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Biomedical subjects

D J Prockop

Publications and source records attributed to D J Prockop.

At least 37 records · Page 2Linked to original sources
PubMed2000

Rapid expansion of recycling stem cells in cultures of plastic-adherent cells from human bone marrow.

Cultures of plastic-adherent cells from bone marrow have attracted interest because of their ability to support growth of hematopoietic stem cells, their multipotentiality for differentiation, and their possible use for cell and gene therapy. Here we found that the cells grew most rapidly when they were initially plated at low densities (1.5 or 3.0 cells/cm(2)) to generate single-cell derived colonies. The cultures displayed a lag phase of about 5 days, a log phase of rapid growth of about 5 days, and then a stationary phase. FACS analysis demonstrated that stationary cultures contained a major population of large and moderately granular cells and a minor population of small and agranular cells here referred to as recycling stem cells or RS-1 cells. During the lag phase, the RS-1 cells gave rise to a new population of small and densely granular cells (RS-2 cells). During the late log phase, the RS-2 cells decreased in number and regenerated the pool of RS-1 cells found in stationary cultures. In repeated passages in which the cells were plated at low density, they were amplified about 10(9)-fold in 6 wk. The cells retained their ability to generate single-cell derived colonies and therefore apparently retained their multipotentiality for differentiation.

Adult↗
PubMed2000

Lack of a phenotype in transgenic mice aberrantly expressing COL2A1 mRNA because of highly selective post-transcriptional down-regulation.

We reported previously that a 1.9-kb 5'-fragment from the human COL1A1 gene drove transcription of a promoterless human COL2A1 gene in tissues of transgenic mice that normally express the COL1A1 but not the COL2A1 gene. In the present study, we have established that the aberrant transcription of the COL2A1 gene did not produce any gross or microscopic phenotype, because the transcripts were not efficiently translated in cells that do not normally express the COL2A1 gene. In two lines of transgenic mice, the mRNA levels from the transgene were 30% to 45% of the mRNA for the proalpha1(I) chain of type I procollagen, the most abundant mRNA in the same tissues. Analysis of collagens extracted from skin of the transgenic mice indicated that triple-helical type II collagen, with the normal pattern of cyanogen bromide peptides, was synthesized from the transgene. However, the level of type II collagen in skin was less than 2% of the level of type I collagen. Hybridization in situ indicated the presence of mRNA for both COL2A1 and COL1A1 in the same cells. Immunofluorescence staining for type II collagen, however, was negative in the same tissues. The results, therefore, indicated that many mesenchymal cells in the transgenic mice had high steady-state levels of the homologous mRNAs for type I and type II procollagen, but only the mRNAs for type I procollagen were efficiently translated.

Animals↗
PubMed2000

Sequence and genomic organization of the human G-protein Golfalpha gene (GNAL) on chromosome 18p11, a susceptibility region for bipolar disorder and schizophrenia.

The sequence and genomic organization of the human Golfalpha (GNAL) gene were determined. The human GNAL gene was found to contain 12 coding exons, and it spans over 80 kb on chromosome 18p11. 5' RACE analysis suggested an additional transcription initiation start site. Sequence analysis of the putative promoter region revealed conserved binding sites for several transcription factors. Sequence analysis of the 3'-untranslated region revealed the presence of two Alu sequences and two polyadenylation signals. 3' RACE analysis confirmed the functionality of the most downstream poly-a signal. The human GNAL was found to be expressed as a single transcript of about 5.9 kb in the brain. One highly informative dinucleotide repeat was found in intron 5. Additionally, a processed pseudogene for asparagine synthetase was found about 6 kb upstream of the GNAL gene. Knowledge of the sequence and structure of the human GNAL gene provides essential information for further analysis of the GNAL locus at chromosome 18p11 which has been linked to bipolar disorder and schizophrenia.

3' Untranslated Regions↗
PubMed2000

Papilin in development; a pericellular protein with a homology to the ADAMTS metalloproteinases.

Papilin is an extracellular matrix glycoprotein that we have found to be involved in, (1) thin matrix layers during gastrulation, (2) matrix associated with wandering, phagocytic hemocytes, (3) basement membranes and (4) space-filling matrix during Drosophila development. Determination of its cDNA sequence led to the identification of Caenorhabditis and mammalian papilins. A distinctly conserved 'papilin cassette' of domains at the amino-end of papilins is also the carboxyl-end of the ADAMTS subgroup of secreted, matrix-associated metalloproteinases; this cassette contains one thrombospondin type 1 (TSR) domain, a specific cysteine-rich domain and several partial TSR domains. In vitro, papilin non-competitively inhibits procollagen N-proteinase, an ADAMTS metalloproteinase. Inhibiting papilin synthesis in Drosophila or Caenorhabditis causes defective cell arrangements and embryonic death. Ectopic expression of papilin in Drosophila causes lethal abnormalities in muscle, Malpighian tubule and trachea formation. We suggest that papilin influences cell rearrangements and may modulate metalloproteinases during organogenesis.

Amino Acid Sequence↗
PubMed2000

Potential use of stem cells from bone marrow to repair the extracellular matrix and the central nervous system.

A subset of stem-like cells from bone marrow that are referred to as marrow stromal cells (MSCs) have been shown to be capable of differentiating into osteoblasts, chondrocytes, adipocytes, myocytes, astrocytes and perhaps neurons. Recently, conditions have been developed where human MSCs can be expanded almost without limit in culture without apparently losing their multipotentiality for differentiation. The cells appear to be potentially useful for the repair of extracellular matrix and the central nervous system.

Animals↗
PubMed1999

Donor variation in the growth properties and osteogenic potential of human marrow stromal cells.

Human marrow stromal cells (MSCs) were isolated from posterior illiac crest marrow aspirates obtained from 17 healthy donors, ages 19-45 years, with no apparent physical disability. First passage hMSCs exhibited growth rates in vitro that varied up to 12-fold between donors. No correlation between growth rate and the age or gender of the donor was evident (P </= 0.05). When hMSCs were cultured without passage for eight days (subconfluent cultures) or 22 days (confluent cultures) in the absence of any osteogenic agonists, levels of alkaline phosphatase enzyme activity varied 40-fold and 10-fold, respectively, between donors. When exposed to osteo-inductive media, donor populations also showed dramatic differences in levels of bone-specific gene induction. Collectively, these data demonstrate that hMSC cultures are composed of a heterogeneous mixture of cells at various stages of differentiation and with distinct osteogenic potentials. Differences in both growth rate and ALP activity were evident in hMSC cultures established from multiple aspirates obtained over a six month period from the same donors. Therefore, it appears that cellular heterogeneity produced by the method of harvest is propagated within and among different donor populations during culture expansion in vitro.

Adult↗
PubMed1999

Multipotential marrow stromal cells transduced to produce L-DOPA: engraftment in a rat model of Parkinson disease.

Bone marrow stromal cells can be used as an alternative source of cells for neural transplantation and repair. Here, the efficacy of genetically modified marrow stromal cells was examined in a rat model of Parkinson disease. Rat marrow stromal cells (rMSCs) and human marrow stromal cells (hMSCs) were genetically engineered by transduction with retroviruses encoding tyrosine hydroxylase (TH) and GTP cyclohydrolase I, the enzyme necessary for production of the tetrahydrobiopterin cofactor for TH (BH4). Transduced cells synthesized 3,4-dihydroxyphenylalanine (L-DOPA) in vitro and maintained their multipotentiality after retroviral transduction. To examine the cells in vivo, transduced rMSCs were injected into the striatum of 6-hydroxydopamine-lesioned rats. L-DOPA and metabolites were detected by microdialysis in the denervated striatum of rats that received doubly transduced rMSCs. Also, there was a significant reduction in apomorphine-induced rotation when compared with controls. The cells engrafted and survived for at least 87 days. However, expression of the transgenes ceased at about 9 days, an observation consistent with reports from other laboratories in which similar retroviruses were used to express transgenes in the brain.

Animals↗
PubMed1999

Marrow stromal cells migrate throughout forebrain and cerebellum, and they differentiate into astrocytes after injection into neonatal mouse brains.

Stem cells are a valuable resource for treating disease, but limited access to stem cells from tissues such as brain restricts their utility. Here, we injected marrow stromal cells (MSCs) into the lateral ventricle of neonatal mice and asked whether these multipotential mesenchymal progenitors from bone marrow can adopt neural cell fates when exposed to the brain microenvironment. By 12 days postinjection, MSCs migrated throughout the forebrain and cerebellum without disruption to the host brain architecture. Some MSCs within the striatum and the molecular layer of the hippocampus expressed glial fibrillary acidic protein and, therefore, differentiated into mature astrocytes. MSCs also populated neuron rich regions including the Islands of Calleja, the olfactory bulb, and the internal granular layer of the cerebellum. A large number of MSCs also were found within the external granular layer of the cerebellum. In addition, neurofilament positive donor cells were found within the reticular formation of the brain stem, suggesting that MSCs also may have differentiated into neurons. Therefore, MSCs are capable of producing differentiated progeny of a different dermal origin after implantation into neonatal mouse brains. These results suggest that MSCs are potentially useful as vectors for treating a variety of central nervous system disorders.

Adipocytes↗
PubMed1999

Complete sequence of the 23-kilobase human COL9A3 gene. Detection of Gly-X-Y triplet deletions that represent neutral variants.

We report the complete sequence of the human COL9A3 gene that encodes the alpha3 chain of heterotrimeric type IX collagen, a member of the fibril-associated collagens with interrupted triple helices family of collagenous proteins. Nucleotide sequencing defined over 23,000 base pairs (bp) of the gene and about 3000 bp of the 5'-flanking sequences. The gene contains 32 exons. The domain and exon organization of the gene is almost identical to a related gene, the human COL9A2 gene. However, exon 2 of the COL9A3 gene codes for one -Gly-X-Y- triplet less than exon 2 of the COL9A2 gene. The difference is compensated by an insertion of 9 bp coding for an additional triplet in exon 4 of the COL9A3 gene. As a result, the number of -Gly-X-Y- repeats in the third collagenous domain remains the same in both genes and ensures the formation of an in-register triple helix. In the course of screening this gene for mutations, heterozygosity for separate 9-bp deletions within the COL1 domain were identified in two kindreds. In both instances, the deletions did not co-segregate with any disease phenotype, suggesting that they were neutral variants. In contrast, similar deletions in triple helical domain of type I collagen are lethal. To study whether alpha3(IX) chains with the deletion will participate in the formation of correctly folded heterotrimeric type IX collagen, we expressed mutant alpha3 chains together with normal alpha1 and alpha2 chains in insect cells. We show here that despite the deletion, mutant alpha3 chains were secreted as heterotrimeric, triple helical molecules consisting of three alpha chains in a 1:1:1 ratio. The results suggest that the next noncollagenous domain (NC2) is capable of correcting the alignment of the alpha chains, and this ensures the formation of an in-register triple helix.

Amino Acid Sequence↗
PubMed1999

An allele of COL9A2 associated with intervertebral disc disease.

Intervertebral disc disease is one of the most common musculoskeletal disorders. A number of environmental and anthropometric risk factors may contribute to it, and recent reports have suggested the importance of genetic factors as well. The COL9A2 gene, which codes for one of the polypeptide chains of collagen IX that is expressed in the intervertebral disc, was screened for sequence variations in individuals with intervertebral disc disease. The analysis identified a putative disease-causing sequence variation that converted a codon for glutamine to one for tryptophan in six out of the 157 individuals but in none of 174 controls. The tryptophan allele cosegregated with the disease phenotype in the four families studied, giving a lod score (logarithm of odds ratio) for linkage of 4.5, and subsequent linkage disequilibrium analysis conditional on linkage gave an additional lod score of 7.1.

Adult↗
PubMed1999

Plastic adherent stromal cells from the bone marrow of commonly used strains of inbred mice: variations in yield, growth, and differentiation.

Bone marrow stroma contains a unique cell population, referred to as marrow stromal cells (MSCs), capable of differentiating along multiple mesenchymal cell lineages. A standard liquid culture system has been developed to isolate MSCs from whole marrow by their adherence to plastic wherein the cells grow as clonal populations derived from a single precursor termed the colony-forming-unit fibroblast (CFU-F). Using this liquid culture system, we demonstrate that the relative abundance of MSCs in the bone marrow of five commonly used inbred strains of mice varies as much as 10-fold, and that the cells also exhibit markedly disparate levels of alkaline phosphatase expression, an early marker of osteoblast differentiation. For each strain examined, the method of isolating MSCs by plastic adherence yields a heterogeneous cell population. These plastic adherent cells also exhibit widely varying growth kinetics between the different strains. Importantly, of three inbred strains commonly used to prepare transgenic mice that we examined, only cells derived from FVB/N marrow readily expand in culture. Further analysis of cultures derived from FVB/N marrow showed that most plastic adherent cells express CD11b and CD45, epitopes of lymphohematopoietic cells. The later consists of both pre-B-cell progenitors, granulocytic and monocytic precursors, and macrophages. However, a subpopulation of the MSCs appear to represent bona fide mesenchymal progenitors, as cells can be induced to differentiate into osteoblasts and adipocytes after exposure to dexamethasone and into myoblasts after exposure to amphotericin B. Our results point to significant strain differences in the properties of MSCs and indicate that standard methods cannot be applied to murine bone marrow to isolate relatively pure populations of MSCs.

Alkaline Phosphatase↗
PubMed1999

Human immunodeficiency virus type 1 Vpr alters bone marrow cell function.

Vpr, a 96 amino acid protein, encoded by the human immunodeficiency virus type I (HIV-1), is important for efficient infection of mononuclear phagocytic cells. These cells are abundant in whole bone marrow, which can easily be cultured in vitro to support hematopoiesis. Our experiments indicate that Vpr plays a role in the potent activation of murine and human mononuclear phagocytic cells within a hematopoietic microenvironment. In murine cultures, avid erythrophagocytosis is triggered by transduction of marrow cells with supernatant derived from PA317 cells transfected with a murine retroviral delivery vector bearing a Vpr expression cassette. Supernatants derived from cells transfected with the same vector carrying sequences for the expression of other relevant viral and nonviral proteins do not induce erythrophagocytosis to any marked degree. The effect on human marrow cells is similar, where treatment promotes adhesion of mononuclear phagocytic cells to culture plates in association with other nucleated and nonnucleated cells that undergo subsequent engulfment. The differential effects of Vpr point and deletion mutants in both marrow culture systems fortify the view that the effect is specific to HIV-1 Vpr. Addition of low molar quantities of purified Vpr to marrow cultures is also capable of promoting cell adhesion and phagocytosis, suggesting that extracellular Vpr is the effector of the phenomenon. Accelerated phagocytosis is a hallmark of promonocyte, monocyte, and macrophage activation and its occurrence within a hematopoietic microenvironment may account for critical in vivo pathogenic features of HIV-1 infection. First, activation of mononuclear phagocytes may promote productive viral infection; and second, premature phagocytosis could provide, at least in part, a molecular explanation for the induction of the idiopathic cytopenias that are typical of individuals infected with HIV-1.

Acquired Immunodeficiency Syndrome↗
PubMed1999

Collagen II containing a Cys substitution for Arg-alpha1-519. Analysis by atomic force microscopy demonstrates that mutated monomers alter the topography of the surface of collagen II fibrils.

A recombinant human procollagen II was prepared that contained a substitution of Cys for Arg at alpha1-519 and that was found in five families with early onset generalized osteoarthritis with or without features of a mild chondrodysplasia. Previously, the presence of mutated monomers in mixtures with wildtype collagen II was shown to increase the lag period for fibril assembly. Also, the fibrils were more loosely packed and some thick fibrils lacked a D-periodic banding pattern. Here we re-examined the fibrils using a combination of transmission electron microscopy and atomic force microscopy. The presence of the mutated monomers increased the diameter of the thin filaments that were consistently formed in association with the thick fibrils of collagen II. In addition, the presence of the mutated monomers increased the depth of the gap regions in all fibrils with a distinct D-periodic banding pattern. The results, therefore, may indicate that the mutated monomers formed two or three additional outer layers of monomers in 0D-period staggers on the surface of the fibrils. Apparently, the mutated monomers were bound on the surface through intermolecular disulfide bonds.

Amino Acid Substitution↗
PubMed1999

Transplantability and therapeutic effects of bone marrow-derived mesenchymal cells in children with osteogenesis imperfecta.

In principle, transplantation of mesenchymal progenitor cells would attenuate or possibly correct genetic disorders of bone, cartilage and muscle, but clinical support for this concept is lacking. Here we describe the initial results of allogeneic bone marrow transplantation in three children with osteogenesis imperfecta, a genetic disorder in which osteoblasts produce defective type I collagen, leading to osteopenia, multiple fractures, severe bony deformities and considerably shortened stature. Three months after osteoblast engraftment (1.5-2.0% donor cells), representative specimens of trabecular bone showed histologic changes indicative of new dense bone formation. All patients had increases in total body bone mineral content ranging from 21 to 29 grams (median, 28), compared with predicted values of 0 to 4 grams (median, 0) for healthy children with similar changes in weight. These improvements were associated with increases in growth velocity and reduced frequencies of bone fracture. Thus, allogeneic bone marrow transplantation can lead to engraftment of functional mesenchymal progenitor cells, indicating the feasibility of this strategy in the treatment of osteogenesis imperfecta and perhaps other mesenchymal stem cell disorders as well.

Bone Density↗
PubMed1999

Mutations in COL11A2 cause non-syndromic hearing loss (DFNA13).

We report that mutation of COL11A2 causes deafness previously mapped to the DFNA13 locus on chromosome 6p. We found two families (one American and one Dutch) with autosomal dominant, non-syndromic hearing loss to have mutations in COL11A2 that are predicted to affect the triple-helix domain of the collagen protein. In both families, deafness is non-progressive and predominantly affects middle frequencies. Mice with a targeted disruption of Col11a2 also were shown to have hearing loss. Electron microscopy of the tectorial membrane of these mice revealed loss of organization of the collagen fibrils. Our findings revealed a unique ultrastructural malformation of inner-ear architecture associated with non-syndromic hearing loss, and suggest that tectorial membrane abnormalities may be one aetiology of sensorineural hearing loss primarily affecting the mid-frequencies.

Amino Acid Sequence↗
PubMed1999

Propagation and senescence of human marrow stromal cells in culture: a simple colony-forming assay identifies samples with the greatest potential to propagate and differentiate.

Marrow stromal cells (MSCs) were isolated from bone marrow obtained by aspirates of the iliac crest of normal volunteers. The cells were isolated by their adherence to plastic and then passed in culture. Some of the samples expanded through over 15 cell doublings from the time frozen stocks were prepared. Others ceased replicating after about four cell doublings. The replicative potential of the cells in culture was best predicted by a simple colony-forming assay in which samples from early passages were plated at low densities of about 10 cells per cm2. Samples with high colony-forming efficiency exhibited the greatest replicative potential. The colonies obtained by plating early passage cells at low density varied in size and morphology. The large colonies readily differentiated into osteoblasts and adipocytes when incubated in the appropriate medium. As samples were expanded in culture and approached senescence, they retained their ability to differentiate into osteoblasts. However, the cells failed to differentiate into adipocytes. The loss of multipotentiality following serial passage in culture may have important implications for the use of expanded MSCs for cell and gene therapy.

Bone Marrow Cells↗