treated arteries expressed reduced levels of embryonic myosin heavy chain and RM-4, an inflammatory cell marker. In the presence of platelet-derived growth factor (PDGF-BB), conditioned medium from MSCs increased p27 protein levels and significantly attenuated VSMC proliferation in culture. Furthermore, MSC-conditioned medium suppressed the expression of inflammatory cytokines and RM-4 in PDGF-BB-treated VSMCs. Thus, perivascular administration of MSCs may improve restenosis after vascular injury through paracrine effects that modulate VSMC inflammatory phenotype.     

Cardiac stem cell therapy to modulate inflammation upon myocardial infarction

Background

After myocardial infarction (MI) a local inflammatory reaction clears the damaged myocardium from dead cells and matrix debris at the onset of scar formation. The intensity and duration of this inflammatory reaction are intimately linked to post-infarct remodeling and cardiac dysfunction. Strikingly, treatment with standard anti-inflammatory drugs worsens clinical outcome, suggesting a dual role of inflammation in the cardiac response to injury. Cardiac stem cell therapy with different stem or progenitor cells, e.g. mesenchymal stem cells (MSC), was recently found to have beneficial effects, mostly related to paracrine actions. One of the suggested paracrine effects of cell therapy is modulation of the immune system.

Scope of review

MSC are reported to interact with several cells of the immune system and could therefore be an excellent means to reduce detrimental inflammatory reactions and promote the switch to the healing phase upon cardiac injury. This review focuses on the potential use of MSC therapy for post-MI inflammation. To understand the effects MSC might have on the post-MI heart the cellular and molecular changes in the myocardium after MI need to be understood.

Major conclusions

By studying the general pathways involved in immunomodulation, and examining the interactions with cell types important for post-MI inflammation, it becomes clear that MSC treatment might provide a new therapeutic opportunity to improve cardiac outcome after acute injury.

General significance

Using stem cells to target the post-MI inflammation is a novel therapy which could have considerable clinical implications. This article is part of a Special Issue entitled Biochemistry of Stem Cells.     

Cell membrane and bioactive factors derived from mesenchymal stromal cells: Cell-free based therapy for inflammatory bowel diseases

Inflammatory bowel diseases (IBD) are chronic inflammatory disorders of the gastrointestinal tract associated with multifactorial conditions such as ulcerative colitis and Crohn’s disease. Although the underlying mechanisms of IBD remain unclear, growing evidence has shown that dysregulated immune system reactions in genetically susceptible individuals contribute to mucosal inflammation. However, conventional treatments have been effective in inducing remission of IBD but not in preventing the relapse of them. In this way, mesenchymal stromal cells (MSC) therapy has been recognized as a promising treatment for IBD due to their immunomodulatory properties, ability to differentiate into several tissues, and homing to inflammatory sites. Even so, literature is conflicted regarding the location and persistence of MSC in the body after transplantation. For this reason, recent studies have focused on the paracrine effect of the biofactors secreted by MSC, especially in relation to the immunomodulatory potential of soluble factors (cytokines, chemokines, and growth factors) and extracellular vehicles that are involved in cell communication and in the transfer of cellular material, such as proteins, lipids, and nucleic acids. Moreover, treatment with interferon-γ, tumor necrosis factor-α, and interleukin-1β causes MSC to express immunomodulatory molecules that mediate the suppression via cell-contact dependent mechanisms. Taken together, we present an overview of the role of bioactive factors and cell membrane proteins derived from MSC as a cell-free therapy that can improve IBD treatment.     

Mesenchymal stem cell‐derived exosomes ameliorate erection by reducing oxidative stress damage of corpus cavernosum in a rat model of artery injury

Erectile dysfunction (ED) is a common ageing male's disease, and vascular ED accounts for the largest proportion of all types of ED. One of the mechanisms of vascular ED in the clinic is arterial insufficiency, which mainly caused by atherosclerosis, trauma and surgical. Moreover, oxidative stress damage after tissue ischemia usually aggravated the progress of ED. As a new way of acellular therapy, mesenchymal stem cell‐derived exosomes (MSC‐Exos) have great potential in ED treatment. In the current study, we have explored the mechanism of MSC‐Exos therapy in a rat model of internal iliac artery injury‐induced ED. Compared with intracavernous (IC) injection of phosphate‐buffered saline after artery injury, of note, we observed that both mesenchymal stem cells (MSCs) and MSC‐Exos through IC injection could improve the erectile function to varying degrees. More specifically, IC injection MSC‐Exos could promote cavernous sinus endothelial formation, reduce the organization oxidative stress damage, and improve the nitric oxide synthase and smooth muscle content in the corpus cavernosum. With similar potency compared with the stem cell therapy and other unique advantages, IC injection of MSC‐ Exos could be an effective treatment to ameliorate erectile function in a rat model of arterial injury.     

Melatonin preconditioning is an effective strategy for mesenchymal stem cell‐based therapy for kidney disease

Based on multiple studies in animal models, mesenchymal stem cell (MSC)‐based therapy appears to be an innovative intervention approach with tremendous potential for the management of kidney disease. However, the clinical therapeutic effects of MSCs in either acute kidney injury (AKI) or chronic kidney disease (CKD) are still under debate. Hurdles originate from the harsh microenvironment in vivo that decreases the cell survival rate, paracrine activity and migratory capacity of MSCs after transplantation, which are believed to be the main reasons for their limited effects in clinical applications. Melatonin is traditionally regarded as a circadian rhythm‐regulated neurohormone but in recent years has been found to exhibit antioxidant and anti‐inflammatory properties. Because inflammation, oxidative stress, thermal injury, and hypoxia are abnormally activated in kidney disease, application of melatonin preconditioning to optimize the MSC response to the hostile in vivo microenvironment before transplantation is of great importance. In this review, we discuss current knowledge concerning the beneficial effects of melatonin preconditioning in MSC‐based therapy for kidney disease. By summarizing the available information and discussing the underlying mechanisms, we aim to improve the therapeutic effects of MSC‐based therapy for kidney disease and accelerate translation to clinical application.     

WJSC 6th Anniversary Special Issues（2）:Mesenchymal stem cells Neurotrauma and mesenchymal stem cells treatment:From experimental studies to clinical trials

Mesenchymal stem cell(MSC)therapy has attracted the attention of scientists and clinicians around the world.Basic and pre-clinical experimental studies have highlighted the positive effects of MSC treatment after spinal cord and peripheral nerve injury.These effects are believed to be due to their ability to differentiate into other cell lineages,modulate inflammatory and immunomodulatory responses,reduce cell apoptosis,secrete several neurotrophic factors and respond to tissue injury,among others.There are many pre-clinical studies on MSC treatment for spinal cord injury(SCI)and peripheral nerve injuries.However,the same is not true for clinical trials,particularly those concerned with nerve trauma,indicating the necessity of more well-constructed studies showing the benefits that cell therapy can provide for individuals suffering the consequences of nerve lesions.As for clinical trials for SCI treatment the results obtained so far are not as beneficial as those described in experimental studies.For these reasons basic and pre-clinical studies dealing with MSC therapy should emphasize the standardization of protocols that could be translated to the clinical set with consistent and positive outcomes.This review is based on pre-clinical studies and clinical trials available in the literature from 2010 until now.At the time of writing this article there were 43 and 36 pre-clinical and 19 and 1 clinical trials on injured spinal cord and peripheral nerves,respectively.     

Direct intercellular communications dominate the interaction between adipose-derived MSCs and myofibroblasts against cardiac fibrosis

The onset of cardiac fibrosis post myocardial infarction greatly impairs the function of heart. Recent advances of cell transplantation showed great benefits to restore myocardial function, among which the mesenchymal stem cells (MSCs) has gained much attention. However, the underlying cellular mechanisms of MSC therapy are still not fully understood. Although paracrine effects of MSCs on residual cardiomyocytes have been discussed, the amelioration of fibrosis was rarely studied as the hostile environment cannot support the survival of most cell populations and impairs the diffusion of soluble factors. Here in order to decipher the potential mechanism of MSC therapy for cardiac fibrosis, we investigated the interplay between MSCs and cardiac myofibroblasts (mFBs) using interactive co-culture method, with comparison to paracrine approaches, namely treatment by MSC conditioned medium and gap co-culture method. Various fibrotic features of mFBs were analyzed and the most prominent anti-fibrosis effects were always obtained using direct co-culture that allowed cell-to-cell contacts. Hepatocyte growth factor (HGF), a well-known anti-fibrosis factor, was demonstrated to be a major contributor for MSCs’ anti-fibrosis function. Moreover, physical contacts and tube-like structures between MSCs and mFBs were observed by live cell imaging and TEM which demonstrate the direct cellular interactions.     

Methods to produce induced pluripotent stem cell-derived mesenchymal stem cells: Mesenchymal stem cells from induced pluripotent stem cells

Mesenchymal stem cells (MSCs) have received significant attention in recent years due to their large potential for cell therapy. Indeed, they secrete a wide variety of immunomodulatory factors of interest for the treatment of immune-related disorders and inflammatory diseases. MSCs can be extracted from multiple tissues of the human body. However, several factors may restrict their use for clinical applications: the requirement of invasive procedures for their isolation, their limited numbers, and their heterogeneity according to the tissue of origin or donor. In addition, MSCs often present early signs of replicative senescence limiting their expansion in vitro, and their therapeutic capacity in vivo. Due to the clinical potential of MSCs, a considerable number of methods to differentiate induced pluripotent stem cells (iPSCs) into MSCs have emerged. iPSCs represent a new reliable, unlimited source to generate MSCs (MSCs derived from iPSC, iMSCs) from homogeneous and well-characterized cell lines, which would relieve many of the above mentioned technical and biological limitations. Additionally, the use of iPSCs prevents some of the ethical concerns surrounding the use of human embryonic stem cells. In this review, we analyze the main current protocols used to differentiate human iPSCs into MSCs, which we classify into five different categories: MSC Switch, Embryoid Body Formation, Specific Differentiation, Pathway Inhibitor, and Platelet Lysate. We also evaluate common and method-specific culture components and provide a list of positive and negative markers for MSC characterization. Further guidance on material requirements to produce iMSCs with these methods and on the phenotypic features of the iMSCs obtained is added. The information may help researchers identify protocol options to design and/or refine standardized procedures for large-scale production of iMSCs fitting clinical demands.     

The immunosuppressive effects of human bone marrow-derived mesenchymal stem cells target T cell proliferation but not its effector function

Mesenchymal stem cells (MSC) are non-haematopoietic stem cells that are capable of differentiating into tissues of mesodermal origin. MSC play an important role in supporting the development of fetal and adult haematopoiesis. More recently, MSC have also been found to exhibit inhibitory effect on T cell responses. However, there is little information on the mechanism of this immunosuppression and our study addresses this issue by targeting T cell functions at various level of immune responses. We have generated MSC from human adult bone marrow (BM) and investigated their immunoregulatory function at different phases of T cell responses. MSC showed the ability to inhibit mitogen (CD3/CD28 microbeads)-activated T cell proliferation in a dose-dependent manner. In order to evaluate the specificity of this immunosuppression, the proliferation of CD4⁺ and CD8⁺ cells were measured. MSC equally inhibit CD4⁺ and CD8⁺ subpopulations of T cells in response to PHA stimulation. However, the antiproliferative effect of MSC is not due to the inhibition of T cell activation. The expression of early activation markers of T cells, namely CD25 and CD69 were not significantly altered by MSC at 24, 48 and 72 h. Furthermore, the immunosuppressive effect of MSC mainly targets T cell proliferation rather than their effector function since cytotoxicity of T cells is not affected. This work demonstrates that the immunosuppressive effect of MSC is exclusively a consequence of an anti-proliferative activity, which targets T cells of different subpopulations. For this reason, they have the potential to be exploited in the control of unwanted immune responses such as graft versus host disease (GVHD) and autoimmunity.     

Mesenchymal stem cells: From bench to bedside

Human mesenchymal stem cells (hMSCs) have tremendous promise for use in a variety of clinical applications. The ability of these cells to self-renew and differentiate into multiple tissues makes them an attractive cell source for a new generation of cell-based regenerative therapies. Encouraging results from clinical trials have also generated growing enthusiasm regarding MSC therapy and related treatment, but gaps remain in understanding MSC tissue repair mechanisms and in clinical strategies for efficient cell delivery and consistent therapeutic outcomes. For these reasons, discoveries from basic research and their implementation in clinical trials are essential to advance MSC therapy from the laboratory bench to the patient's bedside.     

Comparative osteogenic transcription profiling of various fetal and adult mesenchymal stem cell sources

Human mesenchymal stem cells (MSC) from adult and fetal tissues are promising candidates for cell therapy but there is a need to identify the optimal source for bone regeneration. We have previously characterized MSC populations in first trimester fetal blood, liver, and bone marrow and we now evaluate their osteogenic differentiation potential in comparison to adult bone marrow MSC. Using quantitative real-time RT-PCR, we demonstrated that 16 osteogenic-specific genes (OC, ON, BSP, OP, Col1, PCE, Met2A, OPG, PHOS1, SORT, ALP, BMP2, CBFA1, OSX, NOG, IGFII) were expressed in both fetal and adult MSC under basal conditions and were up-regulated under osteogenic conditions both in vivo and during an in vitro 21-day time-course. However, under basal conditions, fetal MSC had higher levels of osteogenic gene expression than adult MSC. Upon osteogenic differentiation, fetal MSC produced more calcium in vitro and reached higher levels of osteogenic gene up-regulation in vivo and in vitro. Second, we observed a hierarchy within fetal samples, with fetal bone marrow MSC having greater osteogenic potential than fetal blood MSC, which in turn had greater osteogenic potential than fetal liver MSC. Finally, we found that the level of gene expression under basal conditions was positively correlated with both calcium secretion and gene expression after 21 days in osteogenic conditions. Our findings suggest that stem cell therapy for bone dysplasias such as osteogenesis imperfecta may benefit from preferentially using first trimester fetal blood or bone marrow MSC over fetal liver or adult bone marrow MSC.     

Effect of mesenchymal stem cells on cytochrome-c release and inflammation in colon cancer induced by 1,2-dimethylhydrazine in Wistar albino rats

Colon cancer is one of the most common causes of deaths by cancer worldwide. Stem cells have immunosuppressive properties that promote tumor targeting and circumvent obstacles currently in gene therapy. Bone marrow stem cells are believed to have anticancer potential. The transplantation of mesenchymal stem cells (MSCs), a type of bone marrow stem cells, has been considered a potential therapy for patients with solid tumors due to their capability to enhance the immune response; MSC transplantation has received renewed interest in recent years. The present study aimed to evaluate the antiapoptotic effects of the MSCs on 1,2-dimethylhydrazine (DMH)-induced inflammation in the rat model of colorectal cancer. The rats were randomly allocated into four groups: control, treated with MSCs, induced by DMH, and induced by DMH and treated with MSCs. The MSCs were intra-rectally injected, and DMH was subcutaneously injected at 20 mg/kg body weight once a week for 15 weeks. The administration of MSCs into rats starting from day 0 of the DMH injection was found to enhance the histopathological picture. The MSC treatment resulted in fewer inflammatory cells than in the DMH group. Therefore, our findings suggest that BMCs have antitumor effects by modulating the cellular redox status and down-regulating the pro-inflammatory genes. Thus, BMCs may provide therapeutic value for colon cancer treatment.     

Allogeneic mesenchymal stem cells: agents of immune modulation

Adult mesenchymal stem cells possess a remarkably diverse array of immunosuppressive characteristics. The capacity to suppress the regular processes of allogeneic rejection, have allowed the use of tissue mismatched cells as therapeutic approaches in regenerative medicine and as agents of immune deviation. This review describes recent advances in understanding the mechanistic basis of mesenchymal stromal or stem cells (MSC) interaction with innate immunity. Particular emphasis is placed on the effect of Toll-like receptor signalling on MSC and a hypothesis that innate immune signals induce a 'licensing switch' in MSC is put forward. The mechanisms underlying MSC suppression of T cell responses and induction of regulatory populations are surveyed. Conflicting data regarding the influence of MSC on B cell function are outlined and discussed. Finally the limits to MSC mediated immune modulation are discussed with reference to the future clinical application of novel cell therapies.     

Human umbilical cord blood‐derived mesenchymal stem cells (hUCB‐MSC) inhibit the proliferation of K562 (human erythromyeloblastoid leukaemic cell line)

hUCB‐MSC (human umbilical cord blood‐derived mesenchymal stem cells) offer an attractive alternative to bone marrow‐derived MSC for cell‐based therapy by being less invasive a source of biological material. We have evaluated the effect of hUCB‐MSC on the proliferation of K562 (an erythromyeloblastoid cell line) and the cytokine secretion pattern of hUCB‐MSC. Co‐culturing of hUCB‐MSC and K562 resulted in inhibition of proliferation of K562 in a dose‐dependent manner. However, the anti‐proliferative effect was reduced in transwells, suggesting the importance of direct cell‐to‐cell contact. hUCB‐MSC inhibited proliferation of K562, arresting them in the G₀/G₁ phase. NO (nitric oxide) was not involved in the hUCB‐MSC‐mediated tumour suppression. The presence of IL‐6 (interleukin 6) and IL‐8 were obvious in the hUCB‐MSC conditioned media, but no significant increase was found in 29 other cytokines. Th1 cytokines, IFNα (interferon α), Th2 cytokine IL‐4 and Th17 cytokine, IL‐17 were not secreted by hUCB‐MSC. There was an increase in the number of hUCB‐MSC expressing the latent membrane‐bound form of TGFβ1 co‐cultured with K562. The anti‐proliferative effect of hUCB‐MSC was due to arrest of the growth of K562 in the G₀/G₁ phase. The mechanisms underlying increased IL‐6 and IL‐8 secretion and LAP (latency‐associated peptide; TGFβ1) by hUCB‐MSC remains unknown.     

Mechano-growth factor induces migration of rat mesenchymal stem cells by altering its mechanical properties and activating ERK pathway

Mechano-growth factor (MGF) generated by cells in response to mechanical stimulation has been identified as a mechano effector molecule, playing a key role in regulating mesenchymal stem cell (MSC) function, including proliferation and migration. However, the mechanism(s) underlying how MGF-induced MSC migration occurs is still unclear. In the present study, MGF motivated migration of rat MSCs (rMSCs) in a concentration-dependent manner and optimal concentration of MGF at 50 ng/mL (defined as MGF treatment in this paper) was demonstrated. Notably, enhancement of mechanical properties that is pertinent to cell migration, such as cell traction force and cell stiffness were found to respond to MGF treatment. Furthermore, MGF increased phosphorylation of extracellular signal-regulated kinase (ERK), ERK inhibitor (i.e., PD98059) suppressed ERK phosphorylation, and abolished MGF-induced rMSC migration were found, demonstrating that ERK is involved molecule for MGF-induced rMSC migration. These in vitro evidences of MGF-induced rMSC migration and its direct link to altering rMSC mechanics and activating the ERK pathway, uncover the underlying biomechanical and biological mechanisms of MGF-induced rMSC migration, which may help find MGF-based application of MSC in clinical therapeutics.     

Internalization of nanopolymeric tracers does not alter characteristics of placental cells

In the cell therapy scenario, efficient tracing of transplanted cells is essential for investigating cell migration and interactions with host tissues. This is fundamental to provide mechanistic insights which altogether allow for the understanding of the translational potential of placental cell therapy in the clinical setting. Mesenchymal stem/stromal cells (MSC) from human placenta are increasingly being investigated for their potential in treating patients with a variety of diseases. In this study, we investigated the feasibility of using poly (methyl methacrylate) nanoparticles (PMMA‐NPs) to trace placental MSC, namely those from the amniotic membrane (hAMSC) and early chorionic villi (hCV‐MSC). We report that PMMP‐NPs are efficiently internalized and retained in both populations, and do not alter cell morphofunctional parameters. We observed that PMMP‐NP incorporation does not alter in vitro immune modulatory capability of placental MSC, a characteristic central to their reparative/therapeutic effects in vitro. We also show that in vitro, PMMP‐NP uptake is not affected by hypoxia. Interestingly, after in vivo brain ischaemia and reperfusion injury achieved by transient middle cerebral artery occlusion (tMCAo) in mice, iv hAMSC treatment resulted in significant improvement in cognitive function compared to PBS‐treated tMCAo mice. Our study provides evidence that tracing placental MSC with PMMP‐NPs does not alter their in vitro and in vivo functions. These observations are grounds for the use of PMMP‐NPs as tools to investigate the therapeutic mechanisms of hAMSC and hCV‐MSC in preclinical models of inflammatory‐driven diseases.     

Enhanced infarct myocardium repair mediated by thermosensitive copolymer hydrogel-based stem cell transplantation

Mesenchymal stem cell (MSC) transplantation by intramyocardial injection has been proposed as a promising therapy strategy for cardiac repair after myocardium infarction. However, low retention and survival of grafted MSCs hinder its further application. In this study, copolymer with N-isopropylacrylamide/acrylic acid/2-hydroxylethyl methacrylate-poly(ɛ-caprolactone) ratio of 88:9.6:2.4 was bioconjugated with type I collagen to construct a novel injectable thermosensitive hydrogel. The injectable and biocompatible hydrogel-mediated MSC transplantation could enhance the grafted cell survival in the myocardium, which contributed to the increased neovascularization, decreased interstitial fibrosis, and ultimately improved heart function to a significantly greater degree than regular MSC transplantation. We suggest that this novel hydrogel has the potential for future stem cell transplantation.