Govardhana Rao Yannam

Differentiation and Transplantation of Human Embryonic Stem Cell–Derived Hepatocytes

BACKGROUND & AIMS The ability to obtain unlimited numbers of human hepatocytes would improve the development of cell-based therapies for liver diseases, facilitate the study of liver biology, and improve the early stages of drug discovery. Embryonic stem cells are pluripotent, potentially can differentiate into any cell type, and therefore could be developed as a source of human hepatocytes. METHODS To generate human hepatocytes, human embryonic stem cells were differentiated by sequential culture in fibroblast growth factor 2 and human activin-A, hepatocyte growth factor, and dexamethasone. Functional hepatocytes were isolated by sorting for surface asialoglycoprotein-receptor expression. Characterization was performed by real-time polymerase chain reaction, immunohistochemistry, immunoblot, functional assays, and transplantation. RESULTS Embryonic stem cell-derived hepatocytes expressed liver-specific genes, but not genes representing other lineages, secreted functional human liver-specific proteins similar to those of primary human hepatocytes, and showed human hepatocyte cytochrome P450 metabolic activity. Serum from rodents given injections of embryonic stem cell-derived hepatocytes contained significant amounts of human albumin and alpha1-antitrypsin. Colonies of cytokeratin-18 and human albumin-expressing cells were present in the livers of recipient animals. CONCLUSIONS Human embryonic stem cells can be differentiated into cells with many characteristics of primary human hepatocytes. Hepatocyte-like cells can be enriched and recovered based on asialoglycoprotein-receptor expression and potentially could be used in drug discovery research and developed as therapeutics.

Hepatic irradiation augments engraftment of donor cells following hepatocyte transplantation

Engraftment of donor hepatocytes is a critical step that determines the success of hepatocyte transplantation. Rapid and efficient integration of donor cells would enable prompt liver repopulation of these cells in response to selective proliferative stimuli offered by a preparative regimen. We have earlier demonstrated that hepatic irradiation (HIR) in combination with a variety of hepatotrophic growth signals, such as partial hepatectomy and hepatocyte growth factor, can be used as a preparative regimen for liver repopulation of transplanted hepatocytes. In this study, we investigated the effects of HIR on engraftment of transplanted dipeptidyl peptidase IV (DPPIV)–positive hepatocytes in congeneic DPPIV‐deficient rats. HIR‐induced apoptosis of hepatic sinusoidal endothelial cells (SEC) within 6 hours of HIR resulted in dehiscence of the SEC lining in 24 hours. Although there was no change of the number of Kupffer cells after HIR, colloidal carbon clearance decreased 24 hours post HIR, indicating a suppression of phagocytic function. DPPIV+ donor cells were transplanted 24 hours after HIR (0–50 Gy). There was an HIR dose‐dependent increase in the donor hepatocyte mass engrafted in the liver parenchyma. The number of viable transplanted hepatocytes present in hepatic sinusoids or integrated in the parenchyma was greater in the HIR‐treated group at 3 and 7 days after transplantation compared with the sham controls. Finally, we validated these rodent studies in cynomolgus monkeys, demonstrating that a single 10‐Gy dose of HIR was sufficient to enhance engraftment of donor porcine hepatocytes. These data indicate that transient disruption of the SEC barrier and inhibition of the phagocytic function of Kupffer cells by HIR enhances hepatocyte engraftment and the integrated donor cell mass. Thus, preparative HIR could be potentially useful to augment hepatocyte transplantation. (HEPATOLOGY 2009;49:258‐267.)

Spontaneous hepatic repopulation in transgenic mice expressing mutant human α1-antitrypsin by wild-type donor hepatocytes

α1-Antitrypsin deficiency is an inherited condition that causes liver disease and emphysema. The normal function of this protein, which is synthesized by the liver, is to inhibit neutrophil elastase, a protease that degrades connective tissue of the lung. In the classical form of the disease, inefficient secretion of a mutant α1-antitrypsin protein (AAT-Z) results in its accumulation within hepatocytes and reduced protease inhibitor activity, resulting in liver injury and pulmonary emphysema. Because mutant protein accumulation increases hepatocyte cell stress, we investigated whether transplanted hepatocytes expressing wild-type AAT might have a competitive advantage relative to AAT-Z-expressing hepatocytes, using transgenic mice expressing human AAT-Z. Wild-type donor hepatocytes replaced 20%-98% of mutant host hepatocytes, and repopulation was accelerated by injection of an adenovector expressing hepatocyte growth factor. Spontaneous hepatic repopulation with engrafted hepatocytes occurred in the AAT-Z-expressing mice even in the absence of severe liver injury. Donor cells replaced both globule-containing and globule-devoid cells, indicating that both types of host hepatocytes display impaired proliferation relative to wild-type hepatocytes. These results suggest that wild-type hepatocyte transplantation may be therapeutic for AAT-Z liver disease and may provide an alternative to protein replacement for treating emphysema in AAT-ZZ individuals.

Human Hepatocytes and Hematolymphoid Dual Reconstitution in Treosulfan-Conditioned uPA-NOG Mice

Human-specific HIV-1 and hepatitis co-infections significantly affect patient management and call for new therapeutic options. Small xenotransplantation models with human hepatocytes and hematolymphoid tissue should facilitate antiviral/antiretroviral drug trials. However, experience with mouse strains tested for dual reconstitution is limited, with technical difficulties such as risky manipulations with newborns and high mortality rates due to metabolic abnormalities. The best animal strains for hepatocyte transplantation are not optimal for human hematopoietic stem cell (HSC) engraftment, and vice versa. We evaluated a new strain of highly immunodeficient nonobese diabetic/Shi-scid (severe combined immunodeficiency)/IL-2Rγc(null) (NOG) mice that carry two copies of the mouse albumin promoter-driven urokinase-type plasminogen activator transgene for dual reconstitution with human liver and immune cells. Three approaches for dual reconstitution were evaluated: i) freshly isolated fetal hepatoblasts were injected intrasplenically, followed by transplantation of cryopreserved HSCs obtained from the same tissue samples 1 month later after treosulfan conditioning; ii) treosulfan conditioning is followed by intrasplenic simultaneous transplantation of fetal hepatoblasts and HSCs; and iii) transplantation of mature hepatocytes is followed by mismatched HSCs. The long-term dual reconstitution was achieved on urokinase-type plasminogen activator-NOG mice with mature hepatocytes (not fetal hepatoblasts) and HSCs. Even major histocompatibility complex mismatched transplantation was sustained without any evidence of hepatocyte rejection by the human immune system.

The microenvironment in hepatocyte regeneration and function in rats with advanced cirrhosis.

In advanced cirrhosis, impaired function is caused by intrinsic damage to the native liver cells and from the abnormal microenvironment in which the cells reside. The extent to which each plays a role in liver failure and regeneration is unknown. To examine this issue, hepatocytes from cirrhotic and age‐matched control rats were isolated, characterized, and transplanted into the livers of noncirrhotic hosts whose livers permit extensive repopulation with donor cells. Primary hepatocytes derived from livers with advanced cirrhosis and compensated function maintained metabolic activity and the ability to secrete liver‐specific proteins, whereas hepatocytes derived from cirrhotic livers with decompensated function failed to maintain metabolic or secretory activity. Telomere studies and transcriptomic analysis of hepatocytes recovered from progressively worsening cirrhotic livers suggest that hepatocytes from irreversibly failing livers show signs of replicative senescence and express genes that simultaneously drive both proliferation and apoptosis, with a later effect on metabolism, all under the control of a central cluster of regulatory genes, including nuclear factor κB and hepatocyte nuclear factor 4α. Cells from cirrhotic and control livers engrafted equally well, but those from animals with cirrhosis and failing livers showed little initial evidence of proliferative capacity or function. Both, however, recovered more than 2 months after transplantation, indicating that either mature hepatocytes or a subpopulation of adult stem cells are capable of full recovery in severe cirrhosis. Conclusion: Transplantation studies indicate that the state of the host microenvironment is critical to the regenerative potential of hepatocytes, and that a change in the extracellular matrix can lead to regeneration and restoration of function by cells derived from livers with end‐stage organ failure. (HEPATOLOGY 2011)

IL-23 in Infections, Inflammation, Autoimmunity and Cancer: Possible Role in HIV-1 and AIDS

The growing family of interleukin (IL)-12-like cytokines produced by activated macrophages and dendritic cells became the important players in the control of infections, development of inflammation, autoimmunity and cancer. However, the role of one of them—heterodimer IL-23, which consists of IL12p40 and the unique p19 subunit in HIV-1 infection pathogenesis and progression to AIDS, represent special interest. We overviewed findings of IL-23 involvement in control of peripheral bacterial pathogens and opportunistic infection, central nervous system (CNS) viral infections and autoimmune disorders, and tumorogenesis, which potentially could be applicable to HIV-1 and AIDS.

Acute transverse myelitis and paralysis in a kidney–pancreas recipient

Acute transverse myelitis (ATM), a group of neurologic disorders with an incidence of one to four cases per million people per year, is characterized by focal inflammation of the spinal cord resulting in sensory, motor, and autonomic dysfunction. ATM may be caused by infection, spinal cord infarction, pre-existing inflammatory disease (e.g., SLE), or de novo autoimmunity (idiopathic) [1]. Acute transverse myelitis typically presents with thoracic sensory loss and focal acute inflammation of the spinal cord visible on MRI. Examination of lumbar cerebrospinal fluid (CSF) reveals significant pleocytosis without evidence for infection. Motor loss involves the lower extremities bilaterally but can include the upper extremities in severe cases. These symptoms reach peak severity between 4 h and 21 days after onset [2]. The diagnosis of ATM includes ruling out radiation exposure, vascular events, connective tissue disorders, and infection. MRI of the brain and spine is necessary to rule out compressive etiologies. Laboratory studies of CSF is required to rule out syphilis, Lyme disease, mycoplasma, human immunodeficiency virus, human lymphotropic virus-1, or other viral infection. CSF examination may detect proteins that may aid in making the diagnosis and predicting the severity of ATM [1–4]. Treatment for autoimmune ATM includes high-dose steroids, antimetabolites, and plasmapheresis [5]. Approximately equal proportions of ATM patients recover with little neurologic deficit, moderate disability (ambulation with assistance), or permanent severe disability [2–4,6]. Our patient is a 35-year-old African-American male with Type I diabetes and a strong family history of autoimmune diseases. He underwent deceased donor simultaneous kidney and pancreas transplantation with T-cell depleting rabbit anti-thymocyte globulin (rATG) induction (total of 7 mg/kg) with methylprednisolone (total of 15 mg/kg). Maintenance immunosuppression consisted of mycophenolate mofetil and tacrolimus started on postoperative day (POD) 1 and 5 respectively. On POD 10, he developed a urinary tract infection with Enterobacter aerogenes for which ciprofloxacin was initiated. On POD 16, he developed a right lower lobe pneumonia, was intubated, and treated with imipenemcilastatin and linezolid. Continued fever after resolution of pneumonia led to exploratory laparotomy without significant findings. On POD 25, physical examination revealed poor muscle tone in both upper and lower extremities. An MRI showed normal brain and lumbar spine but increased T2 signal of the spinal cord from C5–6 to T4 consistent with myelitis (Fig. 1a and b). CSF revealed a greatly elevated protein level (709 mg/dl) and subsequent CSF cultures and antigen testing were all negative. Intravenous methylprednisolone (500 mg b.i.d.) was started and initially the patient defervesced, but within 4 days he was persistently febrile (>38 C) despite wide-spectrum antibiotics and high-dose steroids. Multiple follow-up bacterial cultures, virologic and serologic studies were negative. On POD 27, the patient appeared to improve somewhat and was extubated. Neurologic examination revealed a lack of pinprick sensation from chest to thighs, inability to move lower extremities, and gross but not fine motor movement in upper extremities. The patient remained febrile, and a follow-up MRI performed 7 days later (Fig. 1c and d) showed significant progression. A followup lumbar puncture showed decline of CSF protein to 243 mg/dl and despite continued negative microbial, viral, and serologic studies the patient remained febrile. Follow-up MRI on POD 52 (Fig. 1e and f) showed progression of transverse myelitis to T10-11 and an electromyographic study showed severe axonal sensorimotor polyneuropathy. Plasmapheresis was initiated every other day for a total of seven treatments. He reported temporary improvement in sensation in his abdomen and legs while receiving plasmapheresis. After his last treatment, high-dose steroids were restarted and subsequently tapered to 5 mg of oral prednisone daily. A repeat MRI on POD 67 showed no further progression or resolution of the transverse myelitis. The patient eventually became afebrile and was discharged 81 days after transplantation with a T2–3 complete paraplegia and partial involvement of the left C8–T1 dermatomes and myotomes. To date, the patient has shown no further recovery despite a long course of steroids. MRI performed 8 months later showed extensive