Response to Frederic Bretzner et al. “Target Populations for First-In-Human Embryonic Stem Cell Research in Spinal Cord Injury”

We address concerns raised in this issue by Bretzner et al. (2011) by explaining the rationale for including subjects with subacute, neurologically complete spinal cord injuries in the Phase 1 trial of GRNOPC1. We also present elements of the informed consent process that minimize the likelihood of therapeutic misconception

MPSS profiling of human embryonic stem cells

BACKGROUND: Pooled human embryonic stem cells (hESC) cell lines were profiled to obtain a comprehensive list of genes common to undifferentiated human embryonic stem cells. RESULTS: Pooled hESC lines were profiled to obtain a comprehensive list of genes common to human ES cells. Massively parallel signature sequencing (MPSS) of approximately three million signature tags (signatures) identified close to eleven thousand unique transcripts, of which approximately 25% were uncharacterised or novel genes. Expression of previously identified ES cell markers was confirmed and multiple genes not known to be expressed by ES cells were identified by comparing with public SAGE databases, EST libraries and parallel analysis by microarray and RT-PCR. Chromosomal mapping of expressed genes failed to identify major hotspots and confirmed expression of genes that map to the X and Y chromosome. Comparison with published data sets confirmed the validity of the analysis and the depth and power of MPSS. CONCLUSIONS: Overall, our analysis provides a molecular signature of genes expressed by undifferentiated ES cells that can be used to monitor the state of ES cells isolated by different laboratories using independent methods and maintained under differing culture condition

Ten-year safety of pluripotent stem cell transplantation in acute thoracic spinal cord injury.

OBJECTIVE: The purpose of this study was to evaluate the safety of oligodendrocyte progenitor cells (LCTOPC1) derived from human pluripotent stem cells administered between 7 and 14 days postinjury to patients with T3 to T11 neurologically complete spinal cord injury (SCI). The rationale for this first-in-human trial was based on evidence that administration of LCTOPC1 supports survival and potential repair of key cellular components and architecture at the SCI site. METHODS: This study was a multisite, open-label, single-arm interventional clinical trial. Participants (n = 5) received a single intraparenchymal injection of 2 × 106 LCTOPC1 caudal to the epicenter of injury using a syringe positioning device. Immunosuppression with tacrolimus was administered for a total of 60 days. Participants were followed with annual in-person examinations and MRI for 5 years at the time of this report and will be followed with annual telephone questionnaires for 6 to 15 years postinjection. The primary endpoint was safety, as measured by the frequency and severity of adverse events related to the LCTOPC1 injection, the injection procedure, and/or the concomitant immunosuppression administered. The secondary endpoint was neurological function as measured by sensory scores and lower-extremity motor scores as measured by the International Standards for Neurological Classification of Spinal Cord Injury examinations. RESULTS: No unanticipated serious adverse events related to LCTOPC1 have been reported with 98% follow-up of participants (49 of 50 annual visits) through the first 10 years of the clinical trial. There was no evidence of neurological decline, enlarging masses, further spinal cord damage, or syrinx formation. MRI results during the long-term follow-up period in patients administered LCTOPC1 cells showed that 80% of patients demonstrated T2 signal changes consistent with the formation of a tissue matrix at the injury site. CONCLUSIONS: This study provides crucial first-in-human safety data supporting the pursuit of future human embryonic stem cell-derived therapies. While we cannot exclude the possibility of future adverse events, the experience in this trial provides evidence that this cell type can be well tolerated by patients, with an event-free period of up to 10 years. Based on the safety profile of LCTOPC1 obtained in this study, a cervical dose escalation trial was initiated (NCT02302157)

Evidence for two levels of DNA folding in histone-depleted HeLa interphase nuclei

The long-range order of the DNA in interphase nuclei was investigated by sedimentation studies. From these studies, two discreet sedimentation forms have been characterized for histone-depleted HeLa nuclei. Type I structures, obtained by extraction of isolated nuclei with a buffer containing 2 m-NaCl, have an s value of 18,000 S. Histone-depletion with the polyanions dextran sulfate/heparin produces slightly slower type I structures of 15,000 S. When the thiols, β-mercaptoethanol or dithiothreitol, or the metal chelators, 1,10-phenanthroline or neocuproine, are included during histone extraction, the interphase DNA is further unfolded, generating type II structures of 8500 S. Both forms are destroyed by proteolytic agents, but are insensitive to RNAase A. Fluorescence studies of type I and type II structures show that both retain the spherical shape of nuclei, with type II structures having a more expanded DNA halo than that of type I. It is suggested that metalloprotein interactions are important in one level of the DNA folding of type I structures, since the chelators 1,10-phenanthroline and neocuproine bring about formation of type II structures. Metal-depleted nuclei, prepared in buffers containing β-mercaptoethanol, provide a means for identifying the metal involved. Such nuclei generate type II structures upon histone-depletion. Addition of as little as 1 × 10−6m-CuSO4 or 1 × 10−6m-CaCl2 to metal-depleted nuclei restores the capacity to generate type I structures. The restoration of the type I complex is reversible following exposure of nuclei to CuSO4, but is irreversible after CaCl2 treatment. These experiments suggest that one level of DNA compaction in histone-depleted nuclei is stabilized by either Cu or Ca.</p

Non-histone proteins and long-range organization of HeLa interphase DNA

Two forms of histone-depleted HeLa nuclei have been characterized (Lebkowski &amp; Laemmli, 1982). In the studies presented here, the proteins associated with both structures are identified. Type I structures, which are obtained by extraction of isolated nuclei with a buffer containing 2 m-NaCl or dextran sulfate/heparin, retain 10 to 15% of the total nuclear proteins. These proteins are represented by the three nuclear lamina proteins of 60,000 to 70,000 Mr and numerous high molecular weight species. Histone-extraction in the presence of β-mercaptoethanol leads to more expanded type II structures. Accompanying this further DNA unfolding, is a selective loss of certain proteins that are associated with type I structures. Present in type II structures are 3 to 5% of the total nuclear proteins, almost exclusively represented by the three nuclear lamina proteins and two minor proteins of 64,000 and 200,000 Mr. The proteins of both type I and type II histone-depleted nuclei are comparable to those that remain as residual nuclear structures after histone-depletion of nuclease-digested nuclei. These residual protein structures are termed type I and type II nuclear scaffolds. The selective removal of proteins by β-mercaptoethanol is pH-dependent and maximal at alkaline pH values. It is further demonstrated that type I structures are stabilized by CaCl2. Nuclei incubated with CaCl2 are resistant to extraction with β-mercaptoethanol. In addition, evidence is presented that confirms that β-mercaptoethanol acts to disrupt metalloprotein interactions important for the stabilization of type I structures. The metal chelator, 1,10-phenanthroline, causes solubilization of the same set of proteins as β-mercaptoethanol. DNA binding studies are presented which show that 12 proteins of type I and four proteins of type II scaffolds bind DNA in vitro. The three nuclear lamina proteins are the major DNA binding proteins of both type I and type II scaffolds.</p

Interphase Nuclear Matrix and Metaphase Scaffolding Structures

The protein compositions of purified metaphase chromosomes, nuclei and their residual scaffold and matrix structures, are reported. The protein pattern of nuclei on sodium dodecyl sulphate/polyacrylamide gels is considerably more complex and rich in non-histone proteins than that of chromosomes. Nuclei contain about three to four times more non-histone proteins relative to their histones than chromosomes. Besides the protein components of the peripheral lamina, several protein bands are specific or at least highly enriched in nuclei. Conversely, two proteins X0 (33×10³ Mr) and X1 (37×10³ Mr) are highly enriched in the pattern of metaphase chromosomes. We have compared morphologically the previously defined nuclear matrices type I and II. The type I nuclear matrix is composed of the known lamina proteins, which form the peripheral lamina structure, and a complex series of proteins that form the internal network of the matrix as observed by electron microscopy. This internal network is stabilized similarly to the metaphase scaffolding by metalloprotein interaction. Both the scaffolding and the internal network of the matrix dissociate if thiols or certain metal chelators are used in the extraction buffer. Under these conditions the resulting nuclear structure, called matrix type II, appears empty in the electron microscope, with the exception of some residual nucleolar material. This latter material can be extracted from the internal network by exhaustive treatment of the nuclei with RNase before extraction with high salt. Immunoblotting and activity studies show RNA polymerase II to be tightly bound to the type I, but not to the type II matrix, or to the scaffolding structure. No polymerase II enzyme was detected in isolated metaphase chromosomes. Another nuclear enzyme, poly(ADP-ribose) polymerase is not bound to either of the residual nuclear matrices or to the scaffolding structures. The association of RNA polymerase with the internal network of the nuclear matrix is consistent with the idea that transcription occurs in close association with this structure

Immortalized fibroblast-like cells derived from human embryonic stem cells support undifferentiated cell growth

Human embryonic stem cells (hESCs) have the potential to generate multiple cell types and hold promise for future therapeutic applications. Although undifferentiated hESCs can proliferate indefinitely, hESC derivatives significantly downregulate telomerase and have limited replication potential. In this study we examine whether the replicative lifespan of hESC derivatives can be extended by ectopic expression of human telomerase reverse transcriptase (hTERT), the catalytic component of the telomerase complex. To this end, we have derived HEF1 cells, a fibroblast-like cell type, differentiated from hESCs. Infection of HEF1 cells with a retrovirus expressing hTERT extends their replicative capacity, resulting in immortal human HEF1-hTERT cells. HEF1-hTERT cells can be used to produce conditioned medium (CM) capable of supporting hESC growth under feeder-free conditions. Cultures maintained in HEF1-CM show characteristics similar to mouse embryonic fibroblast CM control cultures, including morphology, surface marker and transcription factor expression, telomerase activity, differentiation, and karyotypic stability. In addition, HEF1-hTERT cells have the capacity to differentiate into cells of the osteogenic lineage. These results suggest that immortalized cell lines can be generated from hESCs and that cells derived from hESCs can be used to support their own growth, creating a genotypically homogeneous system for the culture of hESCs