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Background: The safety and efficacy of subretinally transplanted human central nervous system stem cells (HuCNS-SCR) in subjects with geographic atrophy (GA). Methods: Twenty-two eyes of 11 subjects with bilateral GA due to age-related macular degeneration (AMD) were enrolled in this IRB-approved, multicenter, prospective Phase I/II open-label clinical trial. For each subject, the eye with the worst best-corrected visual acuity was selected for treatment and considered the study eye; the fellow eye was treated as a control for this analysis. A total of 0·25 X 106 or 1·0 X 106 HuCNS-SC cells were directly infused into the subretinal space, superotemporal to the fovea near the junctional zone outside the area of GA. Subjects were followed for 12 months and the GA was monitored with fundus autofluorescence (FAF) imaging. Total GA area in both eyes was measured at baseline and month 12 by certified graders and sectoral (clock hour)/directional GA progression rates with respect to the foveal center were computed, and compared between study and control eyes. Findings: Although there was a numerical difference and trend, no statistically significant difference was observed in the overall 12 month enlargement in GA area between study and fellow eyes (1·07 ± 0·84 vs. 2·08 ± 1·97 mm2, P=0·08). However, the month 12 sectoral/directional GA growth rate for the superotemporal region (i.e. toward the location of HuCNS-SC transplantation) showed a significantly slower progression rate in study eyes compared to the same region in fellow eyes (0·06 ± 0·11 vs. 0·21 ± 0·13 mm2, P=0·03). The progression rate in the superotemporal quadrant of the study eye was also significantly slower than in the other three quadrants of the study eye combined (P=0·003). Interpretation: In this Phase I/II trial, HuCNS-SC transplantation was well tolerated and appeared to be associated with slower expansion of the GA lesion in the transplanted quadrant. Trial Registration: NCT01632527. Funding Statement: The clinical study was supported in full by the original trial sponsor, StemCells, Inc. Declaration of Interests: Financial disclosure: Dr. Sadda receives research support from Carl Zeiss, Meditec, Optos, Genentech, and Allergan. He has served as a consultant for Genentech, Allergan, Alcon, Novartis, and Roche. Dr. Birch receives research support from Nightstar, Allergan, AGTC, 4D Molecular Therapeutics, and ProQR Therapeutics. He has served as a consultant for Nightstar, AGTC, Nacuity Pharmaceuticals, Editas Medicine, Acucela, Foundation Fighting Blindness, and ProQR Therapeutics. All other authors declare no conflicts of interest. Ethics Approval Statement: All study procedures were approved by the institutional review boards (IRBs) of the participating clinical centers, and the research adhered to the tenets set forth in the Declaration of Helsinki. Informed consent was obtained from all subjects after explanation of the study procedures and the expected risks and benefits.

Summary The aging population in the U.S. and other developed countries has led to a large increase in the number of patients suffering from degenerative diseases. Transplantation surgery has been a successful therapeutic option for certain patients; however, the availability of suitable donor organs and tissues significantly limits the number of patients who can benefit from this approach. Regenerative medicine has witnessed numerous recent and spectacular advances, making the repair or replacement of dysfunctional organs and tissues an achievable goal. Public-private partnerships and government policies and incentives would further catalyze the development of universally available donor tissues, resulting in broad medical and economic benefits. This article describes a Regenerative Medicine Grand Challenge that the Alliance for Regenerative Medicine recently shared with the White House's Office of Science and Technology Policy in response to a White House call to action in scientific disciplines suggesting that the development of “universal donor tissues” should be designated as a Regenerative Medicine Grand Challenge. Such a designation would raise national awareness of the potential of regenerative medicine to address the unmet needs of many diseases and would stimulate the scientific partnerships and investments in technology needed to expedite this goal. Here we outline key policy changes and technological challenges that must be addressed to achieve the promise of a major breakthrough in the treatment of degenerative disease. A nationalized effort and commitment to develop universal donor tissues could realize this goal within 10 years and along the way result in significant innovation in manufacturing technologies. Significance Regenerative therapies, in which dysfunctional or degenerating cells, tissues, or organs are repaired or replaced, have the potential to cure chronic degenerative diseases. Such treatments are limited by a shortage of donor organs and tissues and the need for immune suppression to prevent rejection. This article proposes a 21st Century Grand Challenge that would address this significant medical need by coordinating a national effort to convene the multidisciplinary expertise needed to manufacture functional and engraftable cells, tissues, or organs that could be made available to any patient without significant risk of rejection—so-called universal donor tissues.

Summary Human neural stem cells (NSCs) offer therapeutic potential for neurodegenerative diseases, such as inherited monogenic nervous system disorders, and neural injuries. Gene editing in NSCs (GE-NSCs) could enhance their therapeutic potential. We show that NSCs are amenable to gene targeting at multiple loci using Cas9 mRNA with synthetic chemically modified guide RNAs along with DNA donor templates. Transplantation of GE-NSC into oligodendrocyte mutant shiverer-immunodeficient mice showed that GE-NSCs migrate and differentiate into astrocytes, neurons, and myelin-producing oligodendrocytes, highlighting the fact that GE-NSCs retain their NSC characteristics of self-renewal and site-specific global migration and differentiation. To show the therapeutic potential of GE-NSCs, we generated GALC lysosomal enzyme overexpressing GE-NSCs that are able to cross-correct GALC enzyme activity through the mannose-6-phosphate receptor pathway. These GE-NSCs have the potential to be an investigational cell and gene therapy for a range of neurodegenerative disorders and injuries of the central nervous system, including lysosomal storage disorders., Graphical Abstract, Highlights • Human neural stem cells are amenable to CRISPR-Cas9-mediated gene targeting • Truncated CD19 surface marker allows for enrichment of gene-targeted NSCs to >90% • Gene-targeted NSCs engraft, migrate, and differentiate in immunodeficient mice • GALC-engineered overexpressing NSCs cross-correct Krabbe disease fibroblasts in vitro, Molecular Biology; Neuroscience; Bioengineering; Biotechnology; Cell Biology; Biological Sciences Research Methodologies

Purpose The loss of retinal pigment epithelial (RPE) cells is a feature common to age-related macular degeneration (AMD) and retinitis pigmentosa (RP) and multiple early phase clinical trials are underway testing the safety of RPE cell replacement for these diseases. We examined whether transplantation of human neural stem cells into the subretinal space could enhance the endogenous proliferative capacity of the host RPE cell to regenerate. Methods Human central nervous system stem cells (HuCNS-SC) were isolated from enzymatically treated brain tissue using flow cytometry. Pigmented dystrophic Royal College of Surgeons (RCS) and S334ter-4 rats treated with oral bromodeoxyuridine (BrdU) received a unilateral subretinal injection of 1.0 × 105 HuCNS-SC cells at either postnatal day 21 or 60. Animals were sacrificed at 90, 120, and 150 days of age. Eyes were fixed processed for cryostat sectioning. Sections were immunostained with Stem101, Ku80, RPE65, OTX1/2, BrdU, and CRALBP antibodies and analyzed via confocal microscopy. Results RCS rats that received transplantation of HuCNS-SC had significantly more (approximately 3-fold) Ki67-positive or BrdU-labelled host RPE cells adjacent to the HuCNS-SC graft than controls. Significantly increased host RPE cell proliferation as a result of HuCNS-SC transplantation also was confirmed in S334ter-line 4 transgenic rats with higher proliferation observed in animals with longer posttransplantation periods. Conclusions These results suggest that controlled proliferation of endogenous RPE by HuCNS-SC may provide another mechanism by which RPE cell diseases could be treated. Translational relevance Engaging the capacity for endogenous RPE cell regeneration in atrophic diseases may be a novel therapeutic strategy for degenerative diseases of the RPE and retina.

Stem cells, which are clonogenic cells with self-renewal and multilineage differentiation properties, have the potential to replace or repair damaged tissue. We have directly isolated clonogenic human central nervous system stem cells (hCNS-SC) from fresh human fetal brain tissue, using antibodies to cell surface markers and fluorescence-activated cell sorting. These hCNS-SC are phenotypically 5F3 (CD133) + , 5E12 + , CD34 − , CD45 − , and CD24 −/lo . Single CD133 + CD34 − CD45 − sorted cells initiated neurosphere cultures, and the progeny of clonogenic cells could differentiate into both neurons and glial cells. Single cells from neurosphere cultures initiated from CD133 + CD34 − CD45 − cells were again replated as single cells and were able to reestablish neurosphere cultures, demonstrating the self-renewal potential of this highly enriched population. Upon transplantation into brains of immunodeficient neonatal mice, the sorted/expanded hCNS-SC showed potent engraftment, proliferation, migration, and neural differentiation.

The DNA synthesis inhibitor hydroxyurea (HU) was administered to determine whether it induces changes in the cell-cycle status of primitive hematopoietic stem cells (HSCs)/progenitors. Administration of HU to mice leads to bone marrow accumulation of c-kit+Thy-1.1loLin−/loSca-1+ (KTLS) cells in S/G2/M phases of the cell cycle. HU is a relatively nontoxic, reversible cell-cycle agent that can lead to approximately a threefold expansion of KTLS cells in vivo and approximately an eightfold increase in the number of KTLS cells in S/G2/M. HSCs in HU-treated mice have undiminished multilineage long-term and short-term clonal reconstitution activity.

Treatment with a combination of cytokines and chemotherapy can effectively stimulate the release of hematopoietic stem cells (HSC) into the peripheral blood (PB), which can then be harvested for transplantation. The cell cycle status of the harvested HSC from mobilized PB (MPB) is of interest because of the impact that cell cycling may have on optimizing the conditions for ex vivo expansion, retrovirus-mediated gene transfer, and the engraftment of transplanted tissues. Therefore, we characterized the cell cycling status of mobilized HSC from mice and humans. The murine HSC, which express the phenotype c-kit+ Thy-1.1lo Lin−/lo Sca-1+, were purified from PB, bone marrow (BM), and spleen after the mice were treated with the mobilizing regimen of granulocyte colony-stimulating factor (G-CSF ) or a combination of cyclophosphamide (CTX) and G-CSF. Human HSC (CD34+ Thy-1+ Lin−) and progenitor cells (CD34+ Thy-1− Lin−) were isolated from the BM of untreated healthy volunteers and from MPB of healthy volunteers and patients treated with G-CSF or a combination of CTX and GM-CSF. Cell cycle status was determined by quantitating the amount of DNA in the purified cells after staining with the dye Hoechst 33342. Fluorescence-activated cell sorting analysis of the progenitor cells from the murine and human samples showed an unexpected finding, ie, virtually none of the cells from the MPB was cycling. The G0/G1 status of HSC from MPB was surprising, because a significant proportion of HSC from BM are actively proliferating and, after mobilization, the HSC in the spleen and BM were also actively cycling.

Human neural stem cell transplants have potential as therapeutic candidates to treat a vast number of disorders of the central nervous system (CNS). StemCells, Inc. has purified human neural stem cells and developed culture conditions for expansion and banking that preserve their unique biological properties. The biological activity of these human central nervous system stem cells (HuCNS-SC®) has been analyzed extensively in vitro and in vivo. When formulated for transplantation, the expanded and cryopreserved banked cells maintain their stem cell phenotype, self-renew and generate mature oligodendrocytes, neurons and astrocytes, cells normally found in the CNS. In this overview, the rationale and supporting data for pursuing neuroprotective strategies and clinical translation in the three components of the CNS (brain, spinal cord and eye) are described. A phase I trial for a rare myelin disorder and phase I/II trial for spinal cord injury are providing intriguing data relevant to the biological properties of neural stem cells, and the early clinical outcomes compel further development.

The ability of an infusion of ex vivo expanded hematopoietic cells to ameliorate cytopenia following transplantation of hematopoietic stem cells (HSCs) is controversial. To address this issue, we measured the recovery of circulating leukocytes, erythrocytes, and platelets in lethally irradiated mice transplanted with 10(3) enriched HSCs, with or without their expanded equivalent (EE) generated after 7 days of culture in interleukin-3 (IL-3), IL-6, granulocyte colony-stimulating factor and Steel Factor. Two HSC populations differing in their content of short-term repopulating progenitors were evaluated. Thy-1loLIN-Sca- 1+ (TLS) bone marrow (BM) is enriched in colony-forming cells (CFCs), day 8 and day 12 spleen colony-forming units (CFU-S) (435 +/- 19, 170 +/- 30, and 740 +/- 70 per 10(3) cells, respectively), and stem cells with competitive long-term repopulating potential (> or = 1 per 43 cells). Thy-1loSca-1+H-2Khl cells (TSHFU) isolated from BM 1 day after treatment of donor mice with 5-fluorouracil (5-FU) are also highly enriched in competitive repopulating units (CRU, > or = 1 per 55 cells), but are depleted of CFCs, day 8 and day 12 CFU-S (171 +/- 8, 0 and 15 +/- 4 per 10(3) cells, respectively). Recipients of 10(3) TLS cells transiently recovered leukocytes to > or = 2,000/microL in 12 days, but sustained engraftment required 25 days. Platelets recovered to > or = 200,000/microL in 15 days, and erythrocytes never decreased below 50% of normal. Mice transplanted with 10(3) TSHFU cells recovered leukocytes in 15 days, and platelets and erythrocytes in 18 days. Recipients of unseparated normal or 5-FU-treated BM cells (containing 10(3) TLS or TSHFU cells) recovered safe levels of blood cells in 9 to 12 days, suggesting that unseparated marrow contains early engrafting cells that were depleted by sorting. Upon ex vivo expansion, total cells, CFCs and day 12 CFU-S were amplified 2,062-,83- and 13-fold, respectively, from TLS cells; and 1,279-, 259- and 708-fold, respectively, from TSHFU cells. Expanded cells could regenerate the majority of lymphocytes and granulocytes in primary (17 weeks) and secondary (26 weeks) hosts and were only moderately impaired compared to fresh HSCs. The EE of TSHFU cells was more potent than that of TLS cells, suggesting that more highly enriched HSCs are more desirable starting populations for this application. When mice were transplanted with 10(3) TSHFU cells and their EE, the duration of thrombocytopenia was shortened from 18 to 12 days, and anemia was abolished. Leukocytes were also elevated on days 9 to 12, although sustained recovery was not accelerated. Anemia was also abrogated in recipients of 10(3) TLS cells and their EE. Early platelet counts were slightly higher than with TLS cells alone, but leukocyte recovery was not improved. These data confirm that TLS cells contribute to early and sustained hematopoiesis, and demonstrate a benefit of ex vivo expanded cells in accelerating engraftment of more primitive TSHFU stem cells depleted of progenitors.

Human bone marrow (BM) CD34+ cells were stained with the vital dye, rhodamine 123 (Rh123), and analyzed for their biological properties based on the level of dye retention. Heterogeneous rhodamine staining is seen within the CD34+ population, and the staining patterns differ dramatically between fetal BM (FBM), adult BM (ABM) and mobilized peripheral blood (MPB). Kinetic analysis of the efflux of Rh123 from ABM CD34+ cells showed that efflux of Rh123 was most rapid from the most primitive Thy-1+ subset. The efflux of Rh123 could be inhibited by verapamil, suggesting that rhodamine efflux from primitive hematopoietic cells is primarily due to the P-glycoprotein (P-gp) pump or another intracellular transport system affected by verapamil. When four CD34+ subpopulations were plated onto SyS1 BM stromal cell cocultures after 1 to 2 weeks, only wells plated with CD34+ Thy- 1+Rh123lo (low-level Rh123 retention) or CD34+Thy-1+Rh123mid (mid-level Rh123 retention) cells maintained greater than 50% of cells in an uncommitted CD34+33- stage. CD34+Lin- (lineage-negative) cells were fractionated based on Rh123 dye staining into Rh123hi (high-level Rh123 retention), Rh123mid, and Rh123lo and deposited as single cells into long-term SyS1 BM stromal cell cultures. The Rh123mid fraction had immense early proliferative activity in vitro, but lost the ability to form cobblestone areas after 5 to 6 weeks in culture. In contrast, the Rh123lo fraction proliferated more slowly but sustained long-term in vitro hematopoiesis as evidenced by continued cobblestone area-forming cells (CAFC) activity for at least 6 weeks. The Rh123hi fraction showed a plating efficiency similar to that of the Rh123lo or Rh1123mid fractions but did not extensively proliferative in vitro and did not show evidence of CAFC activity. We predicted from these in vitro results that the Rh123lo subsets possesses long-term engrafting potential. Indeed, on transplantation into the SCID-hu bone assay, all long-term engrafting potential and multilineage differentiation potential resided within the Rh123lo-mid but not Rh123hi subset. Furthermore, human marrow subpopulations derived from chimeric sheep after in utero transplantation with CD34+Thy-1+Lin- cells were reisolated based on Rh123 staining. Again, CD34+Lin- subsets showing Rh123lo-mid had long-term growth in culture, whereas Rh123hiCD34+Lin- cells did not. These results show that, after injection of CD34+Thy- 1+Lin- cells into an in utero microenvironment, primitive CD34+ cells maintain a Rh123 phenotype that correlates with their in vitro CAFC activity. Thus, Rh123 staining is an effective way to define functional subsets of primitive hematopoietic cell populations.

Attempts to maintain or expand primitive hematopoietic stem cells in vitro without the concomitant loss of their differentiative and proliferative potential in vivo have largely been unsuccessful. To investigate this problem, we compared the ability of three cloned bone marrow (BM) stromal cell lines to support the growth of primitive Thy- 1lo Sca-1+H-2Khi cells isolated by fluorescence-activated cell sorting from the BM of Ly-5.2 mice treated 1 day previously with 5-fluo- rouracil. Sorted cells were highly enriched in cobblestone area-forming cells (CAFC), but their frequency was dependent on the stromal cell lines used in this assay (1 per 45 cells on SyS-1; 1 per 97 cells on PA6). In the presence of recombinant leukemia inhibitory factor (LIF), CAFC cloning efficiency was increased to 1 per 8 cells on SyS-1 and 1 per 11 cells on PA6, thus showing the high clonogenicity of this primitive stem cell population. More primitive stem cells with competitive repopulating potential were measured by injecting the sorted cells into lethally irradiated Ly-5.1 mice together with 10(5) radioprotective Ly-5.1 BM cells whose long-term repopulating ability has been “compromised” by two previous cycles of marrow transplantation and regeneration. Donor-derived lymphocytes and granulocytes were detected in 66% of animals injected with 50 sorted cells. To quantitate the maintenance of competitive repopulating units (CRU) by stromal cells, sorted cells were transplanted at limiting dilution before and after being cultured for 2 weeks on adherent layers of SyS-1, PA6, or S17 cells. CRU represented 1 per 55 freshly sorted cells. CRU could be recovered from cocultures supported by all three stromal cell lines, but their numbers were approximately-sevenfold less than on day 0. In contrast, the addition of LIF to stromal cultures improved CRU survival by 2.5-fold on S17 and PA6 cells (approximately two-fold to threefold decline), and enabled their maintenance on SyS-1. LIF appeared to act indirectly, because alone it did not support the proliferation of Thy- 1lo Sca-1+H-2Khi cells in stroma-free cultures. Polymerase chain reaction (RT-PCR) analysis revealed that Interleukin-1beta (IL-1 beta) IL-2, IL-6, granulocyte-colony stimulating factor, granulocyte macrophage-colony stimulating factor, transforming growth factors, LIF, and Steel Factor (SLF) mRNAs were upregulated in SyS-1 within 1 to 6 hours of LIF-stimulation. To determine if increased expression of SLF by LIF-stimulated SyS-1 cells could account for their capacity to support stem cells, sorted calls were cocultured on simian CV-E cells that were transfected with an expression vector encoding membrane-bound SLF, or supplemented with soluble SLF. In both cases, SLF synergized with IL-6 produced endogenously by CV-E cells enabling CAFC growth equivalent to that on LIF-stimulated SyS-1. CAFC development on LIF- stimulated SyS-1 could also be completely abrogated by an anti-SLF antibody. These data provide evidence for a role of LIF in the support of long-term repopulating stem cells by indirectly promoting cytokine expression by BM stroma. Furthermore, we have used quantitative assays to show a maintenance of CRU numbers, with retention of in vivo function following ex vivo culture.

CD43 (the major sialoglycoprotein of leukocytes) is an adhesion molecule broadly expressed on hematopoietic cells. A monoclonal antibody recognizing this molecule induces apoptosis of lineage marker- negative bone marrow hematopoietic progenitor cells (HPCs) that express CD34 at a high density (CD34hiLIN-). However, not all cells within this population undergo apoptosis on stimulation via CD43. Dividing progenitor cells are most highly affected, whereas more primitive quiescent cells survive anti-CD43 monoclonal antibody treatment. These surviving cells (1) are enriched for cobblestone area-forming cells, (2) repopulate fragments for human fetal bone implanted into C.B-17 scid/scid mice, (3) have a potential to differentiate in vivo to myeloid and lymphoid cells, and (4) have a high proliferative potential in long-term stromal cell-free liquid culture. These data indicate that cells with hematopoietic stem cell characteristics are relatively resistant to CD43-mediated apoptosis as compared with HPCs. Thus, CD43 may be specifically involved in the regulation of HPC proliferation.

Over the last ten years there has been increasing use of mobilized peripheral blood (MPB) progenitor cells as grafts for autologous transplantation. Among the cells comprising these MPB autografts is a subpopulation of CD34+Thy-1+Lineage (Lin)-cells, which is enriched for hematopoietic stem cell (HSC) activity. The percentage of CD34+ cells which express Thy-1 is higher in some samples of MPB than in bone marrow (BM). Using myeloid and erythroid cell depletion prior to high speed cell sorting, it is possible to purify sufficient numbers of CD34+Thy-1 +Lin-HSCs from a MPB leuka-pheresis sample for use as an autograft. CD34+Thy-1+Lin-cells will potentially provide a tumor-depleted autograft for cancer patients. This HSC population is also being studied as a potential target for gene transfer for the treatment of patients with HIV, cancer and a variety of genetic disorders.

A high-speed cell sorter capable of a throughput speed 4-5-fold greater than commercially available systems was developed and evaluated as a processing tool for isolating purified hematopoietic stem cell grafts. The clinical high-speed sorter (CHSS) serves as a single-pass, multiparameter processing tool that provides the means to isolate a highly purified population of cells from starting cell populations with extremely low frequencies. The sorter incorporates environmental barriers to create a sterile environment for cell processing. Monoclonal antibodies and reagents produced under good manufacturing practices (GMP) are used to isolate hematopoietic stem cells by means of the CHSS. Using this technology, the CD34+Thy-1+Lin- hematopoietic stem cell population has been isolated from normal adult bone marrow and mobilized peripheral blood. The sorted cells have been shown to be sterile and viable and to retain hematopoietic function.

Interactions of hematopoietic progenitor cells (HPC) with bone marrow stroma, mediated by adhesion molecules, are assumed to be critically important to the regulation of hematopoiesis. However, the specific roles of individual adhesion molecules involved in these interactions are poorly understood. Here, a monoclonal antibody, MEM-59, recognizing CD43, an adhesion molecule highly expressed on HPC, is shown to induce apoptosis in this cell population. This process operates at the single- cell level, and its initiation requires crosslinking of surface CD43 and the presence of cytokines. In contrast to HPC, more differentiated cells originating from this primitive cell population, as well as peripheral lymphocytes, do not undergo apoptosis in response to the CD43-mediated stimulation. Thus, CD43 may function as a negative regulator of early hematopoietic events, delivering a signal for apoptosis of HPC.

Shiverer-immunodeficient (Shi-id) mice demonstrate defective myelination in the central nervous system (CNS) and significant ataxia by 2 to 3 weeks of life. Expanded, banked human neural stem cells (HuCNS-SCs) were transplanted into three sites in the brains of neonatal or juvenile Shi-id mice, which were asymptomatic or showed advanced hypomyelination, respectively. In both groups of mice, HuCNS-SCs engrafted and underwent preferential differentiation into oligodendrocytes. These oligodendrocytes generated compact myelin with normalized nodal organization, ultrastructure, and axon conduction velocities. Myelination was equivalent in neonatal and juvenile mice by quantitative histopathology and high-field ex vivo magnetic resonance imaging, which, through fractional anisotropy, revealed CNS myelination 5 to 7 weeks after HuCNS-SC transplantation. Transplanted HuCNS-SCs generated functional myelin in the CNS, even in animals with severe symptomatic hypomyelination, suggesting that this strategy may be useful for treating dysmyelinating diseases.

Currently, there are few effective therapies for the treatment of neurodegenerative diseases or injuries to the brain, spinal cord and eye. Human neural stem cell transplants offer the prospect to treat such conditions and represent a potential exciting new medical therapy. A highly purified composition of human neural stem cells has been isolated, expanded and stored as banks of cells, HuCNS-SC®. When transplanted into the brain of immunodeficient rodents, human neural stem cells reside and proliferate in host neurogenic sites, such as the subventricular zone and dentate gyrus of the hippocampus. Their progeny migrate globally throughout the brain and differentiate in a site-appropriate manner into neurons, astrocytes and oligodendrocytes. When transplanted into the spinal cord above and below the injury site, these cells also migrate extensively and differentiate, remyelinate and make synaptic connections with host neurons. HuCNS-SC have also been shown to produce soluble proteins such as housekeeping lysosomal enzymes, neurotrophic factors, and chemokines which may protect damaged host cells and also become mature oligodendrocytes which myelinate dys- or demyelinated host axons. Moreover, these human cells survive long-term in the host brain with no signs of tumor formation or adverse effects and unlike ESC or iPSC, HuCNS-SC do not require pre-differentiation prior to transplant nor do they form teratomas. Therefore, a single transplant of human neural stem cells offers the prospect of a durable clinical benefit. Studies transplanting HuCNS-SC into animal models of human diseases or injury have been performed to assess the cells’ biological properties including their impact on these specific targets. These preclinical efficacy studies have demonstrated protection of host cells and/or improvements in specific functional deficits and provided the foundation for the neuroprotection and neural replacement strategies to support initiation of our clinical studies. Three clinical studies have been initiated to date. The first clinical study in Batten disease, a fatal lysosomal storage disease, has been completed and the surviving patients are now ~4-5 years post-transplant with no safety concerns. A trial in PMD, a fatal myelination disorder, is completing and will examine evidence of de novo myelin formation from transplanted HuCNS-SC. A trial in chronic spinal cord injury has completed dosing of the most severely injured patients and will now enrol those with incomplete thoracic injuries. The Company has recently filed regulatory documents to begin a trial in dry AMD. Preclinical studies in a rat model of retinal degeneration, the RCS rat, have shown cone photoreceptor protection following subretinal transplants of HuCNS-SC. The clinical data derived from these studies should facilitate future clinical testing of HuCNS-SC cells in a broad range of other neurological disorders including Alzheimer’s disease, stroke, and cerebral palsy. In addition to its pioneering efforts in the discovery and development of its proprietary human neural stem cell, the Company’s scientists have also identified a rare cell population from non ideal human adult livers that display the key hallmarks of a therapeutic product for treating liver diseases. Efforts are underway to define optimal expansion conditions to create cell banks of liver-engrafting cells.

The utilization of neural stem cells and their progeny in applications such as disease modelling, drug screening or safety assessment will require the development of robust methods for consistent, high quality uniform cell production. Previously, we described the generation of adherent, homogeneous, non-immortalized mouse and human neural stem cells derived from both brain tissue and pluripotent embryonic stem cells (Conti et al., 2005; Sun et al., 2008). In this study, we report the isolation or derivation of stable neurogenic human NS (hNS) lines from different regions of the 8-9 gestational week fetal human central nervous system (CNS) using new serum-free media formulations including animal component-free conditions. We generated more than 20 adherent hNS lines from whole brain, cortex, lobe, midbrain, hindbrain and spinal cord. We also compared the adherent hNS to some aspects of the human CNS-stem cells grown as neurospheres (hCNS-SCns), which were derived from prospectively isolated CD133(+)CD24(-/lo) cells from 16 to 20 gestational week fetal brain. We found, by RT-PCR and Taqman low-density array, that some of the regionally isolated lines maintained their regional identity along the anteroposterior axis. These NS cells exhibit the signature marker profile of neurogenic radial glia and maintain neurogenic and multipotential differentiation ability after extensive long-term expansion. Similarly, hCNS-SC can be expanded either as neurospheres or in extended adherent monolayer with a morphology and marker expression profile consistent with radial glia NS cells. We demonstrate that these lines can be efficiently genetically modified with standard nucleofection protocols for both protein overexpression and siRNA knockdown of exogenously expressed and endogenous genes exemplified with GFP and Nestin. To investigate the functional maturation of neuronal progeny derived from hNS we (a) performed Agilent whole genome microarray gene expression analysis from cultures undergoing neuronal differentiation for up to 32 days and found increased expression over time for a number of drugable target genes including neurotransmitter receptors and ion channels and (b) conducted a neuropharmacology study utilizing Fura-2 Ca2+ imaging which revealed a clear shift from an initial glial reaction to carbachol to mature neuron-specific responses to glutamate and potassium after prolonged neuronal differentiation. Fully automated culture and scale-up of select hNS was achieved; cells supplied by the robot maintained the molecular profile of multipotent NS cells and performed faithfully in neuronal differentiation experiments. Here, we present validation and utility of a human neural lineage-restricted stem cell-based assay platform, including scale-up and automation, genetic engineering and functional characterization of differentiated progeny. (C) 2011 Elsevier Ltd. All rights reserved.

We have identified a rare (0.05-0.1%) subset of human fetal bone marrow cells that contains multipotent hematopoietic precursors. The population of human precursor cells that express Thy-1 and CD34 but no known lineage markers is enriched for clonogenic activity that establishes long-term, multilineage (myelomonocytic and B lymphoid) cultures on mouse marrow stromal lines. Further, the Thy-1+CD34+ subset that takes up little of the fluorescent mitochondrial dye rhodamine 123 contains virtually all the cells that establish long-term cultures. In human fetal thymus transplanted into SCID (severe combined immunodeficiency) mice, Thy-1+CD34+ fetal bone marrow cells differentiate into T lymphocytes. In two of nine cases, allogeneic Thy-1+CD34+ cells could engraft intact human fetal bone marrow grown in SCID mice, resulting in donor-derived myeloid and B cells. By extrapolation, the rare human Thy-1+Lin-CD34+ cell population contains pluripotent hematopoietic progenitors; we propose that it is highly enriched for candidate hematopoietic stem cells.

SummaryInfantile neuronal ceroid lipofuscinosis (INCL) is a fatal neurodegenerative disease caused by a deficiency in the lysosomal enzyme palmitoyl protein thioesterase-1 (PPT1). Ppt1 knockout mice display hallmarks of INCL and mimic the human pathology: accumulation of lipofuscin, degeneration of CNS neurons, and a shortened life span. Purified non-genetically modified human CNS stem cells, grown as neurospheres (hCNS-SCns), were transplanted into the brains of immunodeficient Ppt1−/− mice where they engrafted robustly, migrated extensively, and produced sufficient levels of PPT1 to alter host neuropathology. Grafted mice displayed reduced autofluorescent lipofuscin, significant neuroprotection of host hippocampal and cortical neurons, and delayed loss of motor coordination. Early intervention with cellular transplants of hCNS-SCns into the brains of INCL patients may supply a continuous and long-lasting source of the missing PPT1 and provide some therapeutic benefit through protection of endogenous neurons. These data provide the experimental basis for human clinical trials with these banked hCNS-SCns.

Direct isolation of human central nervous system stem cells (CNS-SC) based on cell surface markers yields a highly purified stem cell population that can extensively expand in vitro and exhibit multilineage differentiation potential both in vitro and in vivo. The CNS-SC were isolated from fetal brain tissue using the cell surface markers CD133(+), CD34(-), CD45(-), and CD24(-/lo) (CD133(+) cells). Fluorescence-activated cell sorted (FACS) CD133(+) cells continue to expand exponentially as neurospheres while retaining multipotential differentiation capacity for10 passages. CD133(-), CD34(-), and CD45(-) sorted cells (approximately 95% of total fetal brain tissue) fail to initiate neurospheres. Neurosphere cells transplanted into neonatal immunodeficient NOD-SCID mice proliferated, migrated, and differentiated in a site-specific manner. However, it has been difficult to evaluate human cell engraftment, because many of the available monoclonal antibodies against neural cells (beta-tubulin III and glial fibrillary acidic protein) are not species specific. To trace the progeny of human cells after transplantation, CD133(+)-derived neurosphere cells were transduced with lentiviral vectors containing enhanced green fluorescent protein (eGFP) expressed downstream of the phosphoglycerate kinase promoter. After transduction, GFP(+) cells were enriched by FACS, expanded, and transplanted into the lateral ventricular space of neonatal immunodeficient NOD-SCID brain. The progeny of transplanted cells were detected by either GFP fluorescence or antibody against GFP. GFP(+) cells were present in the subventricular zone-rostral migrating stream, olfactory bulb, and hippocampus as well as nonneurogenic sites, such as cerebellum, cerebral cortex, and striatum. Antibody against GFP revealed that some of the cells displayed differentiating dendrites and processes with neurons or glia cells. Thus, marking human CNS-SC with reporter genes introduced by lentiviral vectors is a useful tool with which to characterize migration and differentiation of human cells in this mouse transplantation model.

It seems like bone marrow is the source of many surprises (Figure 5(Figure 5). Transplanted bone marrow cells are observed to give rise to hepatocytes, muscle (Ferrari et al., 1998xMuscle regeneration by bone marrow-derived myogenic progenitors. Ferrari, G., Cusella-De Angelis, G., Coletta, M., Paolucci, E., Stornaiuolo, A., Cossu, G., and Mavilio, F. Science. 1998; 279: 1528–1530Crossref | PubMed | Scopus (2191)See all References(Ferrari et al., 1998), astrocytes (Kopen et al., 1999xMarrow stromal cells migrate throughout forebrain and cerebellum, and they differentiate into astrocytes after injection into neonatal mouse brains. Kopen, G.C., Prockop, D.J., and Phinney, D.G. Proc. Natl. Acad. Sci. USA. 1999; 96: 10711–10716Crossref | PubMed | Scopus (1322)See all References(Kopen et al., 1999), neurons (Brazelton et al. 2000xFrom marrow to brain (expression of neuronal phenotypes in adult mice) . Brazelton, T.R., Rossi, F.M., Keshet, G.I., and Blau, H.M. Science. 2000; 290: 1775–1779Crossref | PubMed | Scopus (1441)See all References, Mezey et al. 2000xTurning blood into brain (cells bearing neuronal antigens generated in vivo from bone marrow) . Mezey, E., Chandross, K.J., Harta, G., Maki, R.A., and McKercher, S.R. Science. 2000; 290: 1779–1782Crossref | PubMed | Scopus (1528)See all References), or primitive mesenchymal cells (Pereira et al., 1995xCultured adherent cells from marrow can serve as long-lasting precursor cells for bone, cartilage, and lung in irradiated mice. Pereira, R.F., Halford, K.W., O'Hara, M.D., Leeper, D.B., Sokolov, B.P., Pollard, M.D., Bagasra, O., and Prockop, D.J. Proc. Natl. Acad. Sci. USA. 1995; 92: 4857–4861Crossref | PubMed | Scopus (737)See all References(Pereira et al., 1995). Subsequent studies have shown that an enriched bone marrow (BM) population that contains HSC based on Hoechst dye exclusion (side population) can differentiate to muscle (Gussoni et al., 1999xDystrophin expression in the mdx mouse restored by stem cell transplantation. Gussoni, E., Soneoka, Y., Strickland, C.D., Buzney, E.A., Khan, M.K., Flint, A.F., Kunkel, L.M., and Mulligan, R.C. Nature. 1999; 401: 390–394Crossref | PubMed | Scopus (1486)See all References(Gussoni et al., 1999). The biological significance of these data could be interpreted by the possibility that some tissue-specific stem cells, perhaps mislabeled as HSC, are multipotent stem cells possessing a broader range of differentiation potential. Alternatively, tissue-specific stem cells may be more plastic than thought and are able to dedifferentiate or be reprogrammed depending on the environment. However, because many different types of stem cells may also exclude Hoechst dye, this population may have contained other types of stem cells as well. A rigorous demonstration of true transdifferentiation or multipotency would require clonal analysis where the progeny of a single stem cell could demonstrably give rise to blood, muscle, and hepatocytes. The HSC → liver experiment was remarkable in that the isolated KTLS cells all expressed CD45 (Lagasse et al., 2000xPurified hematopoietic stem cells can differentiate into hepatocytes in vivo. Lagasse, E., Connors, H., Al-Dhalimy, M., Reitsma, M., Dohse, M., Osborne, L., Wang, X., Finegold, M., Weissman, I.L., and Grompe, M. Nat. Med. 2000; 6: 1229–1234Crossref | PubMed | Scopus (1944)See all References(Lagasse et al., 2000), a molecule thought to be limited to hematopoietic cells and whose expression was considered to be the hallmark of hematopoietic commitment. The bone marrow side population described above (Gussoni et al., 1999xDystrophin expression in the mdx mouse restored by stem cell transplantation. Gussoni, E., Soneoka, Y., Strickland, C.D., Buzney, E.A., Khan, M.K., Flint, A.F., Kunkel, L.M., and Mulligan, R.C. Nature. 1999; 401: 390–394Crossref | PubMed | Scopus (1486)See all References(Gussoni et al., 1999) was not tested for CD45 expression.Figure 5Model for Stem Cell DifferentiationThe totipotent stem cell gives rise to the pluripotent embryonic stem cells (ES cells), which give rise to both the germline and perhaps putative somatic stem cells. The somatic stem cells are multipotent stem cells, and tissue-restricted stem cells may derive from multipotent stem cells. Hematopoietic stem cells (HSC), muscle stem cells (Muscle-SC), and neural stem cells (CNS-SC) may be multipotent stem cells or tissue-restricted stem cells that dedifferentiate into multipotent stem cells. Only prospectively isolated HSC have been shown to give rise to both blood and liver (hepatocytes)View Large Image | View Hi-Res Image | Download PowerPoint SlideCultured cells found in skeletal muscle, or muscle cells isolated as the Hoechst side population, when transplanted into lethally irradiated mice gave rise to all major adult blood lineages (Jackson et al., 1999xHematopoietic potential of stem cells isolated from murine skeletal muscle. Jackson, K.A., Mi, T., and Goodell, M.A. Proc. Natl. Acad. Sci. USA. 1999; 96: 14482–14486Crossref | PubMed | Scopus (832)See all References(Jackson et al., 1999). Because the identity of this progenitor has not yet been determined by tissue-specific markers, it might be a muscle stem cell, a multipotent stem cell, or an itinerant HSC. In other studies, we estimate that the flux of HSC from BM → blood → elsewhere probably approaches 105 HSC/day in mice; therefore, itinerant HSC are available (D. E. Wright, unpublished data). Thus, the contamination by hematopoietic progenitors cannot be excluded.In another set of experiments, neurospheres containing neural stem cells have apparently shown extraordinary plasticity. Neurosphere cells have been observed to differentiate into blood cells (Bjornson et al., 1999xTurning brain into blood (a hematopoietic fate adopted by adult neural stem cells in vivo) . Bjornson, C.R., Rietze, R.L., Reynolds, B.A., Magli, M.C., and Vescovi, A.L. Science. 1999; 283: 534–537Crossref | PubMed | Scopus (1198)See all References(Bjornson et al., 1999), into all three germ layers in early chick embyos or mouse blastocyts (Clarke et al., 2000xGeneralized potential of adult neural stem cells. Clarke, D.L., Johansson, C.B., Wilbertz, J., Veress, B., Nilsson, E., Karlstrom, H., Lendahl, U., and Frisen, J. Science. 2000; 288: 1660–1663Crossref | PubMed | Scopus (801)See all References(Clarke et al., 2000), or into muscle cells (Galli et al., 2000xSkeletal myogenic potential of human and mouse neural stem cells. Galli, R., Borello, U., Gritti, A., Minasi, M.G., Bjornson, C., Coletta, M., Mora, M., De Angelis, M.G., Fiocco, R., Cossu, G. et al. Nat. Neurosci. 2000; 3: 986–991Crossref | PubMed | Scopus (377)See all References(Galli et al., 2000). Although these experiments are preliminary and have to be reproduced by other laboratories, it will be interesting to determine if the repopulating cells are committed neural stem cells, or are multipotent stem cells. It is interesting to note that neurospheres are generated from fetal tissues, not adult tissues, and that no experiments have yet been reported on the lineage plasticity of freshly isolated neural stem cells. It is conceivable that neural stem cells acquired their multipotent potential through in vitro culture.So with many tissue-specific stem cells giving rise to developmentally unrelated lineage-specific cells (Figure 5(Figure 5), we might have to rethink the notion of germ layer origins of some of the tissues. Blood and liver are currently believed (and taught) to be derived from two distinct germ layers, mesoderm and endoderm, respectively. Yet the only direct, clonal precursor shown to give rise to hepatocytes in an adult regeneration model is HSC. Can we exclude an HSC origin of liver during embryogenesis, perhaps dependent on endodermal buds for their transdifferentiation? And do we have to redefine the notion of tissue-specific stem cells? We need to identify each tissue-specific stem cell and at the clonal level determine the full developmental potential of these cells. But even if the extraordinary plasticity of tissue stem cells does not reflect the in vivo potential these cells exercise during normal development, it will still be scientifically important to study and understand this phenomenon, and, of course clinically it might expand the potential for new cell-based therapies.Strom Chowdhury Fox 1999xHepatocyte transplantation for the treatment of human disease. Strom, S.C., Chowdhury, J.R., and Fox, I.J. Semin. Liver Dis. 1999; 19: 39–48Crossref | PubMedSee all ReferencesStrom Chowdhury Fox 1999

In humans, autologous transplants derived from bone marrow (BM) usually engraft more slowly than transplants derived from mobilized peripheral blood. Allogeneic BM transplants show a further delay in engraftment and have an apparent requirement for donor T cells to facilitate engraftment. In mice, Thy-1.1(lo)Lin-/loSca-1+ hematopoietic stem cells (HSCs) are the principal population in BM which is responsible for engraftment in syngeneic hosts at radioprotective doses, and higher doses of HSCs can radioprotect an allogeneic host in the absence of donor T cells. Using the mouse as a preclinical model, we wished to test to what extent engraftment kinetics was a function of HSC content, and whether at high doses of c-Kit+Thy-1.1(lo)Lin-/loSca-1+ (KTLS) cells rapid allogeneic engraftment could also be achieved. Here we demonstrate that engraftment kinetics varied greatly over the range of KTLS doses tested (100-10,000 cells), with the most rapid engraftment being obtained with a dose of 5,000 or more syngeneic cells. Mobilized splenic KTLS cells and the rhodamine 123(lo) subset of KTLS cells were also able to engraft rapidly. Higher doses of allogeneic cells were needed to produce equivalent engraftment kinetics. This suggests that in mice even fully allogeneic barriers can be traversed with high doses of HSCs, and that in humans it may be possible to obtain rapid engraftment in an allogeneic context with clinically achievable doses of purified HSCs.

Two mutants of the green fluorescent protein (GFP), RSGFP4 and GFPS65T, have been recently created which differ from the wildtype GFP of A. victoria in their excitation maxima. Here we show that human fibroblasts transfected with either of the two mutant GFP genes emit a green fluorescence that is 18-fold brighter than the cells transfected with the wildtype GFP gene. Retroviral vectors expressing the improved GFP gene were also constructed to determine their suitability for stable gene transduction into mammalian cells. The inclusion of the RSGFP4 gene in a retroviral vector did not reduce the viral titer and resulted in a fluorescent signal in viable transduced cells detectable by both fluorescence microscopy and fluorescence-activated cell sorter (FACS) analysis. Therefore, the improved mutant GFP provides a vital marker for monitoring gene transfer and expression in mammalian cells.

The search for human hematopoietic stem cells has been hampered by the lack of appropriate assay systems. Demonstration of the ability of precursor cell candidates to give rise to T cells is of significant difficulty since dissociated in vitro cultured thymus stroma cells lose their ability to sustain thymocyte maturation. To define further the differentiative capacities of the rare human fetal liver and bone marrow cells that express the CD34 surface antigen and exhibit in vitro myeloid and pre-B cell activities, we have microinjected them into HLA-mismatched fetal thymus fragments, partially depleted of hematopoietic cells by low temperature culture. In vitro colonized thymuses have then been allowed to develop upon engraftment into immunodeficient SCID mice. Using this modification of the SCID-hu system, we show that low numbers of fetal CD34+ progenitor cells can repopulate the lymphoid compartment in the human thymus.

The frequency of normal rat peripheral B lymphocytes stained for surface immunoglobulin kappa allotypes a and b in (a X b) F1 heterozygotes was assessed by two-colour immunofluorescence on a fluorescence-activated cell sorter. The upper limit to the frequency of double- stainers was 8% among all kappa-positive cells, though it was not resolved how far cytophilic antibody accounted for these. Cells expressing each allotype singly were isolated and the extent of rearrangement of the genes encoding the joining-kappa segment on the expressed and non-expressed chromosome were independently assessed. The expressed allele was found to be virtually completely rearranged while the non-expressed allele showed approximately 45-60% rearrangement. The implications of this substantial non-productive rearrangement for models of allelic exclusion are discussed.

Experimentation on human stem cells is hampered by the relative paucity of this population and by the lack of assays identifying multilineage differentiation, particularly along the lymphoid lineages. In our current study, phenotypic analysis of low-density fetal bone marrow cells showed two distinct populations of CD34+ cells: those expressing a high density of CD34 antigen on their surface (CD34hi) and those expressing an intermediate level of CD34 antigen (CD34lo). Multiple tissues were used to characterize the in vitro and in vivo potential of these subsets and showed that only CD34hi cells support long-term B lymphopoiesis and myelopoiesis in vitro and mediate T, B, and myeloid repopulation of human tissues implanted into SCID mice. CD34lo cells repeatedly failed to provide long-term hematopoietic activity in vivo or in vitro. These results indicate that a simple fractionation based on well-defined CD34 antigen levels can be used to reproducibly isolate cells highly enriched for in vivo long-term repopulating activity and for multipotent progenitors, including T- and B-cell precursors. Additionally, given the limited variability in the results and the high correlation between in vitro and in vivo hematopoietic potential, we propose that the CD34hi population contains virtually all of the stem cell activity in fetal bone marrow and therefore is the population of choice for future studies in hematopoietic stem cell development and gene therapy.

The adhesion molecule BEN/SC1/DM-GRASP (BEN) is a marker in the developing chicken nervous system that is also expressed on the surface of embryonic and adult hematopoietic cells such as immature thymocytes, myeloid progenitors, and erythroid progenitors. F84.1 and KG-CAM, two monoclonal antibodies to rat neuronal glycoproteins with similarity to BEN, cross-react with an antigen on rat hematopoietic progenitors, but F84.1 only also recognizes human blood cell progenitors. We have defined the antigen recognized by F84.1 as the hematopoietic cell antigen (HCA). HCA expression was detected on 40% to 70% of CD34+ fetal and adult bone marrow cells and mobilized peripheral blood cells. Precursor cell activity for long-term in vitro bone marrow cell culture was confined to the subset of CD34+ cells that coexpress HCA. HCA is expressed by the most primitive subsets of CD34+ cells, including all rhodamine 123lo, Thy-1+, and CD38−/lo CD34+ adult bone marrow cells. HCA was also detected on myeloid progenitors but not on early B-cell progenitors. We also describe here the cloning and characterization of cDNAs encoding two variants of the human HCA antigen (huHCA-1 and huHCA-2) and of a cDNA clone encoding rat HCA (raHCA). The deduced amino acid sequences of huHCA and raHCA are homologous to that of chicken BEN. Recombinant proteins produced from either human or rat HCA cDNAs were recognized by F84.1, whereas rat HCA but not human HCA was recognized by antirat KG-CAM. Expression of either form of huHCA in CHO cells conferred homophilic adhesion that could be competed with soluble recombinant huHCA-Fc. The molecular cloning of HCA and the availability of recombinant HCA should permit further evaluation of its role in human and rodent hematopoiesis.