Transplanting neural progenitor cells to restore connectivity after spinal cord injury.

Spinal cord injury remains a scientific and therapeutic challenge with great cost to individuals and society. The goal of research in this field is to find a means of restoring lost function. Recently we have seen considerable progress in understanding the injury process and the capacity of CNS neurons to regenerate, as well as innovations in stem cell biology. This presents an opportunity to develop effective transplantation strategies to provide new neural cells to promote the formation of new neuronal networks and functional connectivity. Past and ongoing clinical studies have demonstrated the safety of cell therapy, and preclinical research has used models of spinal cord injury to better elucidate the underlying mechanisms through which donor cells interact with the host and thus increase long-term efficacy. While a variety of cell therapies have been explored, we focus here on the use of neural progenitor cells obtained or derived from different sources to promote connectivity in sensory, motor and autonomic systems.

Transplanting neural progenitors into a complete transection model of spinal cord injury.

Neural progenitor cell (NPC) transplantation is a promising therapeutic strategy for spinal cord injury (SCI) because of the potential for cell replacement and restoration of connectivity. Our previous studies have shown that transplants of NPC, composed of neuron- and glia-restricted progenitors derived from the embryonic spinal cord, survived well in partial lesion models and generated graft-derived neurons, which could be used to form a functional relay. We have now examined the properties of a similar NPC transplant using a complete transection model in juvenile and adult rats. We found poor survival of grafted cells despite using a variety of lesion methods, matrices, and delays of transplantation. If, instead of cultured progenitor cells, the transplants were composed of segmental or dissociated segments of fetal spinal cord (FSC) derived from similar-staged embryos, grafted cells survived and integrated well with host tissue in juvenile and adult rats. FSC transplants differentiated into neurons and glial cells, including astrocytes and oligodendrocytes. Graft-derived neurons expressed glutaminergic and GABAergic markers. Grafted cells also migrated and extended processes into host tissue. Analysis of axon growth from the host spinal cord showed serotonin-positive fibers and biotinylated dextran amine-traced propriospinal axons growing into the transplants. These results suggest that in treating severe SCI, such as complete transection, NPC grafting faces major challenges related to cell survival and formation of a functional relay. Lessons learned from the efficacy of FSC transplants could be used to develop a therapeutic strategy based on neural progenitor cells for severe SCI.

Big tau: What, how, where and why.

Over the last 50 years the different isoforms of tau proteins (45-60 kDa) have been a focus of research because of their roles in modulating the dynamic properties of microtubules shaping the structure and function of neurons but also becoming a center of attention in the pathology of neurodegeneration associated with tauopathies. Much less attention has been given to Big tau, a unique isoform containing exon 4a encoding about 250 amino acids to form a much longer projection domain of a protein of 110 kDa. Big tau is expressed in peripheral neurons and selective regions of the central nervous system in a defined transition during postnatal developmental stages. Although Big tau was discovered 30 years ago, there has been a persistent gap of knowledge regarding its physiological properties and pathological implications. This Perspective summarizes the progress so far in defining the structure and expression of Big tau within and outside the nervous system, proposes a role for Big tau in improving axonal transport in projecting axons, considers its potential in averting tau aggregation in tauopathies and highlights the need for further progress.

The unique properties of Big tau in the visual system.

Tau is a microtubule associated protein that plays important roles in regulating the properties of microtubules and axonal transport, as well as tauopathies associated with toxic aggregates leading to neurodegenerative diseases. It is encoded by the MAPT gene forming multiple isoforms (45-60 kDa) by alternative splicing which are developmentally regulated. The high molecular weight (MW) tau isoform of 105 kDa, termed Big tau, was originally discovered in the peripheral nervous system (PNS) but later found in selective CNS areas. It contains an additional large exon 4a generating a long projecting domain of about 250 amino acids. Here we investigated the properties of Big tau in the visual system of rats, its distribution in retinal ganglion cells and the optic nerve as well as its developmental regulation using biochemical, molecular and histological analyses. We discovered that Big tau is expresses as a 95 kDa protein (termed middle MW) containing exons 4a, 6 as well as exon 10 which defines a 4 microtubule-binding repeats (4R). It lacks exons 2/3 but shares the extensive phosphorylation characteristic of other tau isoforms. Importantly, early in development the visual system expresses only the low MW isoform (3R) switching to both the low and middle MW isoforms (4R) in adult retinal ganglion neurons and their corresponding axons. This is a unique structure and expression pattern of Big tau, which we hypothesize is associated with the specific properties of the visual system different from what has been previously described in the PNS and other areas of the nervous system.

Big Tau: What We Know, and We Need to Know.

Tau is a microtubule-associated protein (MAP) that has multiple isoforms generated by alternative splicing of the MAPT gene at a range of 45-60 kDa [low-molecular-weight (LMW) tau] as well as a unique isoform termed Big tau containing an additional exon 4a encoding a large projecting domain of ∼250 aa to form a protein of 110 kDa. Big tau is expressed in adult PNS neurons such as DRG neurons and specific regions of CNS such as the cerebellum in a developmental transition from LMW tau to Big tau during the postnatal period. Despite a conserved size of the 4a exons across the vertebrate phylogeny, there is no sequence homology among different species outside the Mammalia class, which underscores the focus on structural preservation of Big tau. Despite the original discovery of Big tau in the early 1990s, there has been little progress elucidating its physiological properties and pathologic implications. We propose that Big tau may be able to improve axonal transport in projecting axons and speculate on the potential protective properties in preventing tau aggregation in pathologic conditions. This perspective highlights the importance and benefits of understanding of the role of Big tau in neuronal health and disease.

Regulation of Tau Expression in Superior Cervical Ganglion (SCG) Neurons In Vivo and In Vitro.

The superior cervical ganglion (SCG) is part of the autonomic nervous system providing sympathetic innervation to the head and neck, and has been regularly used to prepare postnatal neuronal cultures for cell biological studies. We found that during development these neurons change tau expression from the low molecular weight (LMW) isoforms to Big tau, with the potential to affect functions associated with tau such as microtubule dynamic and axonal transport. Big tau contains the large 4a exon that transforms tau from LMW isoforms of 45-60 kDa to 110 kDa. We describe tau expression during postnatal development reporting that the transition from LMW tau to Big tau which started at late embryonic stages is completed by about 4-5 weeks postnatally. We confirmed the presence of Big tau in dissociated postnatal SCG neurons making them an ideal system to study the function of Big tau in neurons. We used SCG explants to examine the response of SCG neurons to lesion and found that Big tau expression returned gradually along the regrowing neurites suggesting that it does not drives regeneration, but facilitates the structure/function of mature SCG neurons. The structural/functional roles of Big tau remain unknown, but it is intriguing that neurons that express Big tau appear less vulnerable to tauopathies.

Matching mechanical heterogeneity of the native spinal cord augments axon infiltration in 3D-printed scaffolds.

Scaffolds delivered to injured spinal cords to stimulate axon connectivity often match the anisotropy of native tissue using guidance cues along the rostral-caudal axis, but current approaches do not mimic the heterogeneity of host tissue mechanics. Although white and gray matter have different mechanical properties, it remains unclear whether tissue mechanics also vary along the length of the cord. Mechanical testing performed in this study indicates that bulk spinal cord mechanics do differ along anatomical level and that these differences are caused by variations in the ratio of white and gray matter. These results suggest that scaffolds recreating the heterogeneity of spinal cord tissue mechanics must account for the disparity between gray and white matter. Digital light processing (DLP) provides a means to mimic spinal cord topology, but has previously been limited to printing homogeneous mechanical properties. We describe a means to modify DLP to print scaffolds that mimic spinal cord mechanical heterogeneity caused by variation in the ratio of white and gray matter, which improves axon infiltration compared to controls exhibiting homogeneous mechanical properties. These results demonstrate that scaffolds matching the mechanical heterogeneity of white and gray matter improve the effectiveness of biomaterials transplanted within the injured spinal cord.

Grafted neural progenitors integrate and restore synaptic connectivity across the injured spinal cord.

Transplantation of neural progenitor cells (NPC) is a promising therapeutic strategy for replacing neurons lost after spinal cord injury, but significant challenges remain regarding neuronal integration and functional connectivity. Here we tested the ability of graft-derived neurons to reestablish connectivity by forming neuronal relays between injured dorsal column (DC) sensory axons and the denervated dorsal column nuclei (DCN). A mixed population of neuronal and glial restricted precursors (NRP/GRP) derived from the embryonic spinal cord of alkaline phosphatase (AP) transgenic rats were grafted acutely into a DC lesion at C1. One week later, BDNF-expressing lentivirus was injected into the DCN to guide graft axons to the intended target. Six weeks later, we observed anterogradely traced sensory axons regenerating into the graft and robust growth of graft-derived AP-positive axons along the neurotrophin gradient into the DCN. Immunoelectron microscopy revealed excitatory synaptic connections between regenerating host axons and graft-derived neurons at C1 as well as between graft axons and DCN neurons in the brainstem. Functional analysis by stimulus-evoked c-Fos expression and electrophysiological recording showed that host axons formed active synapses with graft neurons at the injury site with the signal propagating by graft axons to the DCN. We observed reproducible electrophysiological activity at the DCN with a temporal delay predicted by our relay model. These findings provide the first evidence for the ability of NPC to form a neuronal relay by extending active axons across the injured spinal cord to the intended target establishing a critical step for neural repair with stem cells.

Differential effects of distinct central nervous system regions on cell migration and axonal extension of neural precursor transplants.

Transplantation of neural precursor cells (NPCs) is a promising therapeutic strategy in CNS injury. However, the adult CNS lacks instructive signals present during development and, depending on the region and type of transplant, may be inhibitory for neuron generation and axonal growth. We examined the effects of the white matter in different regions of the adult CNS on the properties of NPC transplants with respect to cell survival, differentiation, migration, and axonal growth. NPCs were prepared from day 13.5 embryonic spinal cord of transgenic rats that express the human placental alkaline phosphatase (AP) reporter. These NPCs were injected unilaterally into the cervical spinal cord white matter and into the corpus callosum of adult rats and were analyzed immunohistochemically 2 weeks later. NPCs survived in both regions and differentiated into astrocytes, oligodendrocytes, and neurons, with no apparent differences in survival or phenotypic composition. However, in the spinal cord white matter, graft-derived cells, identified as precursors and glial cells, migrated from the injection site rostrally and caudally, whereas, in the corpus callosum, graft-derived cells did not migrate and remained at the injection site. Importantly, graft-derived neurons extended axons from the grafting site along the corpus callosum past the midline, entering into the contralateral side of the corpus callosum. These results demonstrate dramatic differences between white matter regions in the spinal cord and brain with respect to cell migration and axonal growth and underscore the importance of considering the effects of the local CNS environment in the design of effective transplantation strategies.

Evolutionary perspective of Big tau structure: 4a exon variants of MAPT.

The MAPT gene encoding the microtubule-associated protein tau can generate multiple isoforms by alternative splicing giving rise to proteins which are differentially expressed in specific areas of the nervous system and at different developmental stages. Tau plays important roles in modulating microtubule dynamics, axonal transport, synaptic plasticity, and DNA repair, and has also been associated with neurodegenerative diseases (tauopathies) including Alzheimer's disease and frontotemporal dementia. A unique high-molecular-weight isoform of tau, originally found to be expressed in the peripheral nervous system and projecting neurons, has been termed Big tau and has been shown to uniquely contain the large exon 4a that significantly increases the size and 3D structure of tau. With little progress since the original discovery of Big tau, more than 25 years ago, we have now completed a comprehensive comparative study to analyze the structure of the MAPT gene against available databases with respect to the composition of the tau exons as they evolved from early vertebrates to primates and human. We focused the analysis on the evolution of the 4a exon variants and their homology relative to humans. We discovered that the 4a exon defining Big tau appears to be present early in vertebrate evolution as a large insert that dramatically changed the size of the tau protein with low sequence conservation despite a stable size range of about 250aa, and in some species a larger 4a-L exon of 355aa. We suggest that 4a exon variants evolved independently in different species by an exonization process using new alternative splicing to address the growing complexities of the evolving nervous systems. Thus, the appearance of a significantly larger isoform of tau independently repeated itself multiple times during evolution, accentuating the need across vertebrate species for an elongated domain that likely endows Big tau with novel physiological functions as well as properties related to neurodegeneration.

Transplanting Neural Progenitor Cells into a Chronic Dorsal Column Lesion Model.

Cell transplantation therapy is a promising strategy for spinal cord injury (SCI) repair. Despite advancements in the development of therapeutic strategies in acute and subacute SCI, much less is known about effective strategies for chronic SCI. In previous studies we demonstrated that transplants of neural progenitor cells (NPC) created a permissive environment for axon regeneration and formed a neuronal relay across the injury following an acute dorsal column injury. Here we explored the efficacy of such a strategy in a chronic injury. We tested two preparations of NPCs derived from rat spinal cord at embryonic day 13.5: one prepared using stocks of cultured cells and the other of dissociated cells transplanted without culturing. Transplantation was delayed for 4-, 6- and 12-weeks post injury for a chronic injury model. We found that the dissociated NPC transplants survived and proliferated for at least 5 weeks post transplantation, in contrast to the poor survival of transplants prepared from cultured NPC stocks. The dissociated NPC transplants differentiated into neurons expressing excitatory markers, promoted axon regeneration into the injury/transplant site and extended axons from graft-derived neurons into the host. These results support the potential of NPC transplants to form neuronal relays across a chronic SCI, but they also underscore the challenges of achieving efficient cell survival in the environment of a chronic injury.

Magnetic alignment of injectable hydrogel scaffolds for spinal cord injury repair.

Injectable hydrogels for cell delivery and tissue regeneration have several advantages over pre-fabricated scaffolds that require more invasive transplantation procedures, but lack the ability to implement tunable topologies. Here, we describe an approach to create patternable and injectable scaffolds using magnetically-responsive (MR) self-assembling peptide hydrogels, and validate their efficacy to promote and align axon infiltration at the site of a spinal cord injury. In vitro experiments reveal the parameters needed to align the fibers using the application of an external magnetic field. These results indicate that applying a 100-Gauss (G) field to the peptide hydrogels during polymerization causes fiber alignment as measured by electron microscopy, even in the presence of cells. In order to mimic infiltrating axons, neural progenitor cells (NPCs) are seeded on the surface of peptide hydrogels to interrogate the effects of both magnetic alignment and embedding human mesenchymal stem cells (hMSCs) in the scaffold. NPCs infiltrate peptide hydrogels seeded with hMSCs, and exhibit increased alignment and elongation in aligned gels. In order to evaluate these injectable and patternable scaffolds in vivo, hMSC-seeded peptide hydrogels are injected at the site of a contusion spinal cord injury with and without the presence of a magnetic field to align the resulting fibrous network. Measurements of axon growth and orientation as well as inflammation and glial scar formation indicate that these metrics are improved in magnetically aligned hMSC-seeded hydrogels. The results verify that MR hydrogels can dictate the orientation of infiltrating axons, providing a viable means to control the topology of injectable scaffolds.

Spatial and temporal changes in promoter activity of the astrocyte glutamate transporter GLT1 following traumatic spinal cord injury.

After traumatic spinal cord injury (SCI), there is an opportunity for preserving function by attenuating secondary cell loss. Astrocytes play crucial roles in the adult CNS and are responsible for the vast majority of glutamate buffering, potentially preventing excitotoxic loss of neurons and oligodendrocytes. We examined spatial and temporal changes in gene expression of the major astrocyte glutamate transporter GLT1 following moderate thoracic contusion SCI using transgenic BAC-GLT1-eGFP promoter reporter mice. In dorsal column white matter, total intensity of GLT1-eGFP expression per region was significantly reduced following SCI at both lesion epicenter and at rostral and caudal areas where no tissue loss occurred. This regional decrease in GLT1 expression was due to significant loss of GLT1-eGFP(+) cells, partially accounted for by apoptosis of eGFP(+) /GFAP(+) astrocytes in both white and gray matter. There were also decreased numbers of GLT1-eGFP-expressing cells in multiple gray matter regions following injury; nevertheless, there was sustained or even increased regional GLT1-eGFP expression in gray matter as a result of up-regulation in astrocytes that continued to express GLT1-eGFP. Although there were increased numbers of GFAP(+) cells both at the lesion site and in surrounding intact spinal cord following SCI, the majority of proliferating Ki67(+) /GFAP(+) astrocytes did not express GLT1-eGFP. These findings demonstrate that spatial and temporal alterations in GLT1 expression observed after SCI result from both astrocyte death and gene expression changes in surviving astrocytes. Results also suggest that following SCI a significant portion of astrocytes lacks GLT1 expression, possibly compromising the important role of astrocytes in glutamate homeostasis.

Influence of alginate cross-linking method on neurite response to microencapsulated neurotrophin-producing fibroblasts.

Transplantation of cells genetically modified to produce neurotrophins is a promising spinal cord repair strategy. Previously we showed that fibroblasts engineered to produce brain-derived neurotrophic factor (Fb/BDNF) microencapsulated in alginate survive, continue to grow and express bioactive BDNF. We here compared the effects of different alginate crosslinkers on dorsal root ganglia (DRG) neurite growth using alginate-encapsulated Fb/BDNF. Aqueous sodium alginate (±Fb/BDNF) was contacted with different calcium salts, and used as substrate for DRG growth. Length, number and orientation of neurites were measured. Chloride or carbonate salts promoted significantly more neurite growth than sulphate, with or without Fb/BDNF, although encapsulated Fb/BDNF stimulated significantly more neurite growth than cell-free. An Fb/BDNF-derived neurotrophin concentration gradient directionally guided DRG neurite growth. This positive effect of alginate-encapsulated Fb/BDNF on neurite growth/guidance shows promise for enhanced regeneration and guidance of axons towards a specific target in the injured spinal cord.

Preparation of Neural Stem Cells and Progenitors: Neuronal Production and Grafting Applications.

Neural stem cells (NSCs) are a valuable tool for the study of neural development and function as well as an important source of cell transplantation strategies for neural disease. NSCs can be used to study how neurons acquire distinct phenotypes and how the interactions between neurons and glial cells in the developing nervous system shape the structure and function of the CNS. NSCs can also be used for cell replacement therapies following CNS injury targeting astrocytes, oligodendrocytes, and neurons. With the availability of patient-derived induced pluripotent stem cells (iPSCs), neurons prepared from NSCs can be used to elucidate the molecular basis of neurological disorders leading to potential treatments. Although NSCs can be derived from different species and many sources, including embryonic stem cells (ESCs), iPSCs, adult CNS, and direct reprogramming of nonneural cells, isolating primary NSCs directly from fetal tissue is still the most common technique for preparation and study of neurons. Regardless of the source of tissue, similar techniques are used to maintain NSCs in culture and to differentiate NSCs toward mature neural lineages. This chapter will describe specific methods for isolating and characterizing multipotent NSCs and neural precursor cells (NPCs) from embryonic rat CNS tissue (mostly spinal cord) and from human ESCs and iPSCs as well as NPCs prepared by reprogramming. NPCs can be separated into neuronal and glial restricted progenitors (NRP and GRP, respectively) and used to reliably produce neurons or glial cells both in vitro and following transplantation into the adult CNS. This chapter will describe in detail the methods required for the isolation, propagation, storage, and differentiation of NSCs and NPCs isolated from rat and mouse spinal cords for subsequent in vitro or in vivo studies as well as new methods associated with ESCs, iPSCs, and reprogramming.

Promoting directional axon growth from neural progenitors grafted into the injured spinal cord.

Spinal cord injury (SCI) is a devastating condition characterized by disruption of axonal connections, failure of axonal regeneration, and loss of motor and sensory function. The therapeutic promise of neural stem cells has been focused on cell replacement, but many obstacles remain in obtaining neuronal integration following transplantation into the injured CNS. This study investigated the neurotransmitter identity and axonal growth potential of neural progenitors following grafting into adult rats with a dorsal column lesion. We found that using a combination of neuronal and glial restricted progenitors (NRP and GRP) produced graft-derived glutamatergic and GABAergic neurons within the injury site, with minimal axonal extension. Administration of brain-derived neurotrophic factor (BDNF) with the graft promoted modest axonal growth from grafted cells. In contrast, injecting a lentiviral vector expressing BDNF rostral into the injured area generated a neurotrophin gradient and promoted directional growth of axons for up to 9 mm. Animals injected with BDNF lentivirus (at 2.5 and 5.0 mm) showed significantly more axons and significantly longer axons than control animals injected with GFP lentivirus. However, only the 5.0-mm-BDNF group showed a preference for extension in the rostral direction. We concluded that NRP/GRP grafts can be used to produce excitatory and inhibitory neurons, and neurotrophin gradients can guide axonal growth from graft-derived neurons toward putative targets. Together they can serve as a building block for neuronal cell replacement of local circuits and formation of neuronal relays.

Secretion profile of human bone marrow stromal cells: donor variability and response to inflammatory stimuli.

Mesenchymal stem cells (MSC) derived from bone marrow are ideal transplants for a variety of CNS disorders and appear to support recovery after injury by secreting therapeutic factors. There is considerable variability in the secretion profile of MSC derived from different donors and it is known that MSC secretion changes in response to inflammatory stimuli, but no comprehensive analysis has been performed to address these issues. Here we show that MSC from seven donors secrete chemokines and cytokines in variable ranges, with some factors showing high variability. Treatment of cultured MSC with pro-inflammatory cytokines or tissue extracts from injured spinal cord resulted in up-regulation of selected cytokines, whereas treatment with an anti-inflammatory cytokine had little effect, indicating that the secretion profile is tightly regulated by environmental challenges. Patterns of up-regulated cytokines were similar in MSC from different donors suggesting a comparable response to inflammatory stimuli.

Resurrecting the Mysteries of Big Tau.

Tau, a microtubule-associated protein that modifies the dynamic properties and organization of microtubules in neurons and affects axonal transport, shows remarkable heterogeneity, with multiple isoforms (45-65 kDa) generated by alternative splicing. A high-molecular-weight (HMW) isoform (110 kDa) that contains an additional large exon termed 4a was discovered more than 25 years ago. This isoform, called Big tau, is expressed mainly in the adult peripheral nervous system (PNS), but also in adult neurons of the central nervous system (CNS) that extend processes into the periphery. Surprisingly little has been learned about Big tau since its initial characterization, leaving a significant gap in knowledge about how the dramatic switch to Big tau affects the properties of neurons in the context of development, disease, or injury. Here we review what was learned about the structure and distribution of Big tau in those earlier studies, and add contemporary insights to resurrect interest in the mysteries of Big tau and thereby set a path for future studies.

Vascularization of self-assembled peptide scaffolds for spinal cord injury repair.

The disruption of the blood-spinal cord barrier (BSCB) following spinal cord injury contributes to inflammation and glial scarring that inhibits axon growth and diminishes the effectiveness of conduits transplanted to the injury site to promote this growth. The purpose of this study is to evaluate whether scaffolds containing microvessels that exhibit BSCB integrity reduce inflammation and scar formation at the injury site and lead to increased axon growth. For these studies, a self-assembling peptide scaffold, RADA-16I, is used due to its established permissiveness to axon growth and ability to support vascularization. Immunocytochemistry and permeability transport assays verify the formation of tight-junction containing microvessels within the scaffold. Peptide scaffolds seeded with different concentrations of microvascular cells are then injected into a spinal contusion injury in rats to evaluate how microvessels affect axon growth and neurovascular interaction. The effect of the vascularized scaffold on inflammation and scar formation is evaluated by quantifying histological sections stained with ED-1 and GFAP, respectively. Our results indicate that the peptide scaffolds containing microvessels reduce inflammation and glial scar formation and increase the density of axons growing into the injury/transplant site. These results demonstrate the potential benefit of scaffold vascularization to treat spinal cord injury. STATEMENT OF SIGNIFICANCE: This study evaluates the benefit of transplanting microvascular cells within a self-assembling peptide scaffold, RADA-16I, that has shown promise for facilitating regeneration in the central nervous system in previous studies. Our results indicate that vasculature featuring tight junctions that give rise to the blood-spinal cord barrier can be formed within the peptide scaffold both in vitro and in a rat model of a subacute contusion spinal cord injury. Histological analysis indicates that the presence of the microvessels encourages axon infiltration into the site of injury and reduces the area of astrocyte activation and inflammation. Overall, these results demonstrate the potential of vascularizing scaffolds for the repair of spinal cord injury.

Effects of plating density and culture time on bone marrow stromal cell characteristics.

OBJECTIVE: Bone marrow stromal cells (MSC) are multipotent adult stem cells that have emerged as promising candidates for cell therapy in disorders including cardiac infarction, stroke, and spinal cord injury. While harvesting methods used by different laboratories are relatively standard, MSC culturing protocols vary widely. This study is aimed at evaluating the effects of initial plating density and total time in culture on proliferation, cell morphology, and differentiation potential of heterogeneous MSC cultures and more homogeneous cloned subpopulations. MATERIALS AND METHODS: Rat MSC were plated at 20, 200, and 2000 cells/cm(2) and grown to 50% confluency. The numbers of population doublings and doubling times were determined within and across multiple passages. Changes in cell morphology and differentiation potential to adipogenic, chondrogenic, and osteogenic lineages were evaluated and compared among early, intermediate, and late passages, as well as between heterogeneous and cloned MSC populations. RESULTS: We found optimal cell growth at a plating density of 200 cells/cm(2). Cultures derived from all plating densities developed increased proportions of flat cells over time. Assays for chondrogenesis, osteogenesis, and adipogenesis showed that heterogeneous MSC plated at all densities sustained the potential for all three mesenchymal phenotypes through at least passage 5; the flat subpopulation lost adipogenic and chondrogenic potential. CONCLUSION: Our findings suggest that the initial plating density is not critical for maintaining a well-defined, multipotent MSC population. Time in culture, however, affects cell characteristics, suggesting that cell expansion should be limited, especially until the specific characteristics of different MSC subpopulations are better understood.